肿瘤 · 本周文献汇编

TBL1XR1 is frequently mutated in diffuse large B-cell lymphoma (DLBCL), yet its functional impact on tumor microenvironment remains poorly defined. In this study, we characterized TBL1XR1 mutations in a cohort of 1842 newly diagnosed DLBCL patients, identifying mutations in 9.4% of cases (n = 173). These mutations correlated with clinically aggressive features, including older age (> 60 years), high-risk International Prognostic Index, enrichment in non-GCB subtypes, as well as inferior progression-free and overall survival. Mechanistically, TBL1XR1 mutations enhanced H3K27ac levels at the MYC promoter, increased MYC expression and subsequently up-regulated its immune-regulatory targets CD47 and PD-L1. This axis impaired natural killer (NK) cytotoxicity, facilitating immune escape and tumor progression. In a murine A20 B-lymphoma model, tumors harboring Tbl1xr1 mutations exhibited up-regulated MYC, CD47, and PD-L1 expression, resulting in NK cell dysfunction and tumor growth acceleration via the MYC-CD47/PD-L1 axis. Dual blockade of CD47 and PD-L1 restored NK cell-mediated tumor immunity, triggering rapid regression of Tbl1xr1-mutated tumors. Taken together, our findings identified TBL1XR1 mutations as a microenvironment-related mechanism of DLBCL progression and provided clinical rationale of co-targeting CD47 and PD-L1 to treat this genetically defined subset.

Patients with myeloproliferative neoplasms (MPN), including polycythemia vera (PV) and essential thrombocythemia (ET), have an increased risk of second cancers (SC), although determinants of risk remain incompletely defined. We retrospectively analyzed 1968 consecutive patients with PV or ET (median follow-up 11.2 years) to identify predictors of SC. Cumulative incidence functions were estimated using competing-risk methodology, and predictors were assessed using Fine-Gray regression. During follow-up, SC occurred in 404 patients (20%), with non-melanoma skin cancer (NMSC) representing the most frequent subtype. In multivariable models including all cancer types, prior cancer (sHR 2.02, p < 0.001), older age (per 10 years: sHR 1.20, p < 0.001), male sex (sHR 1.39, p = 0.002), and hyperlipidemia (sHR 1.41, p = 0.003) independently predicted SC. After excluding NMSC, prior cancer remained associated with SC risk (sHR 1.48, p = 0.048). Notably, prior NMSC strongly predicted subsequent NMSC (sHR 6.48, p < 0.001), followed by prior non-NMSC cancer (sHR 2.11, p < 0.001), age (sHR 1.29, p < 0.001), and male sex (sHR 1.43, p = 0.018). TET2 mutations showed a borderline association with NMSC risk (sHR 1.73, p = 0.055), while hydroxyurea was not associated with SC, with a non-significant trend toward increased NMSC risk (sHR 1.82, p = 0.110). These findings support risk-adapted cancer surveillance in PV and ET, with particular emphasis on regular dermatologic monitoring.

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, life-threatening hematologic disease with diagnostic delays exceeding 5 years in 24% of cases. We developed and deployed an artificial intelligence algorithm analyzing structured and unstructured electronic health record data across 14 healthcare organizations in Poland. Screening of 1,307,140 patients identified 356 high-risk individuals; of 119 referred for flow cytometry, 13 were diagnosed (positive predictive value: 10.92%; 95% CI, 9.68%-12.30%), comparing favourably to 6.9% conventional screening hit rate. High-risk patients were significantly older (median 69.5 years) with elevated rates of fatigue (76.4% vs 29.19%), anaemia (72.2% vs 7.61%), and myelodysplastic syndrome (49.2% vs 0.24%; all p < 0.001). Only 2.25% presented with haemoglobinuria versus 45-62% in registry cohorts. Retrospective analysis revealed potentially preventable diagnostic delays of 74-1337 days. Monte Carlo feature selection identified Coombs-negative haemolysis and visit frequency as strongest predictors, supporting the potential utility of AI-assisted screening for identifying atypical PNH presentations.

Remarkable progress in the understanding of disease pathogenesis and treatment across hematologic malignancies has been achieved in the past 2 decades. Nevertheless, the reliable elimination of disease remains elusive for many cancers. Chronic lymphocytic leukemia (CLL) exemplifies the needs that must be addressed to close the gap between discovery science and the remaining clinical challenges. In CLL, targeted therapies have substantially prolonged survival and enabled long-term disease control for many patients. However, curative outcomes remain exceptional, particularly in high-risk groups such as those with TP53 disruption, dual resistance to Bruton tyrosine kinase and B-cell lymphoma 2 inhibitors, or transformation to aggressive lymphoma. Recent insights into the interconnection between cancer and immunity have positioned CLL as a model example of cancer-associated immunodeficiency, a realization brought into sharp focus by the severe acute respiratory syndrome coronavirus 2 pandemic during which patients with CLL were at extremely high-risk for infection and poor outcomes. Therefore, complications related to infections, autoimmunity, and secondary cancers continue to contribute substantially to morbidity and mortality, underscoring the need for research on immune dysfunction in CLL. Furthermore, pronounced heterogeneity in disease progression and therapeutic resistance highlight the need for mechanistic studies to clarify these distinct biological patterns. Advances in these areas not only hold the promise of curative therapy for broader patient subgroups in CLL but will also inform innovation in research on other cancers, particularly in establishing a molecular definition of disease and defining those interactions with the underlying and resultant immune deficiencies.

The B-cell maturation antigen (BCMA)-targeting bispecific T-cell engager pavurutamab (AMG 701) directs the cytotoxic T-cell response toward multiple myeloma (MM) cells. This phase 1/1b, open-label, dose-exploration and dose-expansion study evaluated the safety, tolerability, and efficacy of pavurutamab monotherapy in patients with triple-class relapsed/refractory MM (RRMM). Pavurutamab (5-18 000 μg) was administered IV weekly with step-up dosing in week 1. Overall, 172 patients received at least 1 dose of pavurutamab; 73 received the recommended phase 2 dose (RP2D; 18 000 μg), with 2 different step-up dosing regimens in phase 1b. Twelve patients had dose-limiting toxicities, which included cytokine release syndrome (CRS) and increased transaminases; none of which occurred at the RP2D. The most frequently reported treatment-emergent adverse events included CRS (74.4%), anemia (61.0%), neutropenia (47.1%), and hypophosphatemia (45.3%). Grade ≥3 infections were noted in 60 patients (34.9%). The overall response rate (ORR) was 46.5% among all patients and 65.8% (very good partial response [VGPR] or better, 60.3%) among 73 patients treated at the RP2D. At median follow-up of 17.2 months, median duration of response (DOR) was 36.6 months (95% confidence interval [CI], 22.3 to not estimable). Median progression-free survival (PFS) for all patients was 5.5 months (95% CI, 2.8-10.1) and 16.8 months (95% CI, 5.0 to not estimable) with the RP2D. Pavurutamab exposure generally increased in a dose-proportional manner, with high soluble BCMA levels correlating with lower exposure level. The acceptable safety profile, pharmacokinetics, and preliminary efficacy of pavurutamab supports anti-BCMA T-cell engager therapy in heavily pretreated patients with RRMM. This trial was registered at www.clinicaltrials.gov as NCT03287908.

We describe a case of secondary peripheral T-cell lymphoma (PTCL) arising from infused CD19/CD22-4-1BB-CD3ζ-lenti bispecific chimeric antigen receptor (CAR)-T cells in a patient with relapsed or refractory B-cell lymphoma who carried a pathogenic germline BLM p.L107Ffs*36 variant. Integrated genomic and molecular analyses confirmed that the PTCL was clonally derived from the infused CAR-T product and revealed a multistep oncogenic process involving pre-existing driver mutations (BLM), somatic TET2 mutations (p.R1261H and p.E1755*), and genomic instability. Clonal CAR vector integration events in cancer-associated genes (NF1, CBX5, RNF213, etc) were identified as markers of clonal expansion, although no functional evidence supports a direct oncogenic role. We acknowledge that this case lies on a spectrum between clonal lymphoproliferative disorder and overt PTCL, and terminology continues to evolve. This case suggests a need for re-biopsy of suspected relapses after CAR-T therapy and highlights the limitations of conventional product assessment in detecting premalignant clones.

CD7 is a promising target for chimeric antigen receptor (CAR) T-cell therapy in T-cell lymphoid malignancies; however, antigen loss-mediated relapse emerged as a major challenge. Herein, we systematically analyzed the genetic and epigenetic alterations of paired specimens (pre-treatment and relapsed) from 10 patients with T-cell lymphoma/leukemia receiving CD7 CAR-T cells. Overall, we identified three distinct mechanisms underlying CD7 loss: Firstly, frameshift insertion (Pt4; c.164dupG:p.R55fs) or deletion (Pt7; c.122delG:p.G41Efs*19) resulting in truncation of the CD7 transmembrane domain in two of ten patients; Secondly, hypermethylation of the CD7 promoter in seven of ten patients without CD7 mutation; Thirdly, simultaneous occurrence of promoter region hypermethylation and multiple in-frame mutations with predicted functional interference in one of ten patients (Pt2). Collectively, these findings demonstrated that both clonal heterogeneity and epigenetic plasticity drive antigen-negative relapse in T-cell lymphoid malignancies under the selective pressure of CD7 CAR-T therapy.

The impact of chimeric antigen receptor (CAR)-T cell expansion and persistence on clinical outcomes and treatment-related morbidity in patients with relapsed/refractory multiple myeloma (RRMM) remains incompletely defined, in part due to limited availability of standardized CAR-T cell quantification assays. We evaluated CAR-T cell kinetics and their association with efficacy and toxicity in RRMM patients treated with idecabtagene vicleucel (ide-cel) or ciltacabtagene autoleucel (cilta-cel). Using a uniform flow cytometry-based platform (N=90; cilta-cel, n=54; ide-cel, n=36), we observed significantly greater CAR-T cell expansion with cilta-cel than with ide-cel (median 106 vs 49 cells/uL). Peak CAR-T cell expansion was associated with clinical response in the ide-cel cohort but not with cilta-cel, where rapid and excessive expansion was instead associated with an increased risk of delayed neurotoxicities (DNTs), a complication with potential long-term functional consequences (median peak 1,009 vs 96 cells/uL). To identify clinically accessible biomarkers of CAR-T cell expansion, we analyzed absolute lymphocyte count (ALC) as a surrogate biomarker in a larger multicenter cohort (N=532; cilta-cel, n=256; ide-cel, n=276). Higher peak ALC was significantly associated with the development of DNTs, particularly Parkinsonism after cilta-cel. A peak ALC ³3000/uL - or ³2500/uL following a daily twofold increase - predicted elevated DNT risk (sensitivity 81%, specificity 59%). Together, these findings delineate distinct expansion-toxicity relationships in cilta-cel and ide-cel therapy, establish ALC as a practical, uniformly available surrogate for CAR-T cell expansion, and define quantitative thresholds that may enable early recognition of patients at risk for DNT, informing preemptive strategies to mitigate morbidity following cilta-cel.

The clinical course of Mantle Cell Lymphoma (MCL) varies between individual patients. Early detection of risk is crucial to assign MCL patients to novel treatment strategies. Most of the established biomarkers of outcome require specifically trained pathologists or molecular analysis. Here we introduce MAIPI (MCL Artificial Intelligence Prognostic Index), a deep learning algorithm trained only on Hematoxylin and Eosin (H&E) images of diagnostic biopsies of n = 428 MCL patients from clinical trials to assess prognosis. The capability of MAIPI to predict disease outcome was validated in an independent cohort of n = 140 patients treated with immunochemotherapy with and without ibrutinib. MAIPI selects areas of interest by itself and provides prognostic information independent of the MCL International Prognostic Index (MIPI) and Ki67 and without the need of molecular testing or expert pathologists evaluation.

Tetraspanin CD37, highly expressed in mature B-cells, represents an opportunity for therapeutic targeting in B-cell malignancies. GEN3009 (DuoHexaBody-CD37), a humanized biparatopic IgG1 antibody with an E430G hexamerization-enhancing mutation targeting two non-overlapping CD37 epitopes, was shown to induce potent tumor cell killing through enhanced complement-dependent cytotoxicity (CDC) and other fragment crystallizable-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), in vitro and in vivo. GEN3009 was assessed in non-clinical studies and a phase 1 dose-escalation study of B-cell non-Hodgkin's lymphoma (B-NHL). After a non-clinical toxicity study was conducted in cynomolgus monkeys, a phase 1, first-in-human, open-label, multicenter dose-escalation trial of GEN3009 monotherapy enrolled adults with relapsed/refractory (R/R) B-NHL (NCT04358458). A modified Bayesian optimal interval design informed dose escalation and de-escalation with dose levels ranging from 6 mg to 2000 mg. Primary endpoints were the rate of dose-limiting toxicities (DLTs) to determine the recommended phase 2 dose (RP2D) and safety and tolerability. In the toxicity study, 10 mg/kg GEN3009 weekly was identified as the highest non-severely toxic dose. In the phase 1 study, from March 13, 2020, to July 28, 2023, 46 patients with R/R B-NHL received intravenous GEN3009 infusions. Median duration of treatment was 1.2 months (range, 0.0-14.5). No DLTs were observed up to 1600 mg. The most common treatment-emergent adverse events were neutropenia (n=41 (89.1%)), infusion-related reactions (n=39 (84.8%)), and thrombocytopenia (n=18 (39.1%)). Plasma GEN3009 concentrations increased over time with increasing GEN3009 doses, with no apparent accumulation of GEN3009 observed over the course of treatment. Antitumor activity was observed at dose levels of ≥180 mg in both aggressive and indolent NHL. GEN3009 treatment reduced total hemolytic complement activity (CH50) levels in serum, and peak changes in CH50 levels were significantly correlated with clinical response (p=0.008 by Wilcoxon rank-sum test). Based on safety, efficacy, and pharmacokinetics, the RP2D of GEN3009 was determined to be 1200 mg. Preliminary data suggest that GEN3009 monotherapy demonstrated an acceptable safety profile at the RP2D of 1200 mg, with modest clinical activity. These findings provide the first clinical proof-of-concept for hexamerization-potentiated molecules that induce antitumor activity through enhanced CDC.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an orphan, aggressive hematologic malignancy characterized by high CD123 expression. Tagraxofusp (TAG), a CD123-directed toxin, is the only approved therapy. Prior studies demonstrated BPDCN dependence on BCL2, sensitivity to venetoclax (VEN), and reversal of TAG resistance with azacitidine (AZA). We conducted a phase 2 study evaluating combination therapy with TAG, AZA, and VEN in patients with BPDCN (NCT03113643). Patients with previously untreated (1L) or relapsed/refractory (R/R) BPDCN received 28-day cycles of AZA (75 mg/m² days 1-7), VEN (400 mg days 1-21), and TAG (12 µg/kg days 4-6). Eligibility followed TAG guidelines to mitigate risk of capillary leak syndrome (CLS). Twenty-seven patients were enrolled (16 1L, 11 R/R), with median age of 70 years (range 21-81). Composite complete remission (CR/CRi/CRc) rates were 88% in 1L and 64% in R/R cohorts. Median duration of response was not reached in 1L and was 7.2 months in R/R patients. CLS occurred in 15% of patients; most were grade 2. In the 1L cohort, median overall and progression-free survival were not reached; the 2-year overall survival was 65% and 2-year progression-free survival was 53%. Median overall survival in R/R patients was 8.4 months. A high proportion of patients proceeded to allogeneic stem cell transplantation in remission (63% 1L, including 10 of 11 patients age 75 or younger; and 55% R/R). TAG-AZA-VEN is highly active in both untreated and relapsed BPDCN with a predictable and manageable safety profile, supporting its use as a new therapeutic option.

BCMA and GPRC5D-directed therapies have shown high efficacy in the treatment of relapsed or refractory myeloma with multiple new immune therapies now approved. Preclinical studies have shown that expression of BCMA and GPRC5D are heterogenous in malignant plasma cells. We conducted a phase I dose escalation trial of the BCMA CAR T cell therapy MCARH125 alone or in combination with the GPRC5D CAR T cell therapy MCARH109 in patients with heavily pre-treated relapsed or refractory multiple myeloma. Patients were treated across three dose levels and the primary objective of establishing safety of the concurrent infusion. The trial is registered in ClinTrials.gov, NCT05431608 and has now completed accrual. We treated 15 patients across 3 dose levels; this included 6 patients who received MCARH125 alone and 9 patients treated with concurrent infusion of MCARH125 and MCARH109. 1 patient each with MCARH 125 alone and the co-infusion of MCARH125 and MCARH109 developed immune effector cell associated hemophagocytic syndrome (IEC-HS). There were no instances of grade 3 or higher cytokine release syndrome (CRS) or immune effector cell associated neurologic syndrome (ICANS). The overall response for co-infusion was 78% with a median PFS of 18.2 months. Correlative analysis suggests that antigen loss maybe an important mechanism of resistance even with dual-antigen targeting and we show for the first time that expansion of one CAR population can limit expansion of the second population, as dual-CAR treated patients had significantly less BCMA-CAR expansion than BCMA-CAR alone treated patients, despite equivalent BCMA CAR T cell doses at infusion.

Marginal zone lymphoma (MZL) involving multiple muscles and subcutaneous adipose tissue is extremely rare. We present PET/CT findings of pathologically confirmed MZL with muscle and subcutaneous involvement in a 63-year-old man. 18F-FAPI-04 PET/CT demonstrated significantly higher radiotracer uptake and better lesion detectability than 18F-FDG PET/CT in all detectable lesions and, in addition, identified occult right neck muscle involvement not clearly appreciable on 18F-FDG PET/CT. This case highlights the superiority of 18F-FAPI-04 PET/CT over 18F-FDG PET/CT for evaluating MZL with muscle and subcutaneous adipose tissue involvement, especially for mildly FDG-avid lesions.

Retrospective studies suggested that autologous stem cell transplantation (auto-SCT) might achieve comparable or even better outcomes compared with allogeneic SCT for patients with genetic intermediate-risk acute myeloid leukemia (IR-AML) and no detectable measurable residual disease (MRD-). However, prospective studies are lacking comparing auto-SCT and matched sibling donor SCT (MSD-SCT). We initiated a biological assignment comparative study at fourteen institutions in China. Patients with newly diagnosed de novo IR-AML and MRD- within 3 cycles of intensive chemotherapy were eligible for the study if they were 14 to 60 years old and received auto-SCT or MSD-SCT. Patients were assigned to auto-SCT or MSD-SCT based on donor availability. The primary endpoint was the 3-year disease-free survival (DFS). The trial was registered with Clinicaltrials.gov, NCT03620955 and completed. Between November 15, 2018, and March 31, 2022, 708 patients were screened and 518 eligible for analysis, including 259 in the auto-SCT and 259 in the MSD-SCT groups. With a median follow-up time of 55.2 (IQR 47.4-65.2) months, the 3-year DFS was 82.2% (95% CI 77.0-86.4) and 85.7% (95% CI 80.8-89.4) in auto-SCT and MSD-SCT respectively (HR, 1.24 [95% CI, 0.81-1.91]; p=.32). The treatment-adjusted difference was 3.2% (95% CI, - 3.1 to 9.4; p<.001). Because the upper bound of the 95% CI (9.4) did not exceed 10%, noninferiority was shown. The 3-year overall survival (OS) was 90.7% (95% CI 86.5-93.7) and 87.6% (95% CI 83.0-91.1) respectively (HR, 0.78 [95% CI, 0.47-1.30]; p=.34). In subgroup analyses, auto-SCT achieved similar DFS and OS to MSD-SCT among patients who reached MRD- in the first 2 cycles of chemotherapy, while auto-SCT had inferior DFS but similar OS for patients who achieved MRD- after 3 cycles. Auto-SCT achieved non-inferior DFS and similar OS compared with MSD-SCT, suggesting that auto-SCT is a viable and preferable first-line choice for IR-AML patients achieving MRD- within 3 cycles of chemotherapy, particularly those who reached MRD- in the first 2 cycles.

Tagraxofusp is a CD123-targeted therapy comprised of a recombinant human interleukin-3 (IL-3) fused to a truncated diphtheria toxin payload. It is the first approved treatment specifically for patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN). To identify biomarkers of response, bone marrow samples from 12 BPDCN patients who were treated with tagraxofusp in the pivotal phase II trial (NCT02113982) were profiled longitudinally using a gene panel and single-cell RNA sequencing. Residual tumor cells following tagraxofusp expressed lower levels of TXNRD1 that would reduce the efficacy of tagraxofusp. In support of this, enzymatic inhibition of TXNRD1 resulted in higher viability of CAL-1 BPDCN cells following tagraxofusp. Responders had either wild-type or missense TET2 mutations, while transient and non-responders had at least one truncating TET2 mutation. Examples of these mutations within the catalytic domain of TET2 were constructed and transduced into cells. Missense and truncating mutants displayed reduced sensitivities to hypomethylating agents and prolonged S-phase stasis. These results suggest that the levels of TXNRD1 interact with intrinsic TET2 truncating mutations within the bone marrow to modulate patient response to tagraxofusp.

Decentralized clinical trials (DCTs) are clinical studies where part or all trial-related activities occur remotely, reducing or eliminating the need for patients to physically visit a research site. By applying a more patient-centered design, DCTs have the potential to enhance patient access to clinical trials, improve enrollment and retention, and increase convenience, while simultaneously decreasing costs. This perspective explores the growing importance of DCTs, including the opportunity to facilitate engagement of patients with rare diseases and their broader implications for improving access to care. It also examines the challenges of implementing a DCT, comprising logistical complexities, reliance on technology, and regulatory considerations. Drawing upon our direct experience designing and executing a DCT for patients with Clonal Cytopenia of Undetermined Significance, we discuss our study design, share key pitfalls encountered and lessons learned regarding technology integration, site coordination, and patient engagement. Based on these experiences, we offer recommendations for optimizing DCT design, addressing challenges, and guiding future trials to fully realize the potential of decentralized models, ultimately fostering more equitable and patient-centric clinical research.

After five decades of CHOP-based therapy, the treatment landscape for patients with newly diagnosed large B-cell lymphoma (LBCL) is rapidly evolving. Novel agents, including antibody-drug conjugates, bispecific antibodies, targeted therapies, and chimeric antigen receptor (CAR) T-cell therapies, have demonstrated significant activity, raising the potential for improved outcomes from first-line treatment. However, this progress has introduced substantial complexity without a clear framework for clinical decision-making. Ongoing phase 3 trials employ heterogeneous designs, including differing control arms, eligibility criteria, and endpoints, yet share a common limitation: the inconsistent incorporation of biomarker-driven patient selection. As a result, cross-trial comparisons are challenging, and identifying the optimal regimen for an individual patient remains difficult. Although the POLARIX trial established polatuzumab-based therapy (R-CHP-pola) as superior to R-CHOP, many ongoing studies continue to use R-CHOP as the comparator, further complicating interpretation. Moreover, "R-CHOP + X" strategies evaluated in clinically defined populations without biologic stratification may yield modest benefits across heterogeneous groups, limiting their applicability to individual patients. These trends risk producing a fragmented landscape of effective yet non-comparable regimens without a principled framework for patient selection. We thus propose a harmonized, data-driven strategy to shape the next era of LBCL therapy: (1) pooled patient-level analyses; (2) standardized biomarker and minimal residual disease platforms; (3) AI-enabled predictive modeling integrating trial and real-world data; and (4) regulatory approaches promoting collaborative trial design and shared biomarker analyses. Ultimately, progress in LBCL will depend on aligning data, biology, and clinical decision-making to deliver precise, equitable, and effective care.

Orelabrutinib is a potent, irreversible, and highly-selective BTK inhibitor that has been approved for the treatment of relapsed/refractory chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL). This randomized, phase 3 study (ClinicalTrials.gov identifier: NCT04578613) compared orelabrutinib with chemoimmunotherapy in patients with treatment-naïve CLL/SLL. From February 20, 2021, to July 8, 2024, 192 eligible patients were randomly assigned (1:1) to receive either orelabrutinib (91 patients) or chlorambucil plus rituximab (101 patients), comprising the intention-to-treat population. At a median follow-up of 21.4 months (data cutoff, May 17, 2024), the primary endpoint of progression-free survival (PFS) per independent review committee (IRC) was not reached (NR; 95% CI, not estimable [NE]-NE) with orelabrutinib versus 19.4 months (95% CI, 16.6-NE) with chlorambucil plus rituximab (hazard ratio [HR], 0.32; 95% CI, 0.18-0.58; p < 0.0001; crossing the efficacy boundary). The IRC-assessed overall response rate (90.1% vs 79.2%; p = 0.041) and duration of response (HR, 0.30; 95% CI, 0.15-0.60; p = 0.0003) also favored orelabrutinib over chlorambucil plus rituximab. In the safety population, treatment-related adverse events occurred in 82 of 91 patients (90.1%) receiving orelabrutinib and 89 of 98 patients (90.8%) receiving chlorambucil plus rituximab, with 32 (35.2%) and 59 (60.2%) at grade 3 or worse, respectively. Orelabrutinib maintained or improved patient-reported outcomes compared with chemoimmunotherapy. In summary, orelabrutinib significantly improved PFS and response versus chemoimmunotherapy in patients with treatment-naïve CLL/SLL, with a manageable safety profile, supporting it as an effective alternative first-line option.

POEMS syndrome is a life-threatening syndrome due to an underlying plasma cell neoplasm. The major criteria for the syndrome are polyneuropathy, clonal plasma cell disorder (PCD), sclerotic bone lesions, elevated vascular endothelial growth factor, and the presence of Castleman disease. Minor features include organomegaly, endocrinopathy, characteristic skin changes, papilledema, extravascular volume overload, and thrombocytosis. The diagnosis of POEMS syndrome is made with three of the major criteria, two of which must include polyneuropathy and clonal plasma cell disorder, and at least one of the minor criteria. Because the pathogenesis of the syndrome is not well understood, risk stratification is limited to clinical phenotype rather than specific molecular markers. Risk factors include low serum albumin, age, pleural effusion, pulmonary hypertension, and reduced eGFR. For those patients with a dominant plasmacytoma, first line therapy is irradiation. Patients with diffuse sclerotic lesions or disseminated bone marrow involvement should receive systemic therapy. Corticosteroids are temporizing, but alkylators and lenalidomide are the mainstays of treatment, the former either in the form of low dose conventional therapy or as high-dose conditioning for stem cell transplantation. Thalidomide and bortezomib also have activity, but their benefit needs to be weighed against their risk of exacerbating the peripheral neuropathy. Daratumumab combinations also appear promising based on case series. Prompt recognition and institution of both supportive care measures and therapy directed against the plasma cell result in the best outcomes.

Bone marrow failure (BMF) syndromes are heterogeneous diseases characterized by impaired hematopoiesis and a risk of evolution to myelodysplastic syndrome (MDS) and leukemia. We report 6 unrelated individuals with variable BMF phenotypes and hypocellular MDS, presenting at a median age of 10 years (range, 4 weeks to 53 years). Genomic analysis revealed germ line heterozygous variants in mouse double minute 4 (MDM4), including 4 null (frameshift, nonsense, and splice site resulting in premature truncation confirmed by RNA sequencing) and 2 missense variants, of which 1 had previously been associated with a familial BMF syndrome. Mechanistically, MDM4 mutations are loss-of-function mutations leading to enhanced p53 activation. We used CRISPR/Cas9 to delete MDM4 in healthy donor hematopoietic stem and progenitor cells (HSPCs). The resulting MDM4-haploinsufficient HSPCs exhibited increased p53 activity, impaired colony-forming capacity, and reduced engraftment potential in immunodeficient mice. Complementation studies revealed both p53-binding and RING-finger domains as necessary for MDM4-mediated hematopoietic regulation. To study variant effects in a confounder-free genetic background, we introduced patient-specific MDM4 variants into induced pluripotent stem cells (iPSCs). MDM4-mutant iPSCs yielded significantly reduced erythroid and myeloid cells and exhibited increased p53 activity, as evidenced by elevated p21 expression, confirming the role of MDM4 in regulating hematopoiesis through p53. Transcriptome analysis of iPSC-derived hematopoietic cells revealed upregulation of the p53 pathway. Importantly, 1 patient with MDS acquired loss-of-function TP53 mutations, suggesting maladaptive somatic rescue. Our findings establish MDM4 deficiency as a TP53-activating syndrome, with features of BMF and variable hematopoietic manifestations. This study also highlights the critical role of the MDM4-p53 axis in maintaining hematopoietic homeostasis.

Acute myeloid leukaemia (AML) is an aggressive blood cancer characterized by the unregulated proliferation of immature myeloblasts. Gene mutations have been shown to have a large effect on pathogenesis, inter-tumour heterogeneity and clinical outcomes in AML1-8; however, the role of epigenetic alterations in these respects has been investigated less extensively. Here we use ATAC-seq (assay for transposase-accessible chromatin with sequencing) in a cohort of 1,563 individuals with a recent diagnosis of AML (the 'eCHROMA' cohort) to show that AML can be classified into 16 subgroups on the basis of chromatin accessibility profiles. Multiomics analyses of gene mutations, the transcriptome, DNA methylation and histone marks show that these ATAC subgroups exhibit distinct driver mutations, differentiation states, gene expression, DNA methylation and super-enhancer profiles, and are also associated with clinical outcomes. These findings were validated in independent cohorts. Single-cell ATAC sequencing reveals that all leukaemic cells in each subgroup share a common chromatin accessibility profile, which suggests that subgroup-specific epigenomic fingerprints underlie the ATAC-based classification. Mechanistically, the subgroups have distinct gene-regulatory networks that are driven by the activities of key transcription factors in haematopoiesis, and in which subgroup-specific super-enhancers have a pivotal role. Multiomics single-cell analysis further reveals deregulated trajectories of differentiation coupled with chromatin accessibility and gene expression. Notably, ATAC subgroups have an independent prognostic effect, compared with genomic classification, and are associated with particular drug sensitivities. In summary, ATAC-based chromatin profiling, combined with multiomics data, provides insights into AML pathogenesis beyond genomics and constitutes a valuable resource for AML research.

WNK2 is a known tumor suppressor in a set of solid tumors, including glioblastoma multiforme and pancreatic ductal adenocarcinoma. WNK2 functions are largely unknown beyond the kinase domain (KD)-dependent inhibition of ERK1/2 signaling. Waldenström's Macroglobulinemia (WM) is an indolent, yet incurable, B cell lymphoma characterized by highly recurrent MYD88 (MYD88MUT) and CXCR4 (CXCR4MUT) mutations that trigger sustained NF-κB and ERK1/2 signaling. Although not expressed in healthy B cells, WNK2 is a top dysregulated gene in MYD88MUT WM. To study WNK2 regulation and signaling, we performed multi-omics analyses, including bulk and PacBio Iso-Seq RNA-Seq and methylome, in 264 untreated WM and functional studies in cell lines and primary WM cells. Aberrant expression of WNK2 emerged as a near universal feature of early-stage WM and a hallmark of plasma cell-like MYD88MUT WM. We identified novel isoforms that carried a shared aberrant splicing event, either contained or lacked the KD and were highly expressed by the tumor cells, unlike the canonical full-length isoforms. Functionally, WNK2/S-NK1, the most expressed of the KD-lacking isoforms in WM, triggered a pro-inflammatory cascade that activated ERK1/2 and NF-κB signaling. We observed a similar, cancer-specific upregulation of WNK2 in a set of solid tumors, including the highly aggressive cholangiocarcinoma. Our findings reveal an undocumented oncogenic function for WNK2 driven by novel, cancer-specific isoforms and provide a framework for its further investigation as a determinant of disease progression in MYD88MUT WM and a novel therapeutic target in hematological and solid tumor oncology.

Multiple myeloma progresses from precursor states to active disease, and studying tumor microenvironment (TME) evolution across these stages is key to understanding immune dysregulation and therapeutic resistance. Here, we integrated paired single-cell RNA, T-, and B-cell receptor sequencing data from bone marrow samples of 235 patients spanning the disease spectrum. This dataset revealed dynamic changes in the abundance and functional states of diverse immune populations, including T-, natural killer, B-, and myeloid cells. Using non-negative factorization of cell-subset composition, we identified five reproducible TME subtypes, or "ecotypes," defined by coordinated cellular architectures. These ecotypes captured structured variation beyond disease stage and were associated with distinct cell-cell communication networks, cytokine signaling landscapes, transcription factor programs, and shared gene modules, reflecting coordinated immune adaptation to microenvironmental constraints. By linking tumor features with immune changes and ecotype distributions, we identified context-dependent associations influenced by both disease biology and treatment effects on the TME. We found that an ecotype enriched for bone marrow-resident populations and limited immune infiltration was associated with tumor expansion and inferior clinical outcomes, whereas ecotypes reflecting T-cell functional states showed distinct associations with immunotherapy response and survival. Our study provides a comprehensive single-cell immune atlas of multiple myeloma and its precursors, offering insights into TME organization and progression, and may guide development of stage-specific or ecotype-targeted immunotherapies.

Chromosome copy number variations are poorly understood drivers of human malignancies. -7/del(7q) is common in acute myeloid leukemia, confers a poor prognosis, and is thought to harbor several tumor suppressors. Previously, we identified the histone methyltransferase KMT2C as a tumor suppressor in this region. Here, through a differentiation CRISPR screen in hematopoietic stem and progenitor cells, we find that the mitochondrial iron transporter ABCB8 is essential for their differentiation. ABCB8 deficiency accelerates leukemogenesis in vivo and disrupts iron homeostasis, reducing cytoplasmic iron availability and impairing iron-dependent enzymes, including the histone demethylase KDM6A. Consequently, ABCB8 loss elevates H3K27me3 levels, repressing differentiation genes in an iron- and KDM6A-dependent manner. Notably, ABCB8 and KMT2C, neighboring genes on 7q, cooperatively regulate H3K27me3 to suppress leukemogenesis. Our findings reveal ABCB8 as a tumor suppressor in -7/del(7q) acute myeloid leukemia and uncover an epigenetic collaboration between neighboring tumor suppressors, driven by iron-mediated chromatin remodeling.

Chimeric antigen receptor (CAR)-based immunotherapy has evolved from early linear receptor designs into increasingly sophisticated biological signal processing systems. While conventional CAR-T cell therapies have achieved remarkable clinical success, particularly in hematologic malignancies, their broader application is constrained by limited scalability, manufacturing complexity, and treatment-related toxicities. These challenges have driven the expansion of CAR engineering beyond T cells to alternative immune cell types, including natural killer (NK) cells and macrophages, as well as the diversification of genetic delivery and control strategies. Concurrently, advances in synthetic biology have reframed CARs not merely as static receptors but as programmable immune circuits capable of integrating multiple inputs, executing logical operations, and generating context-dependent outputs. Such circuit-based designs enable precise regulation of immune activation, improved discrimination between tumor and healthy tissues, and enhanced functional persistence within heterogeneous and immunosuppressive microenvironments. In this review, we conceptualize CAR-based immunotherapy as a form of biological signal processing and systematically trace its transition from linear CAR architectures to programmable immune circuits. We summarize engineering strategies for both ex vivo and in situ reprogramming of immune cells, compare viral and nonviral gene delivery platforms, and discuss key design principles underlying circuit CARs across different disease contexts.

Severe thrombocytopenia is a known complication of menin inhibitor therapy; however, the mechanism underlying this effect is unknown. In this issue of Cancer Cell, Wen et al. demonstrate that menin is a key component of normal megakaryopoiesis and establish menin as a therapeutic vulnerability in myeloproliferative neoplasms.

Menin inhibition, an approved therapy for KMT2A-rearranged and NPM1 mutant acute leukemia, is accompanied by decreased platelet counts in 15-20% of heavily pre-treated patients. While studying the mechanism underlying this effect, we discovered that menin inhibition reduced the numbers of megakaryocyte progenitors in human CD34+ cultures and in mice. Because megakaryocytes are key drivers of myeloproliferative neoplasms (MPNs), we investigated the extent to which menin inhibition ameliorates MPN phenotypes. We found that the menin inhibitor revumenib has potent anti-tumor activity, synergizes with ruxolitinib, and shows only subtle effects on healthy mice. Moreover, revumenib suppressed megakaryopoiesis of primary MPN patient specimens in vitro and in vivo. Importantly, genetic knockout of MEN1 and its target MEF2C phenocopied the action of revumenib, confirming an on-target effect of the drug. Together, we reveal menin as a dependency in proliferative megakaryocytes and support further evaluation of menin inhibition as a potential therapy for MPNs.

The internal tandem duplications in FMS-like tyrosine kinase 3 (FLT3-ITD) are associated with poor prognosis in acute myeloid leukemia (AML), yet its kinase-independent mechanisms remain unclear. To investigate kinase-independent immunosuppressive mechanisms in FLT3-ITD AML, we integrated single-cell RNA sequencing from 2 data sets and multiparameter flow cytometry data from 104 primary patient samples and identified CD8+ T-cell exhaustion as a hallmark of the FLT3-ITD immune microenvironment. Mechanistically, FLT3-ITD acts as a mutation-specific scaffold that assembles a ternary complex with protein kinase C iota (PKCι) and STAT1, as demonstrated by coimmunoprecipitation and colocalization. This complex enables PKCι-mediated phosphorylation of STAT1, at serine 727 (S727), thereby driving CD276 transcription independent of the canonical tyrosine 701 (Y701) site. Chromatin immunoprecipitation, electrophoretic mobility shift assays, promoter-reporter assays, and phosphosite-mutant constructs confirmed that S727 phosphorylation is necessary and sufficient for CD276 transactivation. Multiplex immunohistochemistry of bone marrow validated coelevation of pS727-STAT1 and CD276 in FLT3-ITD blasts, accompanied by CD8+ T-cell depletion. Functionally, CD276 upregulation induced profound CD8+ T-cell exhaustion, characterized by reduced cytotoxicity, impaired proliferation, diminished interferon-γ (IFN-γ) production, and elevated inhibitory checkpoint expression. Targeting CD276 restored CD8+ T-cell function by 1.2- to 1.7-fold (cytotoxicity), 1.4- to 1.7-fold (proliferation), 1.5- to 1.8-fold (IFN-γ secretion), and 25.4% to 67.6% (checkpoint expression) in ex vivo coculture. In patient-derived xenograft models, cotreatment with an FLT3 inhibitor (quizartinib) and CD276-targeting agents led to 72.9% to 80.4% tumor burden reduction and enhanced CD8+ T-cell function, outperforming quizartinib monotherapy. These findings define a scaffolded PKCι-pS727-STAT1 signaling axis that promotes immune evasion in FLT3-ITD AML, supporting combined FLT3, and CD276 targeting as a promising translational strategy in this aggressive leukemia subtype.

Patients with acute leukemias harboring translocations involving gene lysine methyltransferase 2A (KMT2A) have a poor prognosis due to chemotherapy resistance with rapid relapse following standard treatments. The resulting KMT2A fusion proteins dysregulate gene expression, leading to an upregulation of leukemogenic transcription factors such as HOXA9 and MEIS1, which drives leukemic transformation. Although Menin inhibitors are proving to be promising new therapeutics for patients with KMT2A-rearranged (KMT2Ar) acute leukemia, resistance mechanisms have already been described and new therapeutic approaches for this patient subgroup must be identified. Here, a genome-wide CRISPR/Cas9 screen in a KMT2Ar B-cell acute lymphoblastic leukemia (ALL) cell line identified the deubiquitinase USP22 as a novel regulator of MEIS1 protein stability. USP22 is a member of the Spt-Ada-Gcn5 acetyltransferase (SAGA) multiprotein complex, which has crucial functions in shaping the chromatin landscape and modulating transcription. Genetic depletion of USP22 impaired cellular growth and proliferation in KMT2Ar acute leukemia models. Chromatin immunoprecipitation revealed cooperative binding between USP22 and MEIS1 at critical oncogenic target genes suggesting that USP22 safeguards leukemogenic transcription by protecting MEIS1 from proteasomal degradation. Genetic or chemical inhibition of USP22 led to polyubiquitination of MEIS1 resulting in proteasomal degradation and downregulation of the expression of target genes. Our study identifies USP22 as a novel regulator of MEIS1 protein stability, that could potentially be exploited as a therapeutic target in the future in KMT2Ar leukemias.

Chemotherapy resistance in acute myeloid leukemia (AML) remains a major clinical challenge. Integration of multiomic profiling and in vivo functional genomics revealed splicing dysregulation as a determinant of chemoresistance in AML. We uncovered a network involving the splicing regulator SRRM1 and the CLK1/4 and PAK1 kinase families as vulnerabilities in chemoresistant AML cells. Both kinase families are hyperactivated in chemoresistant cells, promoting SRRM1 phosphorylation and altering its scaffolding function. We also identified a relapse-associated PAK1 variant, c.1429G>T p.(Ala477→Ser), that confers chemotherapy resistance. Combined PAK1 and CLK1/4 inhibition recapitulated the splicing changes induced by SRRM1 loss, preferentially targeting chemoresistant AML and enhancing chemotherapy efficacy in cell lines, primary cells, and mouse models. Last, we pinpointed MAP2K5 as a critical downstream effector because missplicing of exons 17 and 18 of MAP2K5 upon SRRM1 depletion sensitized cells to chemotherapy. Our findings highlight a therapeutic strategy to overcome AML relapse by targeting splicing dysregulation.

Multiple myeloma (MM) is characterized by the production and secretion of large quantities of immunoglobulins, making this malignancy highly dependent on mechanisms that maintain cellular proteostasis. While significant clinical progress has been made by targeting the degradative branch of proteostasis, much less attention has been given to the biosynthetic branch. In this study, we demonstrated that inhibiting COPII-dependent endoplasmic reticulum (ER) export induces cell death in several MM cell lines and primary patient-derived cells. The induction of cell death was dependent on the secretory status of MM cells. Blocking ER export in secretory MM cells caused the accumulation of misfolded proteins, which activated ER-associated degradation (ERAD). Consequently, we observed an ERAD-dependent increase in the levels of free cytosolic amino acids and a subsequent activation of mTORC1 signaling. Simultaneously, we observed mitochondrial dysfunction. These alterations resulted in a mismatch between the increased energy demand due to mTORC1 activation, and the disrupted energy supply from mitochondrial impairment. This energetic imbalance results in homeostatic collapse and cell death of secretory MM cells. The therapeutic potential of the concept was demonstrated in two in vivo myeloma models. These findings suggest that the ER export machinery could be a promising therapeutic target in multiple myeloma.

Colorectal cancer (CRC) metastases frequently recur due to minimal residual disease (MRD) and persistent micrometastases after therapy. Here, we performed spatial multimodal profiling using spot-level and high-resolution spatial transcriptomics, multi-regional whole-genome sequencing following laser-capture microdissection, and high-plex protein imaging to map 49 tumors from 19 patients, encompassing paired primary CRC and matched liver (CLiM) and lung (CLuM) metastases. Phylogenetic reconstruction revealed that liver micrometastases (CLiMi) arose from early clonal divergences and harbored a stem-like, quiescent state consistent with metastatic dormancy. Spatially, we uncovered distinct stromal barriers: macrometastases were encapsulated by myofibroblasts, whereas micrometastases were surrounded by immunosuppressive niches characterized by T cell exhaustion and distinct ligand-receptor signaling networks. Notably, we identified a CLiMi-specific six-gene signature associated with MRD status, disease-free survival, and chemotherapy resistance across multiple independent cohorts. These findings elucidate the spatial evolutionary landscape of CRC metastases and provide tissue-based spatially validated biomarkers for surveillance and therapeutic targeting.

Although vitamin D (VitD) exhibits anti-tumor activity in colorectal cancer (CRC) preclinically, its effects in the human tumor microenvironment (TME) remain unclear. We conducted a randomized, placebo-controlled trial of preoperative high-dose VitD supplementation in stage I-III colon cancer patients to assess its impact on the TME. Forty-two patients received either VitD3 (50,000 IU/day for 7 days, then 10,000 IU/day) or placebo before surgery. Spatial immune-profiling and assessment of VitD receptor (VDR) and CYP27B1 expression were performed on paired tumor samples from 24 patients. VitD significantly increased plasma 25-hydroxyvitamin D levels (P<0.001), increased CD3+CD8+ memory T cells (P=0.03), reduced CD3+CD4+FoxP3+ regulatory T cells (P=0.02) and spatially re-organized the TME, leading to greater T cell and tumor cell proximity. Post-treatment VDR expression was heterogeneous and decreased overall (P=0.02). Spatial transcriptomic profiling of post-treatment resections reflected predominantly repressive VDR activity. These findings support an immunomodulatory role for VitD, warranting further mechanistic investigation.

Fusobacterium nucleatum (Fn) has been implicated in the progression of colorectal cancer (CRC), immune modulation, and resistance to systemic therapies. However, its role as a predictive biomarker of treatment response remains unclear. We conducted a systematic review to evaluate the association between Fn and oncologic treatment outcomes in CRC. This systematic review was performed according to the PRISMA 2020 guidelines and registered in PROSPERO (CRD420261327958). MEDLINE, Scopus, and CENTRAL were searched through March 2026. Eligible studies included adult CRC patients with Fn assessment performed before or during treatment and reporting treatment-related outcomes. Primary endpoints included pathological complete response (pCR), tumour regression grade (TRG), and objective response rate (ORR). Secondary endpoints included disease-free survival (DFS), relapse-free survival (RFS), progression-free survival (PFS), and overall survival (OS). Eleven studies including 1571 patients, were analysed. In locally advanced rectal cancer, four of five studies indicated that higher pre-treatment Fn abundance was associated with poorer pathological response to neoadjuvant chemoradiotherapy. Persistent Fn positivity after treatment was linked to an increased relapse risk. In metastatic CRC receiving immunotherapy, two studies reported enrichment of Fn in non-responders, while one study observed improved outcomes in Fn-positive tumours treated with PD-L1 blockade. In chemotherapy-treated metastatic CRC, Fn abundance was significantly higher in patients with progressive disease. Overall, the quality of evidence was rated low to very low. Fn appears to be associated with treatment resistance across multiple CRC settings and may represent a promising biomarker and therapeutic target. Prospective biomarker-driven studies are warranted.

The primary treatment for non-metastatic colorectal cancer is surgical resection. Despite the use of neoadjuvant and/or adjuvant chemoradiation, up to 30% of patients undergoing surgery for colorectal cancer will develop a postoperative recurrence. Why patients develop postoperative tumors despite all known cancer being resected at the time of surgery is largely unknown, and novel biomarkers that can predict the development of recurrence are lacking. Here, we report a unique bacterial signature present in the gut during the perioperative period that is strongly associated with the development of postoperative tumors. By studying patients undergoing resection for colorectal cancer, we demonstrate that the gut microbiome on the day of surgery is enriched with collagenase-producing bacteria in patients who later develop a recurrence. This bacterial community demonstrated enhanced antimicrobial resistance, was not eradicated by the standardized perioperative bowel preparation, and could promote cancer cell migration and invasion. Our study establishes that microbiota may contribute to postoperative colorectal cancer recurrence and serve as a prognostic biomarker for postoperative oncologic outcomes.

Colorectal cancer (CRC) carries a microbial fingerprint, but how does it generalize across age, geography, and sequencing platforms? In this issue of Cell Host & Microbe, Pekel and colleagues stitch together large-scale stool and tumor data to reveal a universal signal while exposing where stool-based biomarkers fall short.

Numerous studies have linked gut microbiome alterations to colorectal cancer (CRC), but limited sample sizes and study heterogeneity have hampered cross-study comparisons and subgroup analyses. Here, we present a comprehensive single-disease gut microbiome meta-analysis based on consistently re-computed and re-analyzed shotgun and amplicon sequencing profiles (n = 6,779 samples, 27 studies). Association and machine-learning analyses delineate CRC microbiome signatures, which are robustly generalizable across studies and sequencing approaches and nearly identical between early- and late-onset cases. Meta-analysis of the tumor-resident microbiome reveals characteristic tumor-enriched microbes in concordance with fecal signatures that are clearly detectable in early-stage tumors, although their detection in feces becomes moderately higher in late-stage and distal tumors, possibly due to dilution effects in stool. The unified fecal CRC signature inversely associates with dietary fiber intake and is modifiable by dietary interventions. Finally, genome-resolved functional analysis reveals variation in virulence factor carriage and geographic enrichment across Fusobacterium subspecies.

Metastasis is the principal cause of death from colorectal cancer (CRC), yet the cellular states that enable tumor dissemination remain poorly defined. Disseminated tumor cells (DTCs) are rare, transient, and clinically inaccessible, limiting mechanistic insight into their biology. Here we show that CRC cells transiently adopt a wound-healing program normally used by epidermal keratinocytes during tissue repair to enable metastatic dissemination. Using serial orthotopic transplantation of patient-derived organoids to model metastasis, we find that DTCs lose cancer stem cell features and instead express wound-inducible keratins, including KRT17, before metastatic outgrowth. This state is reversible, as cells reacquire primary tumor-like characteristics upon colonization of distant organs. Mechanistically, this transition is associated with reduced EZH2 activity and activation of YAP signaling. Clinically, KRT17⁺ cells localize to the invasive front of primary CRCs and are absent from adjacent normal tissue. These findings uncover unexpected lineage plasticity across distinct developmental origins and identify a transient, targetable state critical for metastatic progression.

Plasticity, the capability of tumor cells to go through phenotypic transitions, promotes colorectal cancer (CRC) progression and treatment resistance. Although plasticity is evident in advanced CRCs, little is known about plasticity in early-stage tumors and tumor stem cells. Here, we demonstrate that a plastic cell state (PCS) is present already in polyps from patients with familial adenomatous polyposis and in mouse intestinal adenomas, in which PCS is associated with PROX1+ tumor stem cells. We furthermore analyzed progressive plasticity upon loss of the canonical wingless-related integration site (Wnt) effector Tcf7 or Lef1 in Apc mutant mice. Deletion of either gene led to emergence of new plastic tumor cell populations, failure of leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) tumor stem cell differentiation into enterocyte-like cells, enhanced Myc pathway activation, and increased tumor cell proliferation and tumorigenesis. Together, we demonstrate that PCS is associated with early CRC development and identify multiple potentially druggable mechanisms activated during progressive tumor cell plasticity.

Kirsten Rat Sarcoma viral oncogene homolog (KRAS) is one of the most frequently activated driver genes across human cancers. We identified a regulatory mechanism where KRAS forms condensates in the cytoplasm through liquid-liquid phase separation (LLPS), driven by farnesylation at the C185 residue within its hypervariable region (HVR). These condensates are associated with advanced stages and poor outcomes in colon cancer. Functionally, KRAS condensates efficiently interact with Ras-converting enzyme 1 (RCE1), promoting RCE1 clustering, enhancing KRAS processing, and facilitating its translocation to the plasma membrane, which amplifies KRAS signaling and promotes tumor growth. Growth factor stimulation further elevates KRAS condensate formation, emphasizing its role in tumor biology. Therapeutically, screening US Food and Drug Administration (FDA)-approved drugs revealed that statins, particularly pitavastatin, disrupt KRAS LLPS by inhibiting farnesylation, effectively suppressing colon cancer growth and enhancing the efficacy of G12Ci treatment. These findings uncover LLPS as a mechanism regulating KRAS activity and provide a promising target for therapeutic intervention.

Given the limited efficacy of existing therapies for metastatic colorectal cancer (mCRC), there is an urgent need for novel strategies. Prohibitin 1 (PHB1) is significantly upregulated in CRC, where it plays a critical role in oxidative phosphorylation (OXPHOS) to meet the heightened energy demands of rapid tumor growth and metastasis. Here, we discover that PHB1 interacts with NADH: ubiquinone oxidoreductase core subunit S1 (NDUFS1), a subunit of the OXPHOS complex, thereby modulating mitochondrial respiratory function. Based on these insights, we developed TD6, a highly potent and selective small-molecule inhibitor of PHB1. In a mouse model of colorectal cancer pulmonary metastasis (CRPM), TD6 treatment significantly prolonged animal survival. Mechanistically, TD6 binds to PHB1 and induces a conformational change in the PHB complex, which reduces PHB-mediated stabilization of NDUFS1. Disruption of the PHB-NDUFS1 interaction promotes NDUFS1 degradation via the lysosomal pathway, leading to impaired activity of mitochondrial complex I (MCI) and reduced OXPHOS function. In summary, PHB1 maintains MCI stability and OXPHOS activity through its interaction with NDUFS1. By targeting PHB1, TD6 effectively disrupts this regulatory axis, demonstrating therapeutic potential against CRPM. This study presents a novel drug candidate and a theoretical foundation for PHB1-based interventions targeting cancer energy metabolism.

Studies have highlighted the important roles of circRNAs in various cancers. However, their specific functions in the lymph node metastasis (LNM) of colorectal cancer (CRC) remain largely unclear. The Arraystar human circRNA microarray was used to identify circular RNAs (circRNAs) associated with LNM in colorectal cancer (CRC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure circHELQ expression in CRC cell lines and tissue specimens. A series of in vitro and in vivo functional assays were subsequently conducted to investigate the role of circHELQ in LNM and CRC progression. Additionally, fluorescence in situ hybridization, dual-luciferase reporter assays, RNA pull-down, and Western blot experiments were carried out to elucidate the underlying regulatory mechanisms of circHELQ in CRC. Our results demonstrated that circHELQ expression was significantly upregulated in CRC and that its expression level was closely correlated with LNM. Gain- and loss-of-function experiments revealed that circHELQ promotes CRC progression and metastasis-including proliferation, migration, invasion, lymphatic vessel formation, and LNM-both in vitro and in vivo. Mechanistically, circHELQ acts as a competing endogenous RNA (ceRNA) that sponges miR-4793-3p, thereby alleviating its suppression of VEGFC. This circHELQ/miR-4793-3p/VEGFC axis drives LNM in CRC by upregulating VEGFC expression. Furthermore, we developed a cancer cell membrane-based siRNA delivery system (cMDS) targeting circHELQ, and these nanoparticles effectively suppressed CRC proliferation and LNM. CircHELQ drives CRC progression and LNM and is a candidate prognostic biomarker. Furthermore, its molecular function renders it a viable target for nanoparticle-based therapeutic strategies against CRC.

Western diet and associated production of secondary bile acids (BAs) have been linked to the development of sporadic colorectal cancer (CRC). Despite observational studies showing that secondary BAs produced by 7α-dehydroxylating (7αDH+) gut bacteria are increased in CRC, a causal proof of their tumour-promoting effects is lacking. Investigate the causal role of BAs produced by 7αDH+ gut bacteria in CRC. We performed feeding studies in a porcine model of CRC combined with multi-omics analyses and gnotobiotic mouse models colonised with 7αDH+ bacteria or a genetically modified strain to demonstrate causality. Western diet exacerbated the CRC phenotype in APC 1311/+ pigs. This was accompanied by increased levels of the secondary BA deoxycholic acid (DCA) and higher colonic epithelial cell proliferation. The latter was counteracted by the BA-scavenging drug colestyramine. Metagenomic analysis across multiple human cohorts revealed higher occurrence of bai (BA inducible) operons from Clostridium scindens and close relatives in faeces of patients with CRC. Addition of these specific 7αDH+ bacteria (C. scindens/Extibacter muris) to defined communities of gut bacteria led to DCA production and increased colon tumour burden in mouse models of chemically or genetically induced CRC. A mutant strain of Faecalicatena contorta lacking 7αDH caused fewer colonic tumours in azoxymethane/dextran sodium sulfate treated mice and triggered less epithelial cell proliferation in human colon organoids compared with wild-type F. contorta. This work provides functional evidence for the causal role of secondary BAs produced by gut bacteria through 7αDH in CRC under adverse dietary conditions, opening avenues for future preventive strategies.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the United States. While the gut microbiota has been shown to influence CRC development, the specific contribution of bacteria to DNA methylation and carcinogenesis remains underexplored. We colonize two groups of GF ApcMin/+ mice with two consortia, one harboring a pks+ E. coli strain with "low-pks" activity (DSMZ) and the second with a clinical isolate exhibiting "high-pks" activity (UM149). These colonized mice are exposed to DSS-induced colitis, and analyzed for tumor burden, DNA methylation, and transcriptional changes. We find that colonization with C13-UM149 leads to more tumors, increased cell proliferation, and higher DNA damage compared to C13-DSMZ (p < 0.05). Methylation analyses show that C13-DSMZ causes extensive promoter hypermethylation and altered gene expression. Differential DNA methylation in mice colonized with either C13-DSMZ or C13-UM149 is associated with changes in pathways controlling tumor suppression, cell proliferation, inflammation, and Wnt signaling. In C13-DSMZ mice, hypermethylation is associated with gene expression involved in tumor suppression in both tumors and normal tissue, whereas hypomethylation is linked to expression of genes promoting Wnt signaling. In C13-UM149 mice, methylation changes were connected to genes involved in epithelial proliferation, extracellular matrix remodeling, and inflammatory responses. These findings demonstrate that intestinal bacteria with distinct pks activities differentially modulate DNA methylation thereby influencing gene expression and tumor development. This highlights bacterial modulation of epigenetic responses as a potential mechanism underlying CRC progression.

Chemoresistance remains a major barrier in colorectal cancer (CRC) therapy. Through epigenetic compound screening in patient-derived organoids (PDOs), we identified CX6258.HCl as a potent growth inhibitor. Treatment with CX6258.HCl significantly inhibited cell mitosis and induced apoptosis in CRC cell lines. Mechanistically, CX6258.HCl binds the D687 residue within HASPIN's kinase domain, suppressing H3T3 phosphorylation (H3T3ph). This triggers an epigenetic cascade: loss of H3T3ph upregulates demethylase KDM5B pre-mRNA, depleting H3K4me3 at promoters of Kinesin family member (KIFC1/KIF10/KIF14). Consequently, microtubule dynamics are disrupted, leading to mitotic arrest. Target specificity was validated genetically via HASPIN-D687A mutation. In vivo, CX6258.HCl suppressed CRC xenograft growth and further enhanced 5-FU-mediated tumor suppression without obvious histological injury in major organs. Clinically, elevated H3T3ph levels in human CRC tissues were associated with Ki67-positive proliferative tumor regions, suggesting that H3T3ph may represent a proliferation-associated marker in CRC. Together, our findings identify the HASPIN/H3T3ph-KDM5B-H3K4me3-KIF axis as a targetable antimitotic pathway and support therapeutic inhibition of HASPIN/H3T3ph as a potential strategy for CRC.

Primary sclerosing cholangitis (PSC) is a chronic, progressing cholestatic disease that often co-occurs with inflammatory bowel disease (PSC-IBD). PSC-IBD affecting the colon (PSC-ulcerative colitis or PSC-UC) resembles clinical UC, but is characterised by less severe disease flares, right-colon predominance, and a greater lifetime risk of colorectal cancer than UC alone. To elucidate differences in the underlying biology between PSC-UC and UC, here we combine single-cell mRNA and antigen receptor sequencing, 16S ribosomal RNA gene analysis and spatial transcriptomics on biopsies from four colon regions of patients with PSC-UC and UC during endoscopic remission or at the time of relapse. We show that the PSC-UC colon, compared to healthy control (HC) or UC colon, harbours distinct and region-specific mucosal-adherent microbial communities and an enrichment of activated CD8 T and γδ T cells at the right colon, even in the absence of histological inflammation. By contrast, a TMEM176B+ mast cell population that may be pro-tumourigenic is enriched in the colon during disease relapse in both PSC-UC and UC colon. These results highlight that the PSC-UC and UC colonic mucosa are fundamentally different while sharing similar cell programmes during active disease. Our data thus provide insights to guide tailored clinical management and precision therapies.

Probiotics and their metabolites (postbiotics) have gained increasing attention because of their anticancer potential. This study evaluated the effects of postbiotics derived from Lactobacillus rhamnosus (LR) on colorectal cancer (CRC) cells. Sterile LR culture filtrates were applied to SW480 and highly invasive SW480-I5 cells to assess cell viability, proliferation, migration, invasion, apoptosis, and epithelial-mesenchymal transition (EMT)-associated signaling. LR filtrates reduced cell viability in a dose-dependent manner and significantly suppressed migration and invasion, particularly in SW480-I5 cells. In addition, LR treatment inhibited proliferation and promoted apoptosis in both cell lines. Mechanistically, LR filtrates downregulated EMT-associated transcription factors (ZEB2, Snail, and Twist), mesenchymal markers (N-cadherin and Vimentin), and MMP3 expression. These findings demonstrate that LR-derived postbiotics exert inhibitory effects on colorectal cancer-associated malignant phenotypes and may represent promising adjunctive candidates for CRC modulation.

Spatial transcriptomics (ST) assays are transforming our understanding of tumor heterogeneity, but their high cost limits their application in large-scale biomarker discovery. Here, we present "Path2Space," a deep-learning model that predicts spatial gene expression directly from histopathology slides. Trained on extensive breast cancer ST data, Path2Space robustly predicts the spatial expression of thousands of genes, outperforming 21 established methods. Charting the tumor microenvironment (TME) of 976 breast cancer TCGA (The Cancer Genome Atlas) tumors, it accurately infers cell-type abundances and identifies three spatially defined breast cancer subgroups with distinct survival outcomes. Notably, the derived low-cost spatial TME landscapes enable more accurate predictions of patient response to chemotherapy and trastuzumab compared with costly conventional bulk-sequencing-based biomarkers. Path2Space thus offers a scalable, fast, and cost-effective alternative to molecular assays. It opens avenues for large cohort treatment biomarker discovery and translationally relevant insights into tumor biology, with potential applicability across many cancer indications.

The World Health Organization (WHO) Global Breast Cancer Initiative aims to attain meaningful global breast cancer mortality reductions by 2040-a target that hinges on improvements in patient outcomes and survival. So far, however, data on cancer survival remain limited in low- and middle-income countries. The WHO estimated population-based age-standardized 5-year net survival for women diagnosed with breast cancer between 2017 and 2021 across all 194 Member States, providing a global benchmark for monitoring breast cancer outcomes. Here we found that median 5-year net survival varied widely across WHO regions during 2017-2021: 39.1% (95% uncertainty interval 34.1-44.7%) in the African Region, 61.0% (51.4-69.8%) in the Eastern Mediterranean Region, 66.3% (57.7-73.7%) in the South-East Asia Region, 81.1% (78.6-83.5%) in the Western Pacific Region, 84.0% (82.8-85.1%) in the European Region and 88.5% (86.7-90.1%) in the Region of the Americas. Persisting disparities in survival reflect profound global inequities; sustained initiatives to narrow gaps in access to diagnosis and treatment for breast cancer are crucial to strengthening health systems. This will enable all countries to ensure optimal outcomes for their patients with breast cancer and achieve Global Breast Cancer Initiative and Sustainable Development Goals targets.

Recently, histopathology foundation models (hFM) have rapidly advanced in size and complexity, achieving excellent performance in cancer diagnosis and biomarker discovery. Here, we specialise pre-trained hFMs to invasive tumour tissue and present three key contributions. First, we systematically evaluate the biological concepts encoded in hFM representations across multiple biological scales. Second, we demonstrate that informed extended pre-training transforms generalist models into tumour-specialised ones encoding richer semantic information, enabling discovery of recurrent tumour archetypes with consistent morphological and molecular identities across patients. Third, we identify dominant tumour archetypes with aberrant gene-expression programs coexisting within tumours and recurring across heterogeneous epithelial cancers, including HER2-positive and triple-negative breast cancer. Crucially, these archetypes exhibit prognostic value, with RNA splicing-associated archetypes consistently predicting poorer outcomes. Our work shows that tumour-specialised hFMs unlock rich molecular and morphological information from routine H&E slides, providing computationally efficient and biologically informed solutions for biological discovery and patient stratification.

Triple-negative breast cancer is an aggressive and heterogeneous breast cancer subtype for which immune checkpoint inhibitors combined with chemotherapy have improved outcomes in selected patients. However, primary and acquired resistance remain common, underscoring the need to identify extrinsic, modifiable determinants of antitumor immunity. Increasing evidence indicates that the gut and tumor-associated microbiota shape systemic and intratumoral immune tone and influence the efficacy of cancer therapies. Beyond microbial composition, microbiota-derived metabolites-including short-chain fatty acids, indole-tryptophan derivatives, bile acids, polyamines, and other small molecules-can act as functional mediators linking microbial ecology to immune-cell programming and tumor biology. These metabolites modulate dendritic cell function, T-cell priming and fitness, myeloid polarization, inflammatory set points, and metabolic pathways within the tumor microenvironment, thereby potentially enhancing or constraining responses to chemoimmunotherapy. Importantly, while some studies propose intratumoral microbial effects, most clinically actionable evidence currently supports systemic gut-derived metabolites and immune tone modulation that secondarily shapes the TNBC tumor microenvironment. In this review, we synthesize current knowledge on (i) the immunobiology of triple-negative breast cancer (TNBC) relevant to microbiota-driven modulation, (ii) mammary and gut microbiome features reported in TNBC, and (iii) mechanistic pathways through which microbial metabolites may regulate antitumor immunity and immune checkpoint inhibitors (ICI) sensitivity. We also discuss methodological considerations for integrating microbiome profiling with metabolomics and immune phenotyping and evaluate emerging opportunities to leverage microbiota-derived metabolites as biomarkers and therapeutic targets. Finally, we highlight translational strategies-including diet, pre/probiotics, antibiotic stewardship, fecal microbiota transplantation, and metabolite-centric ("postbiotic") approaches-and outline priorities for TNBC-focused, prospective multi-omics studies to move from associative signatures toward actionable interventions.

Triple-negative breast cancer (TNBC) is associated with rapid progression and poor sensitivity to monotherapies, necessitating combination strategies to enhance tumor immunogenicity, reprogram the tumor microenvironment (TME), and convert immunologically "cold" tumors into "hot" ones. In this study, we develop a pH-responsive, pyroptosis-inducing nanoreactor, ZnS/ZIF-90:Fe/Vk3 (ZZFV), designed to elicit potent antitumor immunity. Under acidic conditions, ZZFV rapidly degrades, releasing Zn2+, Fe2+, H2S, and Vk3. H2S promotes glucose uptake and consumption while downregulating cytochrome c oxidase subunit IV (COX IV) expression, whereas Zn2+ suppresses lactate dehydrogenase (LDH) activity, collectively disrupting glucose metabolism and exacerbating intracellular acidification. Meanwhile, NAD(P)H quinone oxidoreductase 1 (NQO1), highly expressed in tumor cells, catalyzes the conversion of Vk3 to H2O2, which is subsequently converted to highly toxic •OH through Fe2+-mediated Fenton reactions. Additionally, Zn2+ and H2S synergistically disrupt mitochondrial function, thereby amplifying reactive oxygen species (ROS) production. Excessive ROS triggers Caspase-1-dependent pyroptosis, leading to the liberation of damage-associated molecular patterns. These events promote dendritic cell (DC) maturation, robust T-cell activation, M1 macrophage polarization, and expansion of central memory T-cell populations, thereby conferring durable immune protection. Consequently, ZZFV demonstrates potent antitumor efficacy, effectively overcoming immune tolerance in TNBC and achieving robust, sustained antitumor immunity.

Cancer progression is driven by the accumulation of DNA mutations and aberrant gene regulation. Recent studies have demonstrated that multiple H3 mutations serve as drivers of tumorigenesis. However, the role and significance of various cancer-associated histone H2B mutations in cancer development remain unknown. Here, we investigate H2BE113K, a missense mutation of histone H2B predominantly found in patients with breast cancer. We show that H2BE113K promotes colony formation in breast cancer. Notably, transcriptomic analysis reveals differential expression of genes in various cancer pathways in H2BE113K cells. The loci with elevated gene expression display increased chromatin accessibility, accompanied by H2BE113K enrichment. Depletion of G3BP2, one of the H2BE113K target genes that has been implicated in breast cancer, reduces the colony formation phenotype in H2BE113K cells. In addition, H2BE113K knock-in mice crossbred with an MMTV-PyMT breast cancer model show elevated lung metastasis. Together, our findings provide critical insights in the mechanistic role of H2BE113K in gene regulation, chromatin function, and breast cancer progression.

Maintenance of genome integrity is essential for cellular homeostasis, and its perturbation leads to tumorigenesis. Here, we uncover an unanticipated somatic role for the synaptonemal complex protein SYCP1, previously regarded as strictly meiosis specific, in a broad spectrum of human cancers including breast cancer. Through integrative genomic, proteomic, and functional analyses, we demonstrate that SYCP1 is aberrantly reexpressed in tumor cells, where it actively promotes DNA damage repair, cell cycle progression, and malignant growth. SYCP1 binds chromatin at regulatory elements and directly controls transcriptional programs governing genome maintenance, including key effectors such as CCNB1, PCNA, RAD51C, and H2AX. Loss of SYCP1 impairs DNA repair kinetics, attenuates tumor cell proliferation and migration, and increases sensitivity to chemotherapeutics cisplatin and gemcitabine. Mechanistically, SYCP1 coimmunoprecipitates with chromatin remodeling complexes and transcription factors SP1 and SP2 and modulates their genomic occupancy and oncogenic transcriptional outputs. Clinically, high SYCP1 expression stratifies patients with poor prognosis and therapy resistance across multiple cancer types. Our findings illuminate a previously unrecognized moonlighting function of SYCP1 in somatic cancer cells and position it as a critical chromatin-associated regulator of genome stability, with implications for biomarker development and therapeutic targeting.

Cancer progression involves not only uncontrolled proliferation but also the strategic entry of tumour cells into reversible (quiescent) or irreversible (senescent) states of cell cycle arrest (G0). These states can give rise to rare persister-like cancer cells that survive hostile tumour microenvironment conditions, facilitating drug resistance, metastasis and disease relapse. Despite their importance, identifying and understanding the mechanisms regulating these cell populations remains challenging. We leveraged single-cell and spatially profiled primary breast tumours to quantify G0 arrest and proliferation decisions in cancer cells, revealing molecular and spatial features associated with proliferation-G0 dynamics. We uncovered a G0 persister-like state with reduced copy number alteration burden and hallmarks of dormancy, characterised by transcriptional reprogramming of stress response pathways and increased epithelial-mesenchymal plasticity. Spatial analyses revealed distinct ecological niches: G0 cells inhabited protective niches with complement pathway activity proximal to CXCL10+ macrophages and myofibroblastic cancer-associated fibroblasts (CAFs), whereas proliferative zones were associated with CLEC9A+ dendritic cells and PERK signalling, with distinct drug sensitivities. Our findings highlight key principles underpinning G0-proliferation dynamics and niche specialisation in breast cancer, offering novel insights into the spatial drivers of tumour heterogeneity and evolution.

The functional crosstalk between mitochondria and the endoplasmic reticulum (ER) serves as a critical adaptive mechanism in cancer cells, wherein mitochondrial damage-induced ER stress can paradoxically activate protective mitophagy to restore cellular homeostasis and limit therapeutic efficacy. To subvert this self-repair cycle and amplify immunogenic cell death (ICD), we engineered a mitochondria-targeted biomimetic nanoplatform (Cel-Ca/CQ@OMM) for tumor-selective co-delivery of Celastrol (Cel) and Chloroquine (CQ). The nanosystem leverages homologous mitochondrial membrane functionalization to achieve precise subcellular localization. Celastrol coordinates with calcium ions to form a complex (Cel-Ca) that induces Ca²⁺ overload and reactive oxygen species (ROS) burst, thereby damaging mitochondria and concomitantly triggering lipophagy as a compensatory survival response. The ER, upon contact with damaged mitochondria, activates mitophagy to clear these organelles. By suppressing autophagic flux, CQ simultaneously abrogates both reparative mitophagy and adaptive lipophagy. The resulting cumulative accumulation of damaged mitochondria and lipid droplets perpetuates ER-mitochondria interaction and imposes metabolic burden on the ER, establishing a vicious cycle that progressively amplifies cellular stress through positive feedback regulation, thereby steering the cell toward apoptotic elimination. This dual-inhibition intervention disrupts mitochondrial-ER homeostasis, leading to exacerbated ER stress, enhanced damage-associated molecular pattern (DAMP) release, and robust CD8⁺ T cell-mediated antitumor immunity. This study highlights the amplification of ICD by synergistically blocking the key adaptive pathways of mitophagy and lipophagy, providing a promising approach for Triple-negative breast cancer (TNBC) treatment.

Chromosome instability is highly prevalent in cancer and drives large-scale chromosomal imbalances, known as aneuploidies1-4. How aneuploidy contributes to tumorigenesis remains difficult to study due to the vast numbers of genes affected. Here we established a CRISPR knockout- and activation-linked assay (CRISPR-KOALA), enabling high-throughput bidirectional genetic screens in immunocompetent mouse models of cancer. We developed a compendium of the ten most frequent human chromosome-arm-level alterations in basal-like breast cancer (BLBC), a disease type that is driven by large copy-number alterations (CNAs)5-8. Using CRISPR-KOALA, we screened the mouse orthologues of 3,752 genes on these arms and identified 90 cancer driver genes, the function of the vast majority of which is unknown. These genes drive distinct signalling pathways including MAPK, HIPPO and WNT, reflecting the high degree of BLBC heterogeneity. Manipulating the identified cancer driver genes overcomes the need for CNAs in Trp53-mutant BLBC mouse models. Mechanistically, we identify that PLGRKT is a potent oncogene that lies on chromosome 9p and show that its tumour-promoting activity is associated with highly stress-resistant mitochondria and an increased ability to detoxify reactive oxygen species. Together, our findings reveal that arm-level CNAs can function to select specific driver genes to promote heterogeneous biological processes.

Breast cancer remains the leading cause of cancer-related mortality in women, with conventional chemotherapy often limited by systemic toxicity due to poor drug targeting. Here, we present an integrated microfluidic chip (MMLP) that enables one-step integrated postprocessing, including solvent removal and ligand conjugation. The MMLP chip combines Y-shaped, crescent-shaped, U-shaped, and Tesla-type microchannels, augmented with pillar arrays to enhance mixing efficiency at low flow rates. A dedicated third channel allows simultaneous solvent removal and aptamer conjugation. Through computational fluid dynamics (CFD) simulations, we optimized the flow-rate ratio (lipid phase/aqueous phase/postprocessing phase = 1:5:2) and achieved AS1411 aptamer modification via thiol-maleimide chemistry. The resulting AS1411 aptamer-functionalized liposomes loaded with doxorubicin (D@Lip-Apt) exhibited uniform size (155 ± 3.2 nm, PDI = 0.17) and high drug encapsulation efficiency (76.64 ± 0.52%). In vitro and in vivo studies confirmed the nanoparticles' superior stability and tumor-targeting specificity. This modular microreactor offers exceptional flexibility in tailoring liposome properties for breast cancer therapy and demonstrates scalability for industrial production through dimensional or parallel amplification.

The significant variation in treatment strategies among breast cancer subtypes establishes precise early subtyping as a critical prerequisite for effective therapy. While molecular profiling offers accurate classification, developing a rapid and reliable detection method remains challenging. Hence, we constructed an integrated platform by integrating a DNA tetrahedral probe (DTP) and surface antiadhesive magnetic micro/nanorobots (MNRs), enabling rapid contact between the MNR-DTP system and target analytes. The probe enhances fluorescence via target-triggered strand displacement and CHA-mediated signal amplification, allowing breast cancer subtypes to be identified through distinct dual-color fluorescence patterns. Furthermore, magnetically driven MNRs improve detection efficiency by enhancing mass transfer, promoting mixing, and accelerating probe-target interactions. Experimental results demonstrate that this strategy markedly enhances fluorescence output, enables rapid detection of dual-miRNA signatures in different breast cell lines, and distinguishes expression heterogeneity at the single-cell level. The system shows high specificity and improved sensitivity, with limits of detection of 1.5 pM for miR-21 and 1.17 pM for miR-31. The biocompatibility and stability of the proposed MNR-DTP system make it a promising tool for early breast cancer subtype discrimination and multiplexed target recognition in complex biological settings.

The dense microenvironment of triple-negative breast cancer (TNBC) poses a formidable barrier to nanomedicine penetration, rendering selective tumor retention critical for effective drug delivery. Cell-penetrating peptides (CPPs)-PEG polymers improve SN38 cellular uptake but lack tumor selectivity. We employed a quantitative structure-activity relationship (QSAR)-driven strategy to screen a combinatorial CPPs library for tumor microenvironment responsiveness. We elucidated that proline residues induce peptide bending and steric effects, resulting in PEG shield to limit cargo cellular entry. Eliminating these residues abrogated structural kinks, yielding a linearized peptide topology that effectively pierced the PEG shell to engage anionic membrane receptors, thereby enhancing tumor penetration and intracellular accumulation. Consequently, the optimized TAT12-PEG-SN38 micelles prolonged blood circulation and suppressed TNBC growth and metastasis. Furthermore, combination treatment with JQ-1 attenuated immune evasion. Overall, peptide structure engineering overcomes the PEG barrier, providing a practical strategy for developing novel peptide-drug conjugates.

Despite advances in immunotherapy, triple-negative breast cancer (TNBC) remains a major therapeutic challenge owing to its marked aggressiveness, low immunogenicity, and limited responsiveness to immune checkpoint blockade (ICB). Herein, the esterase-responsive polymeric nanoparticles (EsCu@NPs) are developed. The polymer backbone covalently integrates an L-buthionine sulfoximine prodrug to inhibit glutathione biosynthesis, and the nanoparticle matrix encapsulates an elesclomol-copper complex to promote intratumoral copper accumulation. By concurrently depleting glutathione and perturbing copper homeostasis, EsCu@NPs elicit reinforced cuproptosis stress that is converted into immunogenic cell death. This process reprograms the suppressive tumor microenvironment, characterized by increased DC maturation, elevated CD8⁺ T-cell infiltration, and a reduction in regulatory T cells. These findings support enhanced cuproptosis-driven metalloimmunotherapy as a strategy for treating TNBC. Briefly, in neoadjuvant settings, preoperative EsCu@NPs reduce tumor burden, decrease minimal residual disease, curb postsurgical recurrence and spontaneous lung metastasis, translating into prolonged survival. Notably, combination with anti-PD-1 checkpoint blockade yields synergy, significantly enhancing the ICB responsiveness of TNBC and producing improved therapeutic efficacy. Overall, this study demonstrates that EsCu@NPs enable coordinated cuproptosis and immune reprogramming, expanding ICB efficacy and supporting a translatable neoadjuvant strategy in TNBC.

Despite the rapidly expanding catalog of long noncoding RNAs (lncRNAs), the functional roles of most remain poorly characterized in cancer. In triple-negative breast cancer (TNBC), an aggressive subtype with limited targeted treatment options, defining lncRNA-associated molecular mechanisms could uncover novel therapeutic vulnerabilities. This study aimed to systematically identify TNBC-enriched lncRNAs associated with cellular fitness, and drug response, and to functionally characterize prioritized lncRNA candidates with potential therapeutic relevance. We generated comprehensive lncRNA annotation by integrating GENCODE, BIGTranscriptome, and MiTranscriptome databases. CRISPR-Cas9 deletion screen targeting 1,029 TNBC-enriched lncRNAs was conducted. Functional validation included proliferation assays in 2D and 3D cultures, invasion assays using an organ-on-chip model, and transcriptomic and proteomic profiling following lncRNA knockdown. Candidate lncRNA-protein associations were assessed using proteomic array analysis. miRNA expression profiling, miRanda-based interaction prediction, and Ingenuity Pathway Analysis (IPA) were used to construct candidate competing endogenous RNA (ceRNA)-like regulatory networks and downstream signaling pathways. Our CRISPR screen identified fourteen recurrent candidate lncRNA dependencies across both TNBC models, with TNBC Promoting LncRNA 1 (TPL1) emerging among the top candidates. ASO-mediated TPL1 suppression significantly reduced TNBC cell proliferation, clonogenic growth, three-dimensional growth, and invasive capacity. TPL1 was significantly overexpressed in TNBC tissues, particularly within the basal-like immune-suppressed (BLIS) subtype. RNA-FISH analysis showed nuclear/perinuclear and cytoplasmic TPL1 localization. Transcriptomic and proteomic analyses revealed suppression of pathways related to extracellular matrix-receptor interaction, focal adhesion, cell migration, and PI3K-Akt signaling following TPL1 knockdown. Proteomic array, RBPsuite, TLC-CLIP, miRNA profiling, and transcriptomic integration supported candidate protein-associated and ceRNA-like regulatory mechanisms involving TPL1. This study identifies TPL1 as a functionally relevant lncRNA associated with TNBC growth, invasion, and molecular regulatory programs. These findings support TPL1 as a candidate RNA-targetable vulnerability in TNBC and provide a framework for further mechanistic and translational investigation.

Androgens and androgen receptor (AR) signaling may play a role in lung cancer pathophysiology. Patients with benign prostatic hyperplasia (BPH) may represent a population with relatively lower androgen levels, and AR signaling can be inhibited through androgen pathway manipulation (APM). This study aimed to evaluate the impact of BPH and APM on survival outcomes in lung cancer patients. A total of 4,742 male lung cancer patients diagnosed between 2006 and 2021 were retrospectively analyzed. APM exposure referred to treatment with antiandrogens, 5-alpha reductase inhibitors, or gonadotropin-releasing hormone modulators for 30 days or longer post lung cancer diagnosis. Experimental validation was carried out using cultured A549 cells and a xenograft model in BALB/c nude mice. Among the enrolled patients, 1,219 (25.7%) had BPH, and 121 (2.6%) were exposed to APM. Multivariable Cox analysis showed that both BPH (aHR 0.72) and APM exposure (aHR 0.63) were independently associated with improved overall survival (both p < 0.001). The in vitro study demonstrated that AR was involved in A549 cell proliferation, and that AR inhibition with finasteride decreased AR protein levels and markedly inhibited A549 cell growth. Mechanistically, finasteride promoted AR protein ubiquitination and degradation, thereby destabilizing AR protein, further inhibiting cell cycle progression, and inducing apoptosis in A549 cells. In addition, the in vivo results corroborated finasteride's inhibitory effect on lung cancer growth. These findings suggest that both BPH and APM exposure were associated with improved survival in male patients with lung cancer. Finasteride disruption of AR stability might subsequently inhibit A549 lung cancer cell growth. Overall, these results highlight the potential therapeutic value of targeting androgen and AR signaling in lung cancer treatment.

Neoadjuvant immunochemotherapy (nICT) is increasingly used for resectable non-small cell lung cancer (NSCLC), yet a substantial proportion of patients fail to achieve pathological complete response (pCR). Clinically scalable, minimally invasive biomarkers that predict response and point to actionable resistance mechanisms remain needed. Pretreatment plasma samples collected prior to the first dose were profiled using the Olink proximity extension assay in a Sun Yat-sen University Cancer Center cohort (n=86), randomly split into a training set (n=65) and an internal validation set (n=21). Differentially abundant proteins associated with pCR were identified, and predictive models were developed using logistic regression, random forest, and extreme gradient boosting (XGBoost), followed by internal validation and independent external validation in the Jiangmen Central Hospital cohort (n=46). Survival associations were evaluated by Cox regression. Mechanistic analyses integrated tumor immunohistochemistry/multiplex immunofluorescence, bulk RNA sequencing with immune deconvolution, and functional validation in subcutaneous and orthotopic murine lung cancer models with pharmacologic leukemia inhibitory factor (LIF) blockade. Four candidate proteins (LIF, CXCL1, CX3CL1, and NT-3) showed modest single-marker discrimination for pCR (area under the curve (AUC), 0.611-0.673). Multiprotein models improved prediction, with XGBoost achieving the highest performance (training AUC=0.95; internal validation AUC=0.845 (95% CI 0.65 to 1.00); external validation AUC=0.801 (95% CI, 0.57 to 0.93)). SHapley Additive exPlanations analysis identified LIF as the dominant feature negatively associated with pCR probability. Elevated baseline plasma LIF was associated with inferior overall survival (HR=13.003; 95% CI 1.456 to 116.135; p=0.0217) and progression-free survival (HR=3.75; 95% CI 1.362 to 10.327; p=0.0105) after nICT. High LIF tumors exhibited reduced CD8+ T-cell infiltration and suppressed cytotoxic effector programs. In murine models, LIF blockade synergized with immunochemotherapy to enhance antitumor efficacy, restore CD8+ T-cell effector function, and mitigate T-cell exhaustion. Baseline plasma proteomics shows promise for predicting pCR to nICT in resectable NSCLC and nominates LIF as a predictive, prognostic, and therapeutic candidate mediator of resistance. These findings warrant prospective validation and further investigation of LIF-targeted combination strategies in the neoadjuvant setting.

Chemoimmunotherapy followed by immunotherapy maintenance is the standard first-line treatment for extensive-stage small cell lung cancer (ES-SCLC), yet data regarding efficacy and safety of consolidative thoracic radiotherapy (TRT) are lacking. To determine whether combining consolidative TRT with immunotherapy maintenance in ES-SCLC is safe and improves patients' overall survival (OS) and progression-free survival (PFS). This was a multicenter open-label phase 2 randomized clinical trial recruiting patients from September 2020 to August 2022 in Germany and Austria, with the last follow-up visit in September 2024. Eligible patients had ES-SCLC with at least stable disease after induction chemoimmunotherapy (carboplatin-etoposide-atezolizumab). Although the database was locked in April 2025, a post hoc survival update was performed in April 2026. Data were analyzed from April 2025 to April 2026. Randomized (1:1) to either atezolizumab maintenance combined with consolidative TRT (30 Gy in 10 fractions) (arm A) or atezolizumab maintenance alone (arm B). OS (time from randomization to death due to any cause). Of 96 patients assessed for eligibility, 68 patients were randomized; recruitment was prematurely terminated due to safety concerns. Median OS in the combination arm was numerically shorter compared to atezolizumab only (6.7 months [95% CI, 5.1-9.0] vs 13.4 months [95% CI, 10.7-17.5]; HR, 1.55 [95% CI, 0.90-2.69]; P = .34), while median PFS was similar (2.4 months [95% CI, 1.3-3.9] vs 2.6 months [95% CI, 1.2-3.9]; HR, 0.92 [95% CI, 0.54-1.55]; P = .85). TRT plus atezolizumab was accompanied by higher frequency of severe adverse events (SAEs) (19 patients [61.3%] vs 6 patients [18.2%]; P < .001) and fatal outcomes (6 patients [19.4%] vs 1 patient [3.0%]; P = .04). Safety analysis revealed predominance of infection and respiratory disorder-related SAEs, possibly facilitated by a depletion of lymphocytes observed specifically in patients after TRT, and by a lower single breath diffuse capacity of the lungs for carbon monoxide in patients with fatal AEs in arm A. No further risk factors were identified, given that baseline characteristics were well balanced between both arms and between patients with and without SAEs, including fatal SAEs. In this randomized clinical trial, TRT combined with immunotherapy increased toxic effects in unselected patients with ES-SCLC without survival benefit, presumably due to radiation-induced lymphocyte depletion facilitating infections. Cautious patient selection and further investigation to identify those who may benefit without undue risk are required. ClinicalTrials.gov Identifier: NCT04462276.

Preoperative localization is often required to achieve successful sublobar resection with adequate margins for computed tomography (CT)-detected pulmonary nodules suspicious for early-stage lung cancer. Conventional CT-guided localization involves a multiple-encounter workflow that may cause pain, radiation exposure, and complications. To determine whether a single-encounter augmented reality (AR)-guided percutaneous localization strategy is noninferior to standard multiple-encounter CT-guided localization for achieving successful sublobar resection. This randomized noninferiority trial was conducted at 5 centers in China between August 8, 2024, and September 30, 2025. Among 296 randomized patients, 270 were included in the modified intention-to-treat analysis (134 in AR; 136 in CT). Exclusion criteria included multiple nodules (≥2), unsafe percutaneous access, comorbidities limiting participation, or consent withdrawal. Single-encounter AR-guided percutaneous localization performed in the operating room under general anesthesia vs multiple-encounter CT-guided percutaneous localization performed in the CT suite under local anesthesia followed by transfer to the operating room for surgery. The primary outcome was successful sublobar resection, defined as R0 resection with protocol-defined margin adequacy according to nodule type. Secondary outcomes included localization accuracy, radiation exposure, complications, patient-reported outcomes, and procedural efficiency. The median (IQR) age of the population was 59 (50-67) years, and 172 participants (63.7%) were female. Successful sublobar resection occurred in 132 of 134 AR-guided procedures (98.5%) and 135 of 136 CT-guided procedures (99.3%) (risk difference, -0.8 percentage points; 95% CI, -2.7 to 3.9), meeting the noninferiority criterion. There was no statistically significant difference in localization error between groups (median [IQR]: AR group, 3.0 [0.0 to 5.0]; CT group, 3.0 [2.0 to 6.0]). AR guidance was associated with lower radiation exposure (median [IQR], 456.50 [378.75 to 631.85] vs 1260.11 [1026.48 to 1544.53] mGy · cm; P < .001), lower preoperative pain (median [IQR] numeric rating scale, 0 [0-0] vs 5 [4-6]; P < .001), shorter puncture time (median [IQR], 0.63 [0.50 to 0.83] vs 6.50 [5.00 to 8.75] minutes; P < .001), and shorter localization-to-incision interval (median [IQR], 2.00 [1.50 to 2.00] vs 33.50 [18.00 to 63.00] minutes; P < .001). Pneumothorax occurred in 40 of 136 CT-guided cases (29.4%). In this randomized clinical trial, single-encounter AR-guided localization was noninferior to CT-guided localization for achieving successful sublobar resection and was associated with reduced radiation exposure, lower pain, shorter puncture time, and a shorter localization-to-incision interval, supporting its use as an alternative to CT-guided localization. ClinicalTrials.gov Identifier: NCT06548451.

Accurate prediction of epidermal growth factor receptor (EGFR) mutation status in lung adenocarcinoma (LUAD) with brain metastases (BMs) is crucial for guiding targeted therapy. However, noninvasive and biologically interpretable tools remain limited. In this multicenter radiogenomic study, we analyzed a total of 1303 BMs from 421 LUAD patients across three institutions. 3435 radiomic features were extracted from T1, T2, and contrast-enhanced T1 sequences. A four-task classification framework was developed to predict EGFR mutation status (EGFR+, 19Del, L858R, or sensitizing mutation) using an adaptive LightGBM-based modeling pipeline. The models achieved excellent performance in the internal cohort (AUCs up to 0.95) and were further validated in 94 lesions with pathologically confirmed EGFR status, reaching an accuracy of 83.0%, sensitivity of 84.7%, and specificity of 80.0%. SHAP and LIME analyses revealed that shape-based radiomic features, particularly original_shape_sphericity, were the most important predictors of EGFR mutational subtypes. Then, we conducted transcriptomic analysis on 38 matched surgical specimens. Radiogenomic correlation revealed that sphericity negatively correlated with RNF125 and SLC37A2. Downstream enrichment analysis identified EGFR-associated features linked to DNA replication, sister chromatid segregation, and ERBB signaling. The study demonstrates that radiogenomic modeling, grounded in interpretable biology, holds promise as a non-invasive, clinical strategy for precision stratification of LUAD BMs.

Brain metastases (BrMs) in non-small cell lung cancer (NSCLC) respond poorly to anti-PD-1 monotherapy, but the underlying immune resistance remains incompletely defined. Here we integrate clinical outcome analyses, paired human tissue profiling and syngeneic mouse models to characterize the BrM immune microenvironment. Clinical analyses suggest improved intracranial disease control with nivolumab plus ipilimumab compared with nivolumab alone. Paired human specimens show that BrMs contain fewer cytotoxic T lymphocytes (CTLs) and tertiary lymphoid structures (TLSs) than primary tumors, defining an immune-excluded phenotype. A syngeneic BrM model recapitulates this phenotype and resists anti-PD-1 monotherapy, whereas combined anti-PD-1 and anti-CTLA-4 blockade suppresses tumor growth and prolongs survival. Single-cell RNA sequencing, flow cytometry and immunofluorescence show increased CTL infiltration and effector function after combination therapy. CD8+ T cell depletion abrogates therapeutic benefit, and combination therapy expands T follicular helper-like cells and induces TLS-like structures. These findings manifest increased adaptive immune response of dual checkpoint blockade in NSCLC BrMs.

Neuroendocrine carcinomas, including small cell lung cancers, are notable for their immune desert phenotype, even with the addition of immunotherapies. While this has been attributed to absent antigen presentation, Wang and colleagues demonstrate the existence of a vascular barrier that limits T cell infiltration and physically underlies this phenotype.

Sensory innervation regulates lung physiology and pathology, but its role in lung cancer is poorly understood. We show that lung adenocarcinoma (LUAD) progression locally amplifies nociceptive sensory innervation and activation, which drives the release of a major sensory neuropeptide, calcitonin gene-related peptide (CGRP). CGRP acts on a subset of macrophages, thereby impairing the recruitment of CXCL13+ fibroblasts and blocking tertiary lymphoid structure (TLS) assembly, a key predictor of LUAD prognosis. Local sensory denervation restores TLS formation, enhances B and T cell-dependent immunity, and suppresses tumor growth. Cigarette smoke extract (CSE) further activates this neural circuit to accelerate LUAD progression. In CSE-exposed animals, pharmacologic CGRP blockade sensitizes tumors to immunotherapy and prolongs survival. Together, our findings uncover a neuroimmune axis linking nociceptive neurons, TLS, and LUAD and identify neurogenic inflammation as a mechanism by which smoking promotes lung tumorigenesis independent of somatic mutagenesis.

Small cell lung cancer (SCLC) responds exceptionally well to cytotoxic chemotherapy. However, relapse with the emergence of chemoresistant disease is rapid and accompanied by poor treatment outcomes. To understand the genetic basis of chemoresistance in SCLC, we apply in vivo CRISPR deletion screening to patient-derived xenograft (PDX) models. Top screen hits include genes encoding components of the transcriptional co-activator SAGA (Spt-Ada-Gcn5 acetyltransferase) complex. We demonstrate that deletion of the SAGA deubiquitylase USP22 confers cisplatin-etoposide resistance in two chemosensitive PDX models, and that restoring expression in a PDX model harboring homozygous truncating mutation of USP22 re-sensitizes tumors to chemotherapy. USP22 loss increases gene body histone H2AK119 monoubiquitylation at key regulators of neuronal differentiation and suppresses neural and neuroendocrine gene expression including targets of ASCL1. Chemoresistance following USP22 loss reflects attenuated DNA damage-driven phosphorylation events and apoptosis, in conjunction with increased expression of glycolysis and hypoxia-related genes. Glycolysis program upregulation may reflect a targetable vulnerability, as inhibition of GLUT1 re-sensitizes USP22-null tumors to chemotherapy.

Itaconate is known as an anti-inflammatory metabolite derived from macrophages that dampens immune responses; however, its role in cancer has emerged to be complex. Mansouri et al.1 report a previously unknown anti-cancer feature of (octyl-) itaconate as an inhibitor of glucose-6-phosphate dehydrogenase (G6PD) in the lung tumor microenvironment (TME) in macrophages and cancer cells.

Non-Small-Cell Lung Cancer (NSCLC) often responds poorly to immune checkpoint blockade due to its immunosuppressive, "cold" tumor microenvironment. Activating alternative immune effectors may overcome this limitation. Here we identified lung-enriched γδ T cells as a key compartment in NSCLC and developed a lung-targeted lipid nanovaccine to activate them in situ. Analysis of patient transcriptomic data sets reveals that γδ T cell and CD1d signatures are associated with improved patient survival in NSCLC. Using this insight, we engineered α-galactosylceramide (α-GalCer) and poly(I:C)-loaded lipid nanoparticles that preferentially accumulated in the lung after intravenous administration. In orthotopic NSCLC models, the nanovaccine activated γδ T cells, enhanced functional CD8+ T cell infiltration, remodeled the immunosuppressive tumor microenvironment, and significantly prolonged survival. Depletion of γδ T cells abolished therapeutic benefit, demonstrating that γδ T cells represented the important effector population for this strategy. Furthermore, splenectomy attenuated vaccine efficacy, suggesting a contribution of systemic immune crosstalk to vaccine efficacy. Together, these findings establish a γδ T cell-centered lung-targeted immunotherapy strategy for treating immune-resistant NSCLC.

Non-small-cell lung cancer (NSCLC), the predominant type of lung cancer, is characterized by high invasiveness and significant mortality. Despite its clinical impact, the molecular mechanisms driving its pathogenesis and progression remain poorly understood. This study demonstrates that TMED9 is overexpressed in NSCLC and showed using multiple independent sample sets that its expression level is significantly associated with poor patient prognosis. Gain- and loss-of-function experiments revealed that TMED9 promotes proliferation, invasion, and migration of NSCLC cells in vitro and significantly accelerates tumor growth and metastasis in vivo. Mechanistically, TMED9 interacts with ATG9A and recruits USP5 to facilitate the deubiquitination and stabilization of ATG9A, thereby activating autophagy and driving malignant progression. Notably, genetic depletion of TMED9 enhances the sensitivity of NSCLC cells to osimertinib. Collectively, these findings identify the TMED9-USP5-ATG9A signaling axis as a critical driver of NSCLC malignancy, highlighting TMED9 as a promising therapeutic target.

Tumor-associated macrophages (TAMs) possess both tumor-promoting and tumor-inhibiting roles. Here, we explore TAMs' anti-tumor functions, focusing on the immune responsive gene 1 (IRG1) and its product, itaconate, in lung cancer development. Spatial metabolomics reveals that endogenous itaconate is markedly depleted within lung tumor regions compared with adjacent non-tumor tissue. Single-cell RNA sequencing shows that macrophages are the primary cells expressing IRG1 in human and mouse lung tumors. Both IRG1 knockout and transplantation of IRG1-depleted bone marrow leads to increased lung tumor growth in various mouse lung tumor models. Additionally, 4-octyl itaconate (Octyl Ita) reduces tumor growth in vitro, in vivo, and in ex vivo human tumor precision-cut lung slices. An integrated multi-omics analysis shows that IRG1/itaconate causes a metabolic shift in cancer cell and pro-tumor macrophages, mainly by inhibiting the pentose phosphate pathway (PPP) through targeting glucose-6-phosphate dehydrogenase (G6PD) activity, thereby suppressing cancer cell growth and transforming pro-tumor macrophages into anti-tumor macrophages. Thus, leveraging IRG1/itaconate's tumor-suppressive effects or using Octyl Ita could be a novel lung cancer therapy.

The aim of the study was to evaluate the complete response (CR) rate achieved with selective conventional transarterial chemoembolization (cTACE) using a porous glass membrane emulsification device in hepatocellular carcinoma (HCC). In a multicenter phase II trial (jRCTs052200095) conducted between January 2021 and June 2023, 50 patients with unresectable HCC (tumor diameter ≤5 cm; Child-Pugh A or B) were enrolled. An emulsion of epirubicin and ethiodized oil (Lipiodol) prepared using the emulsification device was injected into the tumor-feeding arteries, followed by embolization with gelatin sponge particles. The primary endpoint was the CR rate at 3 months post-treatment. Secondary endpoints included the CR rate at 1 month and the incidence of adverse events. Outcomes were compared with a historical control cohort from the JIVROSG-1302 study in exploratory analyses. Two patients were excluded after enrollment, leaving 48 patients who received cTACE (safety set). An additional 3 patients were excluded post-treatment, yielding 45 patients for efficacy analysis. The CR rate at both 1 month and 3 months was 97.8% (95% confidence interval: 88.2-99.9%). Serious adverse events (grade ≥3) included elevated AST in 50.0% of patients, elevated ALT in 29.2%, thrombocytopenia in 2.1%, and elevated ALP in 2.1%. The proportion of patients with deterioration in ALBI grade (≥1 grade increase) at 3 months was 17.8%. In exploratory analyses, CR rates were numerically higher than those observed in the historical control. Selective cTACE using a W/O emulsion prepared with a glass membrane pumping emulsification device was associated with high short-term CR rates in patients with HCC, with an acceptable safety profile. These findings support further clinical investigation.

Microvascular invasion (MVI) is a key prognostic factor in hepatocellular carcinoma but is currently only detectable after surgery. Here, we develop MAPUSE, a deep learning model using contrast-enhanced ultrasound (CEUS) to predict MVI non-invasively. We train and test the model on 5148 CEUS videos from 1716 patients across multiple centers. Results show that MAPUSE achieves accurate MVI prediction (AUCs 0.835-0.978) across different tumor sizes, contrast agents, and prospective validations. Transcriptomic analysis links the model's predictions to CD8 + T cell immune infiltration, confirmed via the model's attention maps. In a clinical cohort, patients predicted as MVI-positive can benefit from post-ablation immunotherapy. MAPUSE thus enables preoperative, non-invasive MVI assessment and provides insights into the tumor immune microenvironment, offering a valuable tool for clinical decision-making.

Locoregional therapy (LRT) is frequently used as bridging therapy to transplantation/resection or palliative treatment for hepatocellular carcinoma (HCC). Multiple prediction models have been developed for prognosis and treatment response among patients undergoing LRTs. We aimed to systematically review the methodological quality and performance of clinical risk scores predicting outcomes in patients with HCC treated with LRT. EMBASE and PubMed were searched from inception to 18 March 2026. Our main outcome was the concordance statistic, or area under the receiver operating characteristic curve (AUROC), to predict survival and other tumour-related and liver-related outcomes. 130 studies met the inclusion criteria, resulting in 179 individual scoring systems. The total population was 70 061 patients, 21% female (4-51%), most with Barcelona Clinic Liver Cancer Stage B (39%). Risk scores commonly incorporated tumour parameters, liver function tests, cirrhosis staging and comorbidities. AUROC values ranged from 0.56 to 0.94. 16 studies (12.3%) had low risk of bias, while most had high risk of bias. Of these, the Y-scoring system, Cheng et al nomogram and Li et al nomogram showed the highest discrimination for overall survival in palliative LRT (AUROC >0.87). Separate pooled analyses showed that the Hepatoma Arterial-embolisation Prognostic (HAP) and modified Hepatoma Arterial-embolisation Prognostic II (mHAP-II) scores had the highest performance (pooled AUROC >0.72), while the Six-and-Twelve (0.68) and Albumin-Bilirubin (ALBI) scores (0.60) demonstrated lower performance. Several outcome scoring systems are promising for specific LRTs and populations. However, most models have high risk of bias, underscoring the need for further development and validation.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide, largely due to the limited efficacy of current therapies in advanced stages of the disease. Most cases of HCC develop in the setting of chronic liver disease, particularly cirrhosis, where ongoing cycles of inflammation, hepatocyte death and regeneration foster the gradual accumulation of genetic and epigenetic alterations that promote malignant transformation. These molecular changes contribute to the high degree of tumour heterogeneity observed in HCC, a major factor underlying resistance to current treatments. As a result, sustained clinical responses to existing therapies, such as tyrosine kinase inhibitors, anti-angiogenic agents and immune checkpoint inhibitors, remain uncommon. In this context, a growing body of evidence has identified epigenetic dysregulation as a key driver of tumour progression and therapeutic resistance, highlighting a new frontier for intervention. This review provides clinicians and researchers with a comprehensive overview of the emerging field of epigenetic therapies in HCC, summarising results from both completed and ongoing clinical trials involving the so-called 'epidrugs'. Importantly, we discuss how targeting epigenetic mechanisms may not only suppress tumour growth but also enhance the effectiveness of current therapies by reversing resistance pathways. By translating complex molecular insights into tangible therapeutic strategies, epigenetics is poised to reshape the future of HCC management, offering renewed hope for more durable and personalised treatment responses in a disease where progress is urgently needed.

Long-term outcomes data of chronic hepatitis B (CHB) patients treated with high-genetic barrier nucleos(t)ide analogues (NA) beyond year-10 are scarce. We assessed the incidence and predictors of such long-term outcomes. The long-term PAGE-B cohort included 1644 Caucasians with CHB treated with entecavir/tenofovir. Cumulative incidence was estimated using Kaplan-Meier or cumulative incidence function accounting for competing events. Incidence rates (IR) per 100 person-years (/100 PYs) were estimated. Of 1644 patients, 903 were followed beyond year-10 (mean:14±2 years). The 10- and 15-year cumulative incidence of hepatocellular carcinoma (HCC) was 10.9% and 13.2%, respectively, and the HCC IR was 1.25 before and 0.55/100 PYs after year-10 (P<0.001), with similar findings after inverse probability weighting. The IR of death or liver transplantation (LT) was relatively lower before than after year-10 (1.50 vs 1.95/100 PYs, P=0.069) and similar for liver-related death/LT in the two periods (0.74 vs 0.70/100 PYs, P=0.840). HCC development and baseline platelets were independently associated with LT-free liver-related or overall survival, which was also associated with older age and diabetes. The 10- and 15-year cumulative incidence of HBsAg loss on NA(s) was 8.3% and 14.3%, respectively; the IR of HBsAg loss was 0.94 before year-10 increasing to 1.42/100 PYs after year-10 (P=0.025). HBsAg loss was independently associated with older age and baseline HBeAg-positive. NA therapy was discontinued in 125 (7.6%) patients remaining HBsAg-positive. Despite >10 years of NA therapy, CHB patients remain at risk for HCC, but the incidence rate declines significantly. HCC remains the main determinant of mortality. HBsAg loss rate increases after year-10, but it remains low being higher in older, initially HBeAg-positive patients.

Sialylated alpha-fetoprotein (sAFP) is a very potential marker for the pathogenesis exploration and clinical assessment of hepatocellular carcinoma (HCC). The specific and sensitive quantification of sAFP across multiple aspects is a primary premise. This work constructs a functionalized gold/silver nanocube-encapsulated microgel (Au/AgNC-MG), which can specifically capture sAFP through dual aptamer-based recognition and generate sensitive Raman fingerprints through the heterogeneous bimetallic SERS system. To further improve the specificity and quantifiability of sAFP detection in different scenarios, a series of machine learning (ML) algorithms, including a sAFP classification algorithm, a sAFP image-processing algorithm, and a sAFP-based clinical HCC risk assessment algorithm, were established for sAFP quantification, imaging of single-cell secreted sAFP, and clinical HCC risk assessment from general check-up to cirrhosis clinic patient populations. An online HCC Risk Assessment website is built for the convenience of practical clinical application. The constructed Au/AgNC-MG and established ML algorithms compose a general paradigm for HCC-related laboratory research and clinical applications.

Radiotherapy is standard-of-care treatment for intermediate and advanced hepatocellular carcinoma (HCC); however, resistance remains widespread. Microplastics are detectable in various tumors and may disrupt immune responses, but evidence regarding the role of microplastics in radiotherapy is scarce. Here we show that various types of microplastics are detectable in 52 of the 72 HCC samples we examine, but only polyvinyl chloride (PVC) impairs radiotherapy efficacy. Mechanistically, irradiation enhances histone lactylation, which promotes transcription of HMG-CoA reductase in HCC cells, thereby facilitating cholesterol synthesis and reinforcing CD8+ T cell stemness. PVC alters cholesterol localization and affects CD8⁺ T cell differentiation, which results in the maintenance of an immunologically cold tumor immune microenvironment. A high-cholesterol diet restores CD8+ T-cell stemness and improves therapeutic response to radiotherapy in PVC-infiltrated HCC. Targeting cholesterol metabolism may represent a potential combinatorial strategy to enhance the efficacy of radiotherapy in PVC-infiltrated HCC.

Immune checkpoint blockade (ICB) targeting the programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis has shown promise in hepatocellular carcinoma (HCC), but clinical responses are often limited in durability. Identifying novel drivers of immune evasion is crucial for improving therapeutic strategies. We employed a multi-omics integrative framework to identify HCC-specific immune biomarkers. Functional roles were validated using in vitro and in vivo models, including UDP-glucuronate decarboxylase 1 (UXS1) knockout/overexpression, co-culture assays, and various mouse models. Molecular mechanisms were dissected using immunoprecipitation coupled with mass spectrometry, ubiquitination assays, chromatin immunoprecipitation, luciferase reporter assays, and single-cell RNA sequencing. We identified the glycosyltransferase UXS1 as a novel immune-related prognostic hub gene in HCC. UXS1 was upregulated due to copy number gain and MZF1-driven promoter hypomethylation. Functionally, UXS1 promoted HCC malignancy independently of its canonical enzymatic activity. Mechanistically, UXS1 interacted with the E3 ligase F-box and WD repeat domain-containing 7 (FBXW7) to promote histone-lysine N-methyltransferase 2D (KMT2D) ubiquitination and degradation. We further discovered that KMT2D, acting as a non-catalytic scaffold, recruited the transcriptional repressor CCAAT enhancer binding protein beta (CEBPB) to the PD-L1 promoter to maintain its repression. UXS1-mediated KMT2D degradation disrupted this complex, leading to CEBPB dissociation and consequent PD-L1 transcriptional derepression. In vivo, UXS1 suppressed CD8+ T-cell effector function. UXS1 knockout synergized with anti-PD-1 therapy, and in human HCC, high UXS1 correlated with low KMT2D, high PD-L1, and reduced CD8+ T-cell infiltration. This study defines a novel UXS1-KMT2D-CEBPB-PD-L1 signaling axis, revealing how UXS1 drives immune evasion in HCC via epigenetic reprogramming. Targeting this axis may represent a potential strategy to overcome immune resistance, warranting further clinical investigation.

Nanozyme-based catalytic therapy has emerged as a promising antitumor strategy by leveraging endogenous substrates to generate reactive oxygen species (ROS) and induce oxidative damage in tumors. However, its efficacy is often constrained by inadequate catalytic activity and inefficient in vivo delivery, largely due to the complex tumor microenvironment. To overcome these limitations, we developed a dual-atom nanozyme (DA nanozyme) with precisely paired Fe-Co bimetallic active sites, which exhibits significantly enhanced multienzyme mimetic activities─including oxidase-like (OXD), peroxidase-like (POD), and catalase-like (CAT) functions and glutathione (GSH) depletion. This synergistic catalytic action amplifies oxidative stress and promotes tumor cell death. Furthermore, a biodegradable composite microneedle (MN) patch was engineered for localized delivery of the FeCo DA nanozyme directly to subcutaneous tumor sites. By integrating multienzyme catalysis with near-infrared photothermal therapy, this platform effectively inhibits hepatocellular carcinoma growth and achieves complete tumor eradication in vivo. Collectively, this work provides an innovative and translatable strategy for synergistic antihepatoma therapy through rational nanozyme design and precision tumor-localized delivery.

Local tumor progression and metastasis substantially compromise the therapeutic efficacy of thermal ablation in liver cancer. Although the inflammatory microenvironment contributes, the specific role of neutrophils after ablation remains unclear. This study aimed to clarify the roles of neutrophil extracellular traps (NETs) after thermal ablation and evaluate the translational potential of NETs-targeted strategies. The adverse prognostic impact of thermal ablation-induced neutrophil and inflammatory changes in liver cancer was retrospectively evaluated. Circulating NETs were quantified by ELISA, and their association with local tumor progression was analyzed. An orthotopic liver cancer mouse model and patient-derived neutrophils elucidated the spatiotemporal dynamics and mechanisms of ablation-induced NETs, and pharmacologic inhibition was used to investigate NET formation, function, and translational potential. Thermal ablation rapidly activates neutrophils, elicits a sustained local inflammatory response and NET formation, which is closely associated with poor prognosis in patients with liver cancer. Depletion of neutrophils, inhibition of NET formation, or degradation of thermal ablation-induced NETs markedly suppressed post-ablation tumor progression. Mechanistically, tumor cell-intrinsic reactive oxygen species generated under heat stress contributed to nuclear factor kappa-B (NF-κB) activation, promoting p65 binding to theC-X-C motif chemokine ligand 1 (CXCL1) promoter and transcriptional upregulation of CXCL1. This process drove CXCL1-C-X-C motif chemokine receptor 2 (CXCR2) signaling and led to the formation of a characteristic band-like NETs-enriched zone at the peri-ablational margin. In turn, NETs activated toll-like receptor (TLR)9-dependent NF-κB,mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription 3 (STAT3) signaling in heat-stressed cancer cells, reinforcing a pro-inflammatory feedback loop. Concurrently, NETs impaired CD8+ T-cell responses and fostered an immunosuppressive microenvironment. Together, this coordinated pro-inflammatory and immunosuppressive effect promotes liver cancer progression following thermal ablation, which can be effectively reversed by dual blockade of NF-κB and CXCR2. Our research uncovers a novel mechanism underlying tumor-promoting inflammation after thermal ablation, highlighting the critical role of NETs as mediators through TLR9 activation of downstream inflammatory pathways and the significant potential of NETs-targeted strategies in combination with thermal ablation.

Lymphocyte activation gene 3 (LAG-3) is an immune checkpoint implicated in T cell exhaustion and a potential therapeutic target in glioblastoma (GBM). We conducted a multicenter, open-label, phase 1 study with sequential allocation to evaluate the safety and preliminary activity of the anti-LAG-3 antibody relatlimab, administered alone or with the anti-programmed cell death protein 1 (PD-1) antibody nivolumab, in patients with recurrent GBM. Forty-six patients were treated (23 per cohort). The primary endpoint of safety was met, with maximum tolerated doses of 800 mg relatlimab for monotherapy and 160 mg relatlimab/240 mg nivolumab for combination therapy. Treatment-related grade 3-4 adverse events occurred in 6 of 23 patients receiving combination therapy and were not observed with monotherapy. Neoadjuvant administration was associated with increased intratumoral CD8+ T cell infiltration for both monotherapy and combination therapy. Exploratory analyses suggested that tumors with elevated baseline interferon signaling and increased T cell clonality were enriched among patients with durable responses to combination therapy. Twelve-month overall survival was 34.8% with relatlimab alone and 52.2% with combination therapy; however, this study was not designed to assess efficacy. These findings demonstrate an acceptable safety profile and provide preliminary immunologic and clinical signals supporting further evaluation of LAG-3 blockade in GBM. ClinicalTrials.gov identifier: NCT02658981 .

Radiation necrosis (RN) complicates neuro-oncological care, mimicking tumor recurrence and lacking high-level evidence for standardized management. A European Association for Neuro-Oncology (EANO) expert panel utilized a three-round Delphi process to create a comprehensive expert opinion document based on the available current scientific evidence. A series of statements, derived from the published literature were created by the experts in each field. Consensus was defined as ≥ 80% agreement using a 5-point Likert scale. After three rounds among 20 experts that included adaptation of statements, the Delphi process reached a consensus (≥80% agreement) on 53 statements out of 57. RN occurs in 4% to 30% of patients, typically appearing 6 to 24 months after radiotherapy for primary (glial) or metastatic brain tumors. Experts identified perfusion MRI and amino acid PET as the most suitable imaging modalities for differentiation from tumor recurrence. While histopathology remains the gold standard, identifying viable tumor cells in irradiated gliomas is challenging due to overlapping cytological features with reactive glia. For symptomatic management, corticosteroids may be tried, and bevacizumab is recommended for corticosteroid-refractory cases, with evidence suggesting profound efficacy even at low doses. Surgery is considered effective for rapid symptom relief and definitive diagnosis in accessible lesions. Laser Interstitial Thermal Therapy (LITT) can be considered an additional treatment option for symptomatic RN. Despite the absence of Level 1 evidence, these Delphi-survey-formulated recommendations provide actionable guidance for clinical practice. There is a need for prospective randomized trials focusing on symptomatic RN.

Artificial intelligence (AI)-based models have shown initial promise in imaging brain metastasis; however many lack validation against advanced imaging-informed datasets, precluding external validity and limiting widespread adoption. To overcome these limitations, we performed comprehensive performance testing against reference standard metrics and externally validated an AI algorithm. As part of its FDA-clearance process, performance testing of a previously developed U-Net-based AI model was conducted on a multi-institutional cohort with reference standard established via consensus review by three neuroradiologists. External validation was performed on patients imaged with dual sequences (augmented) as well as an open-access dataset (UCSF-BMSR). Evaluation metrics included sensitivity, false positive (FP) rate, positive predictive value (PPV), Dice Similarity Coefficient (DSC), 95% Hausdorff distance (HD95), normalized surface distance (NSD), and qualitative physician assessment. In the FDA performance testing cohort, the AI algorithm achieved a sensitivity of 90.0% (95% CI: 87.0%-94.0%), DSC of 0.86 (95% CI: 0.83-0.89), and average FP rate of 0.57 lesions. In the augmented and open-access external validation cohort, a sensitivity of 81.4% (95% CI: 73.7%-89.1%) and 85.2% (95% CI: 83.0%-87.4%) with an average number of 0.22 and 1.19 FP lesions and DSCs of 0.70 (95% CI: 0.66-0.73) and 0.78 (95% CI: 0.77-0.78) were calculated, respectively. In the augmented external validation cohort, 46.3% of contours were rated as requiring major revisions. This AI algorithm demonstrated promising performance via three unique datasets. However, given the notable rate of contour revisions, these findings support its clinical role not as an autonomous system, but as a human-in-the-loop tool requiring physician oversight.

Primary tumors of the central nervous system are extremely aggressive and often incurable. While tumor cells are known to interact with their microenvironment, the complexity and temporal dynamics of this interplay and its impacts on tumor progression remains to be fully understood. We addressed this question in a Drosophila model of cancer stem cell-driven tumor which originates during development and grows extensively within a network of cortex glia cells through adulthood. We revealed a biphasic interplay between tumor and cortex glia cells, characterized by morphological, molecular, and functional changes. In early stages, glial cells infiltrate the tumor, display a distinct transcriptional signature, and resist its growth, supported by the intrinsic neuroprotective activity of the c-Jun N-terminal kinase (JNK) signaling pathway. However, cancer stem cell-driven competition takes place, eliminating cortex glia by apoptosis and ultimately unleashing tumor growth. This second phase sees the breakdown of the glial meshwork and adhesions to neurons, along with the downregulation of the JNK pathway and a decline in essential cellular functions. Ultimately, the host tissue collapses, in turn curbing tumor growth. This study uncovers a dynamic and complex interplay between host tissue resistance and tumor-driven competition, which shapes tumor progression.

Glioblastoma is the most aggressive primary brain tumor, with poor prognosis and limited treatment options. Natural killer (NK) cell therapy is a promising immunotherapeutic strategy, yet its efficacy remains limited. We evaluated FT538, a clinical-grade NK product derived from induced pluripotent stem cells (iPSCs), in glioblastoma models. FT538, engineered with a high-affinity non-cleavable CD16 Fc receptor, a membrane-bound IL-15/IL-15Rα fusion protein, and CD38 knockout, was tested against 13 patient-derived glioblastoma stem-like cells (GSCs) in vitro and in orthotopic xenograft models. Intracranial persistence and neurotoxicity were assessed in mice. Surface proteomics identified therapeutic targets, and a B7-H3-targeted tri-specific killer engager (TriKE) was evaluated with FT538 and NKG2C+ adaptive NK cells. GSCs were classified as sensitive (38%), moderately sensitive (38%), or resistant (23%) to FT538. Intracranial administration in mice was well tolerated, persisted for at least 35 days, and caused no neurotoxicity. A single intratumoral dose induced complete regression in sensitive xenografts. Surface profiling identified B7-H3 as a target to overcome resistance. Combination therapy with FT538 and a B7-H3 TriKE enhanced antitumor efficacy in resistant models, an effect also observed with adaptive NK cells. FT538 exhibits potent tumoricidal activity in 77% of GSC lines (NK-sensitive and moderately sensitive), with curative potential in sensitive models, and demonstrates favorable persistence and tolerability in vivo. B7-H3-targeted TriKE restores NK sensitivity in resistant tumors. These findings provide a strong preclinical rationale for further clinical evaluation of FT538, alone or combined with B7-H3-targeted TriKE, for glioblastoma and other solid tumors.

Humoral immunity, mediated by B cells that mature in germinal centers in lymph nodes (LNs), is essential for adaptive immune responses, but its role in antitumor immunity and responses to immunotherapy remain unclear. Here, we show that activation of B cells in tumor-draining deep cervical LNs (dcLNs) is necessary for the efficacy of CTLA-4 (cytotoxic T lymphocyte-associated protein 4) immune checkpoint blockade in glioma in vivo. Anti-CTLA-4 therapy enhanced T follicular helper cell (TFH cell) expansion in dcLNs, leading to germinal center B cell responses, immunoglobulin G (IgG) class switching, and the generation of glioma-reactive antibodies. Glioma-bearing mice lacking antibody-secreting cells did not benefit from CTLA-4 blockade. Distally secreted IgG accumulated in the tumor microenvironment and promoted glioma cell phagocytosis in vivo. These findings define a B cell-dependent mechanism underlying CTLA-4-mediated control of glioma and provide a conceptual framework for future therapeutic strategies in tumor.

The central nervous system is a critical reservoir for cancer metastasis, underscoring the urgent need for effective strategies for targeted drug delivery that minimize systemic side effects. Here, we present a zwitterionic polycarboxybetaine (PCB) designed to achieve selective accumulation within the tumor vasculature of brain metastases, thereby facilitating the rapid release of therapeutics without relying on external stimuli in preclinical models. PCB selectively targets the betaine-γ-aminobutyric acid transporter 1 (BGT1) at the blood-brain-tumor barrier (BBTB), enabling localized enrichment of nanoparticles within the tumor vasculature. The intrinsic mechanical flexibility of PCB activates the Piezo1 ion channel in endothelial cells, resulting in calcium ion influx and enhanced calpain activity, which together facilitate the degradation of VE-cadherin. The burst-release mechanism improves vascular permeability and compromises the integrity of the tumor vascular barrier, allowing for the swift release of encapsulated therapeutics and promoting extensive drug distribution within the tumor microenvironment. Notably, PCB encapsulating either osimertinib or cisplatin demonstrates therapeutic efficacy, intratumoral targeting, and favorable biological safety in disease models. These findings indicate that this zwitterion-functionalized platform effectively surmounts the challenges posed by the BBTB, supporting improved drug delivery and therapeutic efficacy in brain metastases.

Chronic stress has long been associated with poor cancer outcomes, yet the mechanisms linking systemic stress responses to glioblastoma progression remain poorly understood. In this issue of Cancer Cell, Yang et al. identify a stress-induced brain-bone marrow axis that rewires monocyte differentiation toward pro-tumorigenic macrophages, revealing unexpected therapeutic opportunities in glioblastoma.

Glioblastoma (GBM) is a devastating cancer with a dismal prognosis. Current treatment includes temozolomide (TMZ), which is more effective in about 50% of GBMs that have O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. MGMT is a DNA repair protein that reverses TMZ-induced DNA damage. EGFR is a prime oncogene in GBM. Here, we report that EGFR inhibition induced the down-regulation of MGMT in GBM cells, revealing a previously unidentified link between EGFR signaling and response to TMZ. EGFR inhibition led to activation of two transcription factors, activator protein-1 (AP-1), which repressed MGMT transcription, and nuclear factor κB (NF-κB), which up-regulated MGMT transcription. EGFR inhibition also induced AP-1-mediated transcription of miR-616. miR-616 inhibited both MGMT translation and NF-κB activation. Thus, the overall effect of EGFR inhibition was down-regulation of MGMT expression. In addition, we provided an explanation for the prior failure of clinical trials that used concomitant EGFR tyrosine kinase inhibitors (TKIs) and TMZ. TMZ up-regulated MGMT, and concomitant treatment with EGFR TKIs and TMZ failed to down-regulate MGMT. However, pretreatment with EGFR TKIs followed by TMZ efficiently down-regulated MGMT and enhanced TMZ sensitivity in experimental models. Posttreatment tumor tissues from two clinical trials were used to validate these findings. We demonstrated that EGFR inhibitors induced down-regulation of MGMT in posttreatment resected tumor tissues from patients with GBM and the failure of EGFR inhibition to down-regulate MGMT if TMZ was used concomitantly. These data support using EGFR TKIs before TMZ treatment as a therapeutic approach in MGMT unmethylated GBM.

Cuproptosis presents a potential therapeutic avenue for glioblastoma (GBM), yet its efficacy is severely limited by intrinsic and adaptive resistance mechanisms. Here, we identify a critical therapy-induced barrier where standard-of-care interventions, including Temozolomide, radiotherapy, and Tumor Electric-Field Therapy (TEFT), consistently induce a profound downregulation of essential cuproptosis-execution genes such as Ferredoxin 1 (FDX1) and Dihydrolipoamide S-Acetyltransferase (DLAT). This transcriptomic remodeling reveals a universal mechanism of acquired cuproptosis resistance in recurrent GBM, rendering residual tumor cells refractory to copper toxicity despite their elevated metabolic stress. To overcome this maladaptive remodeling, we engineered an electric-field-responsive CuBi2O4 (CBO) nanoplatform to establish an FDX1-independent, upstream-bypass paradigm for copper activation. Crucially, this strategy repurposes TEFT from a purely cytostatic modality into a physical stimulus tool. The external electric field catalyzes a nonenzymatic Cu2+/Cu+ redox cycle specifically within lysosomes. This process generates a lethal copper pool that bypasses the downregulated FDX1 machinery and translocates to mitochondria, where it converges on lipoylated DLAT-associated cuproptosis execution. Validated in orthotopic and recurrent GBM models, this approach enforces robust cytotoxicity and activates the cGAS-STING pathway to reverse immunosuppression. When combined with anti-PD-1 blockade, this TEFT-triggered nanomedicine elicits durable antitumor immunity, offering a versatile strategy to exploit therapy-induced stress states in refractory malignancies.

KRASG12D is the predominant oncogenic driver in pancreatic ductal adenocarcinoma (PDAC). While most investigational KRAS-G12D inhibitors are oral small molecules limited by gastrointestinal toxicities and suboptimal tumor exposure, HRS-4642 is a new, high‑affinity, noncovalent KRAS-G12D inhibitor. Formulated as a liposomal nanoparticle for intravenous administration, it is designed to enhance tumor accumulation and prolong the duration of target inhibition. This phase 1b/2 study evaluated HRS-4642 in combination with nab-paclitaxel and gemcitabine (AG) in patients with advanced KRASG12D-mutant PDAC. As of 5 December 2025, 68 patients were screened and 31 (1 previously treated patient and 30 treatment-naive patients) were enrolled and treated. In the phase 1b portion, no dose-limiting toxicities were observed, and the starting dose (500 mg on day 1 and 1,200 mg on day 8, every 3 weeks) was selected as the recommended phase 2 dose. In the phase 2 portion, with a median follow-up of 12.3 months (95% confidence interval (CI) = 12.2-13.0), the primary endpoint was met-the confirmed objective response rate in 30 treatment-naive patients was 63.3% (95% CI = 43.9-80.1). Grade ≥3 treatment-related adverse events (TRAEs) occurred in 90.3% patients, primarily hematologic toxicities consistent with AG chemotherapy. No TRAEs led to treatment discontinuation or death. In conclusion, HRS-4642 combined with AG demonstrates promising antitumor activity and a manageable safety profile in advanced KRASG12D-mutant PDAC, supporting further investigation. Clinicaltrials.gov registration: NCT06520488 .

Pancreatic ductal adenocarcinoma has a unique tumor microbiome, and the depletion of gut bacteria or fungi using antibiotic/antifungal cocktails has been shown to decrease pancreatic tumor burden in mice. However, functional studies evaluating the role of tumor-associated microbes are few due to the limited availability of clinically relevant microbiota. Here, we describe in detail an effective workflow for the isolation of bacteria and fungi from the duodenum and tumor of pancreatic cancer patients, specifically optimized for cryopreserved, low biomass samples. Using this workflow we also isolated microbiota from normal pancreatic tissue and duodenum from organ donors, and we confirmed the presence of bacteria and fungi isolated from tissue samples with 16S and ITS sequencing analysis. Isolation and sequencing results show distinct similarities between the pancreatic and duodenal microbiomes and highlight unique bacterial strains that survive in the tumor microenvironment. As a proof of concept, we characterized a select Klebsiella oxytoca strain (UMKO1) isolated from a pancreatic tumor, using whole genome sequencing, metabolomics, and ex- vivo tumor cultures to determine its potential impact on the pancreatic tumor microenvironment. In summary, this optimized workflow allows for the isolation of a variety of bacteria and fungi from low biomass, cryopreserved pancreatic and duodenal tissues, which can then be used for functional studies characterizing clinically relevant tumor-associated microbiota.

Nature killer (NK) cell plays a critical role in cancer immunosurveillance and is considered a potent immunotherapeutic tool for many cancers, including pancreatic ductal adenocarcinomas (PDACs). Increasing evidence suggests that cancer occurs with systemic immune perturbations. However, the effects of PDAC tumor burden on systemic NK cells remain poorly understood. PDAC tumor-bearing mice display decreased frequency and dysfunction of NKs the spleens. We identified an increase in Gr-1+ myeloid cells within the spleens, which negatively impacts both endogenous and adoptively transferred NK cell frequency and function. Apolipoprotein E (ApoE), a lipid metabolism regulator, is upregulated in Gr-1+ myeloid cells of tumor-bearing mice, promoting lipid oxidation and reactive oxygen species (ROS) generation. Genetic knockout of Apoe in Gr-1+ myeloid cells abrogate their suppressive effects on NK cell function. Furthermore, treatment with lipid metabolism inhibitors restores endogenous and adoptively transferred NKs' effector function in the spleens and tumor microenvironment. These studies underscore the importance of understanding preexisting systemic alterations in PDAC patients before applying NK cell-based immunotherapies.

The pancreatic tissue is composite and plastic. The epigenetic makeup of different cell types is poorly characterized due to the lack of protocols that enable efficient recovery of epithelial cells. Here, we improve the canonical pipeline for single-cell analysis, optimizing the harvesting of nuclei from hard-to-dissociate tissues. We provide an in-depth mapping of the murine pancreas through paired RNA/ATAC multiomic single-nucleus sequencing, documenting the transcriptomic and chromatin accessibility profiles of every cell in the parenchyma. This enables a superior examination of the exocrine fraction of the pancreas. We described endocrine-exocrine interaction in silico and validated pro-proliferative signals in vitro. At the same time, we assessed pancreatic cellular heterogeneity in a holistic manner. We showed that pancreatic acinar cells can acquire multiple phenotypic states that lead to the functional diversification of pancreatic acini. In particular, we identified ultra-specialized ZG16HIGH cells in vivo and associated them with superior protein synthesis by tracking the incorporation of a synthetic amino acid. Leveraging the pairing of gene expression and chromatin opening, we studied the epigenetic elements that dictate acinar cell specialization and how they are affected by tissue repair after inflammation. We found that a subset of acinar cells shows enhanced sensitivity to inflammatory cues that determine extensive and persistent chromatin opening. The dataset is an open-source framework to interrogate the molecular histology of the murine pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterised by remarkable cellular heterogeneity, which emerges early from the interplay of oncogenic KRAS signalling and inflammatory injury. However, the transcriptional, metabolic and functional properties of these pre-malignant cell states that initiate and drive PDAC progression remain elusive. This study aimed to identify and functionally characterise the critical premalignant cell states that arise from this heterogeneity, to define novel biomarkers and targets for early intervention. Public and in-house scRNA-seq data of pancreatic tumour models were analysed to identify key subpopulations in early cellular heterogeneity. Genetic perturbation in KrasG12D-driven models was performed to assess functional impact. Mechanistic studies used TurboID proximity proteomics, epigenetic profiling and metabolic assays. Clinical relevance was validated in human PDAC cohorts. We identified LY6D as a marker of a distinct, gastric-like cell state that emerges early and persists throughout tumourigenesis. The LY6D+ population exhibits conserved stemness and a unique, pan-stage dependency on oxidative phosphorylation (OXPHOS). Genetic ablation of Ly6d specifically impaired the gastric lineage and delayed tumourigenesis, while its overexpression enhanced tumourigenic and metastatic potential. Mechanistically, the glycosylphosphatidylinositol (GPI)-anchored LY6D protein scaffolds a lipid raft-associated kinase network that drives FOSL1-dependent epigenetic-transcriptional reprogramming. In human PDAC, LY6D+ cells harbour stemness and Epithelial-Mesenchymal Transition (EMT) signatures, and high LY6D expression is an independent prognostic marker of poor survival. Our work defines the LY6D+ gastric-like cell state as a key driver linking early pre-malignant heterogeneity to PDAC initiation and progression. LY6D represents a pan-stage therapeutic target and a candidate biomarker for early detection and therapeutic targeting.

The efficacy of pharmacological glycogen synthase kinase-3β (GSK3β) inhibition is currently being investigated in unselected cohorts of metastatic pancreatic ductal adenocarcinoma (PDAC). Here, we sought to determine the clinical significance of nuclear GSK3β accumulation in patients with resectable PDAC. This study aimed to explore the therapeutic potential and underlying mechanisms of GSK3β pathway disruption in PDAC with enriched nuclear GSK3β levels. We investigated the activation and function of GSK3β and its downstream transcription factor NFATc1 in tumour recurrence, growth and resistance using human PDAC tissues, patient-derived organoids and tumour cells, PDAC explants, cell lines and murine models. GSK3β signalling was disrupted using genetic and pharmacological approaches. Live-cell imaging, proliferation, homologous recombination (HR) repair and comet assays, messenger RNA sequencing and chromatin immunoprecipitation were used to explore GSK3β-NFATc1 signalling-mediated target gene regulation in DNA repair, growth and resistance. Nuclear GSK3β accumulates in a subset of resected PDAC and promotes proliferation and DNA repair through NFATc1. The GSK3βhigh/NFATc1high subtype accounts for 14% of resected PDAC and is associated with rapid tumour recurrence and poor survival. The GSK3β-NFATc1 signalling pathway contributes to cisplatin resistance by inducing BRCA genes transcription, which facilitates HR-mediated DNA double-strand breaks (DSBs) repair. Disruption of the GSK3β-NFATc1 axis impairs HR-driven DSB repair, increasing cisplatin sensitivity in vitro and in preclinical PDAC models. We have identified a highly aggressive GSK3βhigh/NFATc1high subtype that predicts early recurrence, poor survival and cisplatin resistance in PDAC. This subtype reveals new treatment vulnerabilities suggesting that patients with PDAC may benefit from stratification-based tailored treatment strategies.

The biological significance of forkhead box A1 (FOXA1) in non-steroid-driven malignancies, such as pancreatic ductal adenocarcinoma (PDAC), has garnered increasing recognition. It assumes a pivotal role in regulating critical processes such as PDAC cell lineage, metabolism, and metastasis. However, its regulatory mechanisms remain elusive. Here, we demonstrate that histone deacetylase 5 (HDAC5) mediates the deacetylation of FOXA1 at lysine residue 270 (K270), leading to repression of FOXA1's global chromatin occupancy. In HDAC5-loss PDAC, K270 hyper-acetylated FOXA1 is reprogrammed to the transcription start sites (TSSs) of HIF1α-targeted genes, functioning as a pioneer factor of HIF1α signaling. Additionally, we show that HDAC5 antagonizes LSD1-mediated FOXA1 activation by converging on the dynamic equilibrium of acetylation-methylation transition at K270. Pharmacological inhibition of HIF1α/LSD1 suppresses the growth and progression of HDAC5-deficient PDAC in in vivo and in vitro models. Our study reveals the role of FOXA1 as a pioneer factor of HIF1α in PDAC, providing potential therapeutic strategies for HDAC5-deficient PDAC.

Pancreatic ductal adenocarcinoma (PDAC) exhibits profound therapy resistance driven by lysosome-dependent nutrient recycling, metabolic adaptation, and stress tolerance. Current lysosome targeting agents such as chloroquine (CQ)/hydroxychloroquine (HCQ) show limited efficacy due to transient activity and dose-limiting-toxicities. To overcome these limitations, we developed lysostilbenes, a new class of hybrid small molecules combining the CQ pharmacophore with lysosome-disrupting stilbene analogs. Stilbene pharmacophore is the core structural component of resveratrol. Among the synthesized hybrids, lysostilbene-4 emerged as the lead candidate, demonstrating ~30-40-fold greater cytotoxicity against PDAC cells than parent compounds, while sparing nonmalignant cells. At nanomolar concentrations, lysostilbene-4 induced rapid, irreversible lysosomal membrane permeabilization (LMP), initiating a lysosome mitochondria apoptotic cascade via CTSB (cathepsin B) release, BID cleavage, BAX activation, and caspase-mediated apoptosis. In parallel, it abrogated lysosomal recovery by significantly reducing repair, lysophagy, autophagosome maturation, and uncoupling TFEB-driven transcriptional programs from effective lysosome biogenesis. Reduced TFEB mRNA expression correlated with poor overall-survival and disease-free-survival across multiple cancer patients, with a particularly strong association in pancreatic cancer patients. Using TFEB+/+ and TFEB-/- knockout pancreatic cancer cells we establish that lysostilbene-4 exerts severe cytotoxicity by inducing persistent lysosomal-damage and disrupting autophagosome-lysosome assembly, with vulnerability further amplified in TFEB-deficient cells. This finding underscores TFEB as a key determinant of lysosomal-resilience and a potential predictive biomarker. Importantly, lysostilbene-4 was well tolerated in preclinical mouse-models at supra-therapeutic doses without systemic-toxicity. These findings position lysostilbene-4 as a first-in-class lysosome-targeting therapeutic that enforces sustained lysosomal collapse while compromising adaptive recovery-mechanisms, providing a mechanistically precise and safe strategy against PDAC.Abbreviations: ALG: autophagy-lysosome genes; AMPK: AMP-activated protein kinase; CASM: conjugation of ATG8s to single membranes; CTSB: cathepsin B; LGALS3: galectin 3; LMP: lysosomal membrane permeabilization; LS: lysostilbene; MTOR: mechanistic target of rapamycin kinase; PDAC: pancreatic ductal adenocarcinoma; TCGA: The Cancer Genome Atlas; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers due to late diagnosis, early metastasis and resistance to therapy. Cancer stem cells (CSCs) have been implicated in PDAC aggressiveness and treatment failure. MAP kinase-interacting kinase 1 (MNK1) is overexpressed in PDAC and plays a critical role in tumor progression and CSC maintenance. Here, we show the potential of apMNKQ2, a DNA aptamer targeting MNK1, to therapeutically target CSCs and reduce PDAC tumor burden in patient-derived xenografts (PDXs). PDX cell lines and in vivo mouse models were used to assess the effects of apMNKQ2 on cell viability, apoptosis, cell cycle progression, migration, epithelial-to-mesenchymal transition (EMT) and CSC properties. Functional CSC targeting was validated through clonogenic and self-renewal assays as well as extreme limiting dilution analysis. Systemic administration of free apMNKQ2 was tested for biodistribution, pharmacokinetics, toxicity, and antitumor efficacy at escalating doses. apMNKQ2 downregulated MNK1 and anti-apoptotic proteins (MCL1, XIAP), impaired cell proliferation, induced apoptosis, and disrupted cell cycle progression in PDX PDAC cells. Importantly, apMNKQ2 also inhibited migration, mesenchymal properties and angiogenesis in vitro, and lung colonization in vivo. Notably, apMNKQ2 strongly targeted PDAC CSCs, reducing CD24, CD133, CXCR4 and ALDH expression, clonogenicity and in vivo tumor initiation over 600-fold. Free apMNKQ2 (without transfection agents) entered PDAC cells efficiently, retaining anti-CSC activity. Systemic delivery of free apMNKQ2 accumulated in tumors, was well tolerated up to 400 mg/kg and showed no toxicity. Importantly, 10 mg/kg of apMNKQ2 produced strong antitumor effects in PDX models. Increasing the dose 20-fold enhanced tumor uptake but not efficacy, suggesting a therapeutic plateau at 10 mg/kg. MNK1 plays a central role in PDAC progression and CSC maintenance. apMNKQ2 is a potent anti-MNK1 DNA aptamer with robust preclinical activity, including CSC-targeting and anti-invasive effects. Its low toxicity, systemic bioavailability, and efficacy at low doses support further development as a novel therapeutic strategy for PDAC.

This commentary discusses the phase I study of fecal microbiota transplantation plus anti-programmed cell death protein 1 therapy in refractory microsatellite-stable gastric cancer. We suggest that this strategy should be treated as a pharmacomicrobiomic intervention rather than only as an immunotherapy combination. Evidence from fecal microbiota transplantation trials and microbiome immunotherapy studies indicates that antibiotics, proton pump inhibitors, corticosteroids and other microbiome-modifying exposures may affect donor strain engraftment, immune activation, response assessment and safety. In China and other Asian settings, where acid suppression, Helicobacter pylori history, perioperative antibiotic use and nutritional interventions are common, standardized concomitant medication reporting would make future studies more interpretable and transferable.

Aurora kinase A (AURKA) is frequently overexpressed in gastrointestinal cancers. Helicobacter pylori (H. pylori) infection is a significant risk factor for gastric carcinogenesis. LGR5, a stem cell marker in the stomach, plays an important role in gastric tumorigenesis. This study investigates the link between AURKA and LGR5 in response to H. pylori infection in gastric cancer. We analyzed publicly available datasets, gastric cancer cell lines, patient-derived organoids and xenografts (PDOs and PDXs), mouse models, and de-identified human tissue samples. We found a strong association between AURKA and LGR5 expression levels in gastric cancer tissues. In vitro and in vivo analyses showed increased AURKA and LGR5 protein levels in response to H. pylori infection. Using cell models and PDOs, we demontrated an AURKA-dependent induction of LGR5, whereas genetic knockdown or pharmacologic inhibition of AURKA abolished the H. pylori-induced increase in LGR5 by enhancing LGR5 ubiquitination. Mechanistically, AURKA interacted with STAMBP, suppressing LGR5 deubiquitination and thereby promoting its stabilization. Using a tamoxifen-induced conditional knockout (CKO) mouse model of Aurka (Krt19CreErt/Aurkaflox/flox), we detected a lower baseline expression of LGR5 in gastric glands, with reduced induction following H. pylori infection, compared to controls. The Aurka CKO mouse model was crossed with the Tff1-/- model of gastric tumorigenesis to create the Krt19CreErt/Aurkaflox/flox /Tff1-/- mouse model. AURKA knockout, after tamoxifen treatment, significantly reduced the LGR5 protein level and led to a marked decrease in antral thickness. In PDX models, the use of a combination treatment of AMG900 (a pan-AURORA inhibitor) with docetaxel was more effective than monotherapy, lowering LGR5 levels and suppressing tumor growth. In summary, there is a functional link between AURKA and LGR5, mediated by STAMBP to promote stem-like properties and enhance cell survival in gastric tumorigenesis. Targeting the AURKA-LGR5 axis is a potential therapeutic strategy.

Metastasis in gastric cancer requires metabolic reprogramming, but its key drivers remain unclear. Using a CRISPR-Cas9 metabolic knockout screen integrated with patient transcriptomes, we identified the mitochondrial enzyme ALDH6A1 as an anti-invasive factor. ALDH6A1 down-regulation blocked the terminal step of valine catabolism and caused intracellular accumulation of methylmalonic acid (MMA). MMA competitively occupied the α-ketoglutarate (α-KG) cofactor pocket of the histone demethylase KDM5C, suppressing its activity and increasing H3K4 dimethylation (H3K4me2) at promoters of invasion-related genes, including ANGPT2 (angiopoietin-2) and MMP7 (matrix metalloproteinase 7). This epigenetic reprogramming promoted gastric cancer liver metastasis in mice. Pharmacologic ALDH6A1 activation with Alda-1 or systemic MMA clearance with l-carnitine lowered H3K4me2, dampened the invasive program, and reduced metastatic burden. These findings identify the ALDH6A1-MMA axis as a targetable metabolic-epigenetic pathway in gastric cancer metastasis.

The stomach regenerates its glands after environmental insults by reconstructing diverse cell types. In this issue of Developmental Cell, Loe et al.1 show that ARID1A safeguards regeneration and recovery by maintaining cell lineage identity, whereas compromised lineage integrity following Arid1a loss, together with Trp53, can promote aggressive gastric cancer.

Peritoneal dissemination is a major cause of mortality in gastric cancer (GC), yet its molecular underpinnings remain incompletely defined. By integrating single-cell transcriptomic profiling of primary tumors and peritoneal lesions with functional and mechanistic studies, we identified a hypoxia-sensitive GC cell population that emerges during peritoneal metastasis and exhibits heightened autophagic activity and metastatic potential. Hypoxic stress robustly induced the deubiquitinase USP33 in GC cells, and high USP33 expression correlated with adverse clinical outcome. Gain- and loss-of-function assays demonstrated that USP33 enhances autophagy and promotes proliferation, invasion, and survival of GC cells. Proteomic and biochemical analyses revealed that USP33 directly interacts with the autophagy regulator ATG101 and stabilizes it by removing K48-linked polyubiquitin chains at lysine residues 106 and 191, thereby sustaining autophagic flux and facilitating peritoneal colonization. Upstream, hypoxia activated EPAS1/HIF2A, which bound to and transcriptionally upregulated USP33, establishing a hypoxia-responsive EPAS1-USP33-ATG101 axis. Disruption of this axis, either by USP33 silencing or pharmacological inhibition of EPAS1, suppressed autophagy-dependent peritoneal metastasis and prolonged survival in vivo. Collectively, our findings define a mechanistic link between hypoxia, oncogenic autophagy, and peritoneal dissemination in GC, and highlight the EPAS1-USP33-ATG101 axis as a promising therapeutic target in advanced disease.

Exposed constantly to environmental challenges, the stomach undergoes highly reversible cycles of regeneration and recovery. Although abnormal activation of regeneration is known to be associated with cancer, the mechanisms underlying tissue restoration remain unclear. Our single-cell gene expression and chromatin analysis defined cell state dynamics during regeneration and recovery, identifying the Brahma-related gene 1(BRG1)/BRM-associated factor (BAF) chromatin remodeling complex during recovery. Strikingly, deletion of AT-rich interaction domain 1A (Arid1a), a subunit of the BAF complex and the second most frequently mutated gene in gastric cancer, impaired recovery across multiple murine injury models, resulting in a persistent regenerative state. Integrative analyses combining single-cell multiome and chromatin immunoprecipitation sequencing (ChIP-seq) demonstrated that the BAF complex recruits lineage-specific transcription factors such as MIST1 and estrogen related receptor gamma (ERRγ) to regulate enhancers of recovery genes. Notably, deletion of Trp53 in the unresolved regenerative state caused by Arid1a loss is sufficient to drive cancer development and invasion, revealing the epigenetic mechanisms bridging gastric regeneration, recovery, and cancer.

CDH1 is commonly mutated in sporadic diffuse gastric cancer (DGC) and germline CDH1 mutations underlie most cases of the cancer syndrome hereditary DGC. We aimed to develop mouse models of sporadic and hereditary DGC by inactivation of Cdh1 in the mouse stomach. We generated tamoxifen-inducible Cre/loxP mouse models of DGC driven by the Cd44 promoter with a tdTomato reporter. Two models were developed, one with Cdh1-knockout alone (Cd44-Cre/tdTomloxP/loxP/Cdh1loxP/loxP (Cdh1-KO)) and a second more aggressive model with combined Cdh1 and Trp53 knockout (Cd44-Cre/tdTomloxP/loxP/Cdh1loxP/loxP/Trp53loxP/loxP (Cdh1-KO/Trp53-KO)). Cdh1 inactivation alone led to multiple foci of in situ (pTis) signet ring cells (SRCs) within 1 week of induction and intramucosal DGC (stage pT1a) within 2 months. By 9 months, 50% of mice had developed advanced (pT3) DGC. The morphology of most gastric carcinomas was comparable to human DGC, exhibiting poorly cohesive SRC and poorly differentiated cells. Additional Trp53 knockout accelerated cancer development, resulting in pT3 DGC within 3 months. From this point, Cdh1-KO/Trp53-KO mice frequently developed thymic lymphomas and soft tissue sarcomas. DNA sequencing did not find evidence of additional genetic events necessary for cancer progression in either model. Organoids derived from Cdh1-KO and Cdh1-KO/Trp53-KO mice showed a disrupted morphology with SRCs displaced out of the epithelial plane. Transcriptional changes associated with processes including cell-to-cell adhesion, interaction with the actin cytoskeleton and NF-κB signalling were observed. Inactivation of Cdh1 alone in Cd44-expressing cells is sufficient to induce DGC in mice. Tumour growth is significantly accelerated by concurrent Trp53 inactivation.

Melanoma outcomes vary by subgroups. Identify institutional/systemic factors contributing to outcome differences. Retrospective cohort study of melanoma patients seen 1/1987-5/2023 at a VA center and nearby tertiary care center sharing providers. Outcomes include melanoma-specific survival (MSS) and mortality (MSM). Compared to the tertiary center (n=9,682, 5,564 males), VA patients (n=638, 612 males) were older and had a greater proportion of melanoma in situ (p<0.0001). When comparing VA males to males treated at the tertiary center, MSS was increased for localized melanoma (p=0.003), but decreased for regional (p=0.0002) and distant disease (p=0.02). When comparing VA males to VA-insured males at the tertiary center, MSS remained increased for localized disease (p<0.0001), but differences for regional or distant disease were no longer significant. Within the tertiary center, publicly insured patients had increased MSM compared to privately insured (Medicare, hazard ratio [95%CI] 1.46 [1.22,1.75]; Medicaid, 1.67 [1.28,2.18]; Military/VA 1.92 [1.15,3.20]). Smaller number of VA-insured patients. Differences in preventative screening and selective referrals may contribute to increased survival at the VA for early-stage melanoma. Insurance type within and between institutions is associated with differential mortality outcomes for advanced melanoma stages, indicating a need for additional care standardization.

Over the past decade, the development and implementation of novel circulating tumor "liquid biopsies" in clinical practice has revolutionized cancer care. These tools provide sensitive and specific analysis of tumor status which enable non-invasive alternatives to tissue biopsy for real-time tumor monitoring, guided targeted therapy, predicted treatment response, and assessing therapeutic resistance and residual disease (MRD). The FDA has already approved several assays to detect genetic alterations in plasma cell-free DNA (cfDNA) for use as companion diagnostics matched to specific molecularly targeted therapies for cancer, setting the stage for future use in early relapse detection and additional applications which are expected to be incorporated into standard-of-care practices for aggressive cutaneous malignancies. It will therefore be important for the dermatology community to have a working knowledge of liquid biopsy techniques and their relevance to the management of cutaneous malignancies. This review highlights state-of-the-art liquid biopsy tools currently in practice with an emphasis on the development and implementation of circulating biomarkers in skin cancers. It is anticipated that liquid biopsy tools will soon become integrated into evolving standards-of-care for improved management of patients with aggressive cutaneous malignancies.

Genomic studies revealed elevated DNA damage and mutation rates at transcription factor (TF) binding sites in UV-linked cancers. While TFs can promote UV-induced mutagenesis by altering both damage formation and repair, these mechanisms have not been systematically characterized across TFs at high resolution. Using genome-wide UV damage maps from skin fibroblasts, we develop a scalable statistical framework to analyze TF-mediated mutagenic mechanisms across hundreds of TFs. We identify numerous previously unreported TFs that significantly enhance or suppress UV damage formation within their binding sites. A systematic survey of TF-DNA complexes reveals that damage modulation often coincides with TF-induced DNA distortions that either protect against or promote photodimer formation. Additionally, we analyze repair efficiency in TF binding sites at high resolution, identifying TFs likely to compete with repair. Comparisons with skin cancer mutations distinguish mutation enrichment driven by increased damageability versus attenuated repair, revealing the highly contextual nature of TF-mediated mutagenesis.

Trametinib (Trm) is a highly selective mitogen-activated protein kinase kinase (MEK) inhibitor that potently and persistently abrogates extracellular signal-regulated kinase 1/2 activation. Trm initially was used to treat BRAF Val600→Glu (V600E)-mutated melanoma, but its Food and Drug Administration-approved indications are expanding rapidly. Trm generally is well tolerated, but it can cause dose-limiting cardiomyopathy and heart failure. Here, we characterize a mouse model of Trm cardiotoxicity using complementary in vitro approaches to show that Trm induces mitochondrial dysfunction in cardiomyocytes and some cancer cell types. In vivo, Trm caused contractile dysfunction within 3 days and heart failure within 2 weeks. High-resolution respirometry using isolated cardiac mitochondria revealed that Trm compromises oxidative metabolism, in part, through blunted activity of electron transport system complexes. Trm-mediated mitochondrial injury led to the release of mitochondrial damage-associated molecular patterns including mitochondrial DNA in both mice and humans, triggering activation of canonical innate immune pathways including cGAS-STING. In multiple rodent and human cardiomyocyte platforms, Trm diminished mitochondrial respiratory capacity at nanomolar concentrations, but this lesion was reversed by expression of a phosphomimetic signal transducer and activator of transcription 3-S727 construct. We also found that Trm induced mitochondrial dysfunction in some but not all cancer cell lines, identifying a previously unrecognized effect that could contribute to Trm's anticancer efficacy.

Surgical resection remains the primary mode of treatment for melanoma. However, postoperative tumor recurrence and impaired wound healing significantly compromise the patient prognosis. In this study, we developed a microneedle (MN) patch coloaded with HMnO2 and hydroxydaunorubicin (DOX) for the simultaneous management of residual disease and postsurgical wound repair. The MN patch enables efficient transdermal delivery of both agents by penetrating the stratum corneum and targeting the surgical site. Upon near-infrared (NIR) irradiation, HMnO2 mediates photothermal ablation (PTT), effectively ablating residual melanoma cells while DOX suppresses the proliferation of potential micrometastases. Furthermore, the HMnO2/DOX@MN system modulates the local inflammatory microenvironment, promotes angiogenesis, and enhances collagen deposition, thereby accelerating the wound healing. In vivo studies using a murine melanoma model demonstrated that the MN patch significantly reduced postoperative tumor recurrence and improved wound closure rates. This multifunctional microneedle platform presents a promising strategy for integrated postoperative management, following melanoma resection.

Local administration of drug-encapsulated gels has shown promise for cancer treatment. However, intratumoral drug diffusion is hindered by the dense extracellular matrix. Herein, a versatile skin- and tumor-dual-permeable manganese-dimethyl sulfoxide (Mn-DMSO) gel platform, with a nanofiber structure (MnSO4·2H2O·C2H6SO as suggested by single-crystal structure), for cancer chemoimmunotherapy is introduced, which can be locally applied either through peritumoral injection for multiple tumor types or transdermal delivery for superficial tumors. This gel has a thermoresponsive sol-gel phase transition at 37 °C and intrinsic tumoricidal and cGAS-STING activation capabilities. Importantly, in addition to being a drug reservoir and taking advantage of the tissue penetration ability of DMSO, the gel is tumor-permeable and achieves a 2.5-fold enhancement in tumor penetration of drugs compared with free ones. In multiple tumor models, a peritumorally injected Mn-DMSO gel coencapsulated with a chemodrug (gemcitabine) and an immune agonist (R848) effectively triggers cGAS-STING-mediated local and systemic antitumor responses. Furthermore, we demonstrate that topical application of the R848- and gemcitabine-coloaded Mn-DMSO gel on the skin surface enables noninvasive transdermal delivery of chemo-/immunodrugs into tumors, thereby inhibiting tumor growth and metastasis in a melanoma model. Our findings highlight a gel platform that supports both injectable and topical administration for enhanced cancer therapy.

Long-term outcome data for focal therapy using high-intensity focused ultrasound (HIFU) or cryotherapy for nonmetastatic prostate cancer are needed. We report 10-yr cancer control outcomes. Patients with nonmetastatic prostate cancer who underwent primary focal HIFU or cryotherapy and had at least 6 mo follow-up were identified from the UK HIFU Evaluation and Assessment of Treatment (HEAT) and International Cryotherapy Evaluation (ICE) prospectively maintained registries. The intervention included up to two focal ablative sessions. The primary outcome was cancer-specific mortality. Secondary outcomes were all-cause mortality, metastasis, local retreatment, radical treatment, and androgen-deprivation therapy (ADT) use. A total of 3477 patients (HIFU: n = 2897; cryotherapy: n = 580) were included from 14 UK centres (2004-2024). A total of 48%, 23%, and 25% had European Association of Urology (EAU) 2025-version favourable intermediate-, unfavourable-intermediate-, and high-risk disease, respectively. Ten-yr cancer-specific mortality was 0.13% (95% confidence interval [CI] = 0.027-0.45%). Ten-yr all-cause mortality and metastases were 12% (95% CI = 8.8-15%) and 3.3% (95% CI = 2.1-4.9%), respectively. Ten-yr ADT use was 14% (95% CI = 11-17%). Ten-yr local retreatment and radical treatment were 33% (95% CI = 30-37%) and 30% (95% CI = 27-34%), respectively, on an intention-to-treat basis. In a post hoc per-protocol analysis, undertaken to estimate outcomes under stricter adherence to the two focal ablative session protocol, patients who underwent radical treatment despite being potentially eligible for a further focal ablative session were censored at the time of radical treatment; 10-yr local retreatment and radical treatment were 13% (95% CI = 11-16%) and 8.9% (95% CI = 6.9-11%), respectively. The observational study design is the main limitation. Focal therapy using up to two sessions of focal HIFU or cryotherapy could be considered as a first-line treatment for well-selected patients of nonmetastatic prostate cancer alongside radical treatment. Future research priorities should include the development of a dedicated prognostic risk calculator, further prospective assessment of focal therapy for high-risk disease, and improvements in the detection and management of locally recurrent disease.

Magnetic resonance imaging-targeted prostate biopsy relies heavily on costly software-fusion platforms, limiting global accessibility. We aimed to determine whether cognitive fusion (mental registration) is noninferior to software-based fusion targeted biopsy for detecting clinically significant prostate cancer (csPCa). This multicenter, randomized, blinded, noninferiority trial enrolled 648 predominantly biopsy-naive men (prostate-specific antigen 4-20 ng/ml; Prostate Imaging Reporting and Data System ≥3) at nine centers in China. Participants were randomly assigned 1:1 to cognitive fusion or software-based fusion targeted biopsy, followed by systematic biopsy. The primary outcome was csPCa (ISUP grade group ≥2) detection on targeted biopsy, evaluated using generalized estimating equations with a prespecified -10 percentage point noninferiority margin. In the intention-to-treat population (327 cognitive and 321 software), targeted biopsy detected csPCa in 32% and 34% of the patients, respectively. The adjusted risk difference was -1.02 percentage points (90% confidence interval [CI], -5.6 to 3.6), establishing noninferiority. Combined targeted and systematic biopsy detected csPCa in 35-39% of the patients, respectively (difference, -1.50% [90% CI, -6.4 to 3.4]). A significant body mass index (BMI) interaction was observed (p < 0.001): cognitive fusion performed best in men with BMI <24 kg/m2, whereas software-based fusion improved detection in men with BMI ≥24 kg/m2. Adverse event rates were comparable (15% vs 18%). Limitations include conducting the trial predominantly in Asian populations, necessitating cautious geographic extrapolation of specific BMI cutoffs. Cognitive fusion is diagnostically noninferior to software-based fusion within a prespecified -10% margin overall. However, a significant BMI interaction raises serious concerns regarding the diagnostic feasibility of cognitive fusion in obese populations, where software-based fusion is strictly superior. Future prospective trials in both Asian and Western populations must evaluate the BMI effect as a primary end point. ClinicalTrials.gov NCT04271527.

Prostate biopsy is a complex, multi-step procedure lacking standardisation across patient selection, tools, technique, and biopsy strategy, potentially influencing local treatment planning. An international consensus project was initiated to harmonise these aspects. A systematic review informed statement development. A modified Delphi process involving 34 international experts was conducted over three rounds. Panellists evaluated 96, 99, and 112 statements in Rounds 1-3, respectively, grouped into 36 stems. Statements were iteratively revised based on feedback and discussion. Consensus was assessed using a modified RAND appropriateness method. All experts completed the three rounds. Consensus was achieved for 29 of 36 stems (81%). Both biparametric and multiparametric magnetic resonance imaging were endorsed within the diagnostic pathway, provided imaging quality is adequate. Key elements of a targeted plus perilesional biopsy scheme were harmonised. For treatment planning, a targeted ± perilesional biopsy scheme was considered sufficient for most aspects of local treatment planning. However, additional contralateral sampling still seems necessary for specific indications, notably focal therapy candidate selection. Limitations include imbalances in specialty representation and heterogeneity in expertise across topics. The ProBIOPSY consensus defines a contemporary, standardised framework for prostate biopsy. While targeted-based strategies are sufficient for most clinical scenarios, no single biopsy approach provides complete information for all treatment decisions, underscoring the need for indication-specific biopsy tailoring.

The TMPRSS2:ERG gene fusion, present in approximately 50% of prostate cancers in patients of European ancestry, drives oncogenesis through aberrant overexpression of the ERG transcription factor. Despite its role as a truncal oncogenic driver, ERG has been considered undruggable due to the absence of enzymatic activity and apparent lack of ligandable pockets. Here, we demonstrate continued dependency on ERG in metastatic prostate cancer and identify a druggable pocket within its N-terminal Pointed (PNT) domain. Using an inducible shRNA system in TMPRSS2:ERG-positive VCaP cells, we show that ERG depletion causes profound growth inhibition. To therapeutically exploit this vulnerability, we conducted a domain-focused differential scanning fluorimetry screen targeting the ERG PNT domain, followed by structure-activity relationship optimization. This approach yielded PBITE-1 (PNT-Binding Inhibitor of the Transcription factor ERG), a small molecule that selectively binds the ERG PNT domain. NMR chemical-shift perturbation mapping and molecular docking revealed that PBITE-1 engages a discrete, solvent-exposed surface comprising two α-helices and an adjacent flexible loop, defining a ligand-binding pocket within the PNT domain. In cellular models, PBITE-1 directly engaged ERG, selectively inhibited proliferation and invasion, and induced apoptosis in ERG-driven prostate and hematologic malignancies. PBITE-1 potently suppressed growth of ERG-positive mouse and human-derived prostate cancer organoids. Furthermore, PBITE-1 treatment significantly induced tumor cell apoptosis in VCaP xenograft models. These findings establish the ERG PNT domain as ligandable and provide preclinical evidence that ERG is directly targetable by small molecules, enabling future development of ERG-directed inhibitors and targeted protein degraders.

ConspectusProstate cancer (PCa) is the most prevalent malignancy among men worldwide, with its pathogenesis and progression heavily reliant on the sustained activation of the androgen receptor (AR) signaling pathway. The AR, a transcription factor of nuclear receptor superfamily, serves as the most privileged therapeutic target in PCa, as evidenced by the clinical efficacy of first- and second-generation AR antagonists. Current clinically available AR antagonists exclusively target the ligand binding pocket (LBP), suppressing tumor proliferation through competitive inhibition of androgen binding and subsequent blockade of AR signaling transduction. However, their therapeutic utility is invariably limited by acquired resistance mechanisms, including point mutations that alter LBP specificity, AR gene amplification leading to receptor overexpression, and the emergence of constitutively active splice variants that bypass ligand-dependent activation. Thus, the development of novel AR antagonists featuring innovative mechanisms and structural scaffolds is imperative to overcome resistance to antiandrogen therapy. However, the AR exhibits significant structural flexibility, and the lack of antagonist-bound crystal structures has hindered structure-based rational drug design. In this Article, we summarize our advances in elucidating the molecular mechanisms underlying AR conformational regulation and highlight our progress in the structure-based design and development of novel AR antagonists. First, our molecular dynamic (MD) studies collectively elucidate the molecular mechanisms by which the AR ligand binding domain (LBD) regulates its functional states through dynamic conformational changes mediated by distinct allosteric pathways when bound to agonists or antagonists, providing atomic-level insights and structural basis for drug development. Then, we successfully identified structurally diverse lead compounds targeting the LBP through various integrated approaches combining MD simulations, structure-based virtual screening (SBVS), and systematic biological evaluation. These compounds exhibited potent activity against clinically relevant AR mutations F877L, W742C, T878A, and H875Y, demonstrating their potential to overcome mutations-driven resistance. Further, we explored non-LBP mediated strategies for AR antagonism, including: (1) targeting the allosteric binding sites on LBD; (2) identification of novel druggable binding sites; and (3) targeting alternative domains beyond the LBD. As a paradigm-shifting example, we proposed inhibition of AR LBD dimerization as a novel mechanism of action for LBP-targeting AR antagonists. Building upon this insight, we characterized a promising pocket at the dimer interface, designated the Dimerization Interface Pocket (DIP), and developed first-in-class antagonists specifically targeting this site, which exhibit exceptional therapeutic potential. Collectively, these multipronged strategies not only highlight the power of computation-driven approaches in drug discovery but also yield a diverse pipeline of resistance-targeting candidates, directly addressing the unmet clinical need in advanced PCa.

Patients with very-high risk clear cell renal cell carcinoma (ccRCC) remain at substantial risk of recurrence after nephrectomy. However, heterogeneity in benefit from adjuvant immunotherapy observed in the KEYNOTE-564 trial, together with limited prospective data in this population, warrants further investigation. This multicenter, prospective, non-randomized, controlled phase II trial enrolled patients with very-high risk ccRCC following nephrectomy. Patients received either adjuvant penpulimab or routine surveillance, with 1:1 propensity score matching (PSM) applied to balance baseline characteristics. The primary endpoint was disease-free survival (DFS), with secondary endpoints including overall survival (OS) and safety. Plasma proteomic and metabolomic profiling was conducted for exploratory analyses. Given the non-randomized design, baseline imbalances were addressed by PSM, yielding well-balanced cohorts of 87 patients per group. Adjuvant penpulimab was associated with improved DFS compared with surveillance (HR, 0.37; 95% CI 0.16 to 0.89; p=0.026), whereas OS data remained immature. Consistent with Kaplan-Meier estimates, 1-year (94.25% vs 80.46%) and 2-year (88.68% vs 75.38%) DFS rates favored the penpulimab group (log-rank p=0.026). Penpulimab was generally well tolerated, with most treatment-related adverse events being grade 1-2. Plasma proteomic and metabolomic analyses revealed enrichment of immune response-related pathways in patients without disease progression, with circulating biomarkers associated with DFS. Adjuvant penpulimab was associated with improved DFS and a manageable safety profile in patients with very-high risk ccRCC. Plasma-based biomarkers associated with disease progression may inform postoperative risk stratification and warrant further validation. ChiCTR2200062189.

Biomarkers that predict response to combined radiation and anti-PD-1 therapy in clear cell renal cell carcinoma (ccRCC) are poorly defined. RAPPORT trial (NCT02855203) is a prospective phase I/II study of stereotactic ablative body radiotherapy (SABR) and pembrolizumab in 30 patients with oligometastatic ccRCC. The primary endpoint was safety; secondary endpoints included overall survival, time to local progression, distant progression-free survival, objective and disease control rates, duration of response and patient-reported pain. Here, we report the pre-specified exploratory translational analyses. In pre-treatment tumours, responders had greater intra-tumoural cytotoxic T cell infiltration in close proximity to tumour cells, whereas non-responders showed enrichment of immunosuppressive, TGF-β and angiogenic signatures. In peripheral blood post-treatment, responders developed a proliferative burst of activated CD8+ T cells and sustained sharing of pre-existing tumour-enriched TCR clones. These findings identify trafficking and maintenance of pre-existing tumour-enriched T cell clones as determinants of response to SABR plus anti-PD-1 in ccRCC.

Tertiary lymphoid structures (TLSs) correlate with favorable responses to immune checkpoint inhibitors (ICIs) in various cancers, yet many patients with TLS-positive tumors are resistant to treatment. Multi-omic profiling of clear cell renal cell carcinoma (ccRCC) and soft tissue sarcoma tumors (STSs) reveals an upregulation of gamma-aminobutyric acid (GABA)-related signatures in non-responders to ICIs. In ccRCC, TLSs from non-responders located near GABA-producing tumor cells exhibit impaired B cell maturation, reduced IgG production, higher GABA receptor expression, and tricarboxylic acid cycle activation. In vitro, exposure of human B cells to GABA reduces HLA-DR expression, proliferation, and immunoglobulin secretion by receptor-independent and dependent mechanisms. Pharmacological inhibition of GABA-synthesis increases ICI response and immune infiltration, particularly by B cells, in a TLS-positive STS mouse model. Our findings unravel GABA as an immunoregulatory metabolite and provide a rationale for its therapeutic targeting to overcome ICI resistance in patients with TLS-positive tumors.

Macroautophagy/autophagy is an evolutionarily conserved degradation pathway wherein cytoplasmic components are sequestered within double-membrane autophagosomes for lysosomal delivery. The initiation of autophagy is governed by autophagy-related (ATG) proteins, with the ULK1 kinase complex serving as the most upstream regulator. However, how ULK1 senses and integrates metabolic signals via post-translational modifications remains poorly understood. Here, we discover that ULK1 undergoes lactylation at lysine 46, catalyzed by the mitochondrial aminoacyl-tRNA synthetase AARS2, in response to autophagic stimuli. This modification promotes ULK1 kinase activity, leading to enhanced and selective phosphorylation of its downstream substrate ATG14 at Ser29, thereby activating the class III PtdIns3K complex and facilitating autophagosome biogenesis. Furthermore, we demonstrate that AARS2-mediated ULK1 lactylation drives autophagic flux and promotes tumor metastasis in clear cell renal cell carcinoma (ccRCC), and that a cell-penetrating peptide targeting K46 lactylation suppresses ccRCC progression in vitro and in vivo. Our study identifies lactylation as a novel regulatory mechanism controlling autophagy initiation and suggests that targeting AARS2-mediated ULK1 lactylation could be a potential strategy for treating ccRCC.Abbreviations: AARS2: alanyl-tRNA synthetase 2, mitochondrial; ATG14: autophagy related 14; Baf.A1: bafilomycin A1; ccRCC: clear cell renal cell carcinoma; co-IP: co-immunoprecipitation; CPP: cell-penetrating peptide; EBSS: Earle's balanced salt solution; HIF: hypoxia-inducible factor; K46la: lactylation at lysine 46; Kla: lysine lactylation; KO: knockout; LDHA: lactate dehydrogenase A; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; PtdIns3K: class III phosphatidylinositol 3-kinase; PTM: post-translational modification; SQSTM1/p62: sequestosome 1; TCGA-KIRC: The Cancer Genome Atlas-Kidney Renal Clear Cell Carcinoma; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; VHL: von Hippel-Lindau tumor suppressor; WT: wild-type.

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer. Loss of von Hippel-Lindau (VHL) and the consequent activation of hypoxia-inducible factor-α (HIFα, especially HIF2α) plays an essential role in ccRCC initiation and progression. The VHL-HIF2α axis as the main driver for ccRCC may present specific opportunities to control the disease by cotargeting HIF2α with belzutifan and another vulnerability. This study elucidates the synthetic essentiality of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) in VHL-deficient ccRCC. Upregulated in ccRCC in a VHL-HIF2α-dependent manner, TRAIL is selectively essential in ccRCC cells, promoting cell proliferation by activating the p38 MAPK pathway and facilitating G1/S phase transition. Depletion of endogenous TRAIL or inhibition of HIF2α with belzutifan sensitizes ccRCC cell and tumor models to recombinant TRAIL, presenting a promising avenue for combination therapy in ccRCC.

Complete cytoreductive surgery remains one of the strongest determinants of outcome in ovarian cancer, yet surgeons still lack rapid tissue-assessment tools that can be repeated throughout an operation. Here we evaluated whether SpiderMass ambient mass spectrometry, combined with machine-learning models, could support ex vivo ovarian tissue typing and exploratory immune microenvironment mapping. A total of 128 ovarian specimens, from 119 patients, were analyzed to train subtype-classification models from fresh-frozen and formalin-fixed paraffin-embedded (FFPE) material, and 24 independent tissues (from 16 patients) were reserved for blinded region-level testing. Initial PCA-LDA models were improved by screening 24 classifiers; Ridge models reached up to 97% 5-fold cross-validation accuracy on the combined cohort. In blinded analyses, the mixed Ridge model produced the fewest errors, although misclassification remained concentrated in underrepresented endometrioid regions. A dual-input network combining SpiderMass spectra with digitized histology improved internal performance to 99% in 5-fold cross-validation and 100% on a small, blinded image-spectrum set, outperforming the molecular-only branch. Model explanation followed by MALDI-MSI cross-checking identified 26 subtype-associated lipids. We then trained a LightGBM cell-state model from immune and epithelial cell spectra and applied it to SpiderMass imaging data. Spatial predictions were broadly concordant with multiplex MALDI-IHC and highlighted subtype-specific differences in immune-cell distribution. In an exploratory analysis of eight high-grade serous carcinoma samples obtained before chemotherapy, longer survivalappeared to be associated with higher lymphocyte scores, higher M1-like macrophage scores and a higher M1/M2 ratio, whereas shorter survival appeared to be associated with higher cancer-cell scores. These data support SpiderMass as a promising ex vivo platform for ovarian cancer typing and hypothesis-generating immunoscoring, while underscoring the need for prospective intraoperative studies, orthogonal biomarker validation, validation in larger cohorts for exploratory immunoscoring and multicenter patient-level external validation before clinical implementation.

High-grade serous carcinoma tumours present poor survival rates, often associated with immunologically excluded environments driven by hypoxia and extensive extracellular matrix remodelling that disrupt tumour-stromal-immune interactions. Current experimental models fail to fully capture these microenvironmental features, limiting understanding of tumour-immune dynamics and drug development. Here, we present bioengineered patient-derived tumour-immune models to mimic physiologically relevant oxygen levels and extracellular matrix remodelling. Cancer cells are co-cultured with cancer-associated fibroblasts within human plasma-3D matrices or grown on decellularized human ovaries. Immune cells are either included within the 3D constructs to study multi-cellular interactions or challenged to infiltrate the matrices. We demonstrate that intratumoural hypoxia acts as a friend and a foe enhancing the activation and cytotoxicity of CD8 + T cells while inducing stromal/matrix dysregulation associated with impaired immune infiltration. Targeting TGF-β signalling attenuates the hypoxia-driven stromal-mediated immune exclusion. These relevant models may aid the development of targeted therapies to transform immunologically cold tumours into immunogenic to benefit female patients.

Anoikis resistance is crucial for ovarian cancer (OvCa) transcoelomic metastasis, during which exfoliated OvCa cells survive as spheroids before invading the omentum. Here, we demonstrate that cystathionine β-synthase (CBS), an H2S-producing transsulfuration pathway enzyme, is a key determinant of OvCa spheroidal viability and metastatic potential. Analysis of publicly available patient datasets, as well as an in-house tissue microarray, revealed that high CBS expression positively correlates with both poor progression-free survival and clinically observed peritoneal/omental metastasis. Integrated functional and proteomic analyses indicated that CBS silencing induces apoptosis in 2D monolayers. Consistent with this, CBS silencing in spheroids caused apoptosis and disrupted spheroid architecture. Mechanistically, this phenotype was associated with downregulation of oncogenic stemness and epithelial-mesenchymal transition. Further, through proteomic and bioinformatic analyses, we identified ITGB1 to be the hub protein in OvCa spheroidogenesis. Interestingly, knockdown of CBS led to abrogation of the ITGB1-mediated downstream pathway. Moreover, by proteomic and network analyses, we identified SP1 as a key transcriptional regulator of CBS-induced pro-spheroidal transcriptional programs of stemness and invasiveness. Stabilization of SP1 through persulfidation by H2S supplementation restored spheroidal viability and underlying protein signaling. Further, our results reveal that loss of CBS, through ITGB1 repression, disrupts spheroid architecture, leading to reduced metastatic docking on the murine omental surface in vivo. Collectively, these findings identify CBS as a central regulator of anoikis resistance and OvCa transcoelomic metastasis, highlighting the therapeutic potential of targeting CBS-dependent SP1-ITGB1 regulation to control metastatic spread.

Intratumoral heterogeneity is intrinsically comprised of molecular alterations of tumor cells and extrinsically from interconnections with microenvironments. This study explores the spatial heterogeneity of ovarian clear cell carcinoma (OCCC), a rare cancer with significance to East Asian women. We profile 21 primary-metastatic tumor pairs in a discovery cohort and 16 tumors in two validation cohorts using spatial transcriptomic (ST) platforms. Our integrative analysis revealed an inverse relationship between OXPHOS and inflammation along the EMT gradient. OCCC cells undergoing partial EMT have metabolic shifts and lose LCN2 expression, possibly via concomitant down-regulation of SOX9. Conversely, LCN2 expression correlated with OXPHOS-enriched tumor signature, low EMT, and better outcomes in OCCC. Single-cell ST profiling using CosMx further identifies nine spatially distinct cancer cell populations including the LCN2-high cancer subclone with a high epithelial score. SOX9 induction could partially restore epithelial-ness in LCN2-low cells suggesting that plasticity in OCCC is achieved via transcriptional reprogramming. Our findings provide further insights into epithelial-mesenchymal plasticity and the adaptive interactions between cancer cells and their microenvironments in OCCC.

Oral potentially malignant disorders (OPMDs), particularly leukoplakia, represent critical precursors to oral cancer requiring systematic monitoring. However, current diagnostic approaches rely on invasive biopsies and subjective clinical assessment, which limit screening accessibility. Here, we report a deep learning system that provides both highly reliable diagnostic classification and visual explainability for OPMD assessment. Trained on 778 expertly annotated clinical images, our framework first utilizes a diagnostic classifier to distinguish normal mucosa, leukoplakia, and malignant transformation (accuracy: 91.1%, 95% CI: 86.7-94.2%). For cases flagged as abnormal, it conditionally triggers an interpretable segmentation module providing pixel-level lesion delineation (mean average precision: 72.2%). External validation on 193 independent clinical cases demonstrated maintained diagnostic accuracy (90.5%, 95% CI: 85.7-94.0%), with the strict lower confidence bounds significantly outperforming state-of-the-art general vision baselines. The system processes images in 3-5 milliseconds on standard GPUs (and approximately 1 second on conventional CPUs), enabling highly flexible point-of-care deployment. By combining diagnostic accuracy with dual-dimensional (spatial and mechanistic) explainability, this framework addresses key barriers to clinical AI adoption, offering a scalable and practical tool for OPMD screening and longitudinal monitoring in diverse healthcare settings.

The microbes within the tumor microenvironment are one of the important risk factors for tumor development, but the actions of antibiotic-resistant bacteria on the tumor development remain unclear. Methicillin-resistant Staphylococcus aureus (MRSA) is frequently detected in oral cavities of oral squamous cell carcinoma (OSCC) patients, especially after radiotherapy and/or chemotherapy, and is associated with adverse prognosis. However, the effects of MRSA on OSCC development are not clear. This study aims to determine whether and how MRSA‑derived extracellular vesicles (EVs) affect OSCC proliferation and tumor development. EVs were isolated from MRSA and methicillin‑susceptible S. aureus (MSSA). Their effects on OSCC cells were evaluated by proliferation assays, EV internalization, protein cargo analysis, and signaling pathway inhibition. An ectopic tumor mouse model with IL‑8 knockout or CXCR1 blockade was used for in vivo validation. Nano-size EVs derived from MRSA, compared with those from MSSA, could enter OSCC cells and promote cell proliferation and tumor development, while the proteins were the most important cargos. Mechanistically, MRSA EVs activated ERK/c‑Jun signaling pathway to produce the chemokine IL‑8 (CXCL8) in OSCC cells; then the elevated IL‑8 bound to CXCR1 to enhance IL‑2 production and activate JAK/STAT5A pathway. These actions finally promoted OSCC development in an ectopic tumor mouse model, while knockout of IL‑8 or blocking its binding to CXCR1 compromised the impacts of MRSA EVs. Our results highlight the crucial role of antibiotic-resistant bacteria and their extracellular vesicles in tumor development and provide new insights into the relationships between microbes and tumors.

Human papillomavirus (HPV) E7 oncoprotein drives cervical cancer progression through sustained overexpression in HPV16-positive cells, making it an ideal therapeutic target. This study developed a novel affitoxin, ZHPV16E7-GrB, by fusing a cell-penetrating peptide (CPP) to an affibody (ZHPV16E7) specific for HPV16E7 and conjugating it with human granzyme B (GrB), a cytotoxic effector molecule. The fusion ZHPV16E7-GrB was successfully prepared in a prokaryotic system with the specific binding to the HPV16E7 protein and also demonstrated a rapid cellular uptake with a high specificity for HPV16-positive cervical cancer cells. Significantly, ZHPV16E7-GrB induced a obvious pyroptosis characterized by cell membrane bubble-like protrusions, gasdermin E (GSDME) cleavage, release of proinflammatory cytokines (IL-18, IL-1β, HMGB1), and LDH release. Importantly, this pyroptosis occurred independently of caspase-3 activation, as confirmed by caspase-3 knockdown experiments, and was highlighted GSDME as the key executor of pyroptosis. Similarly, experimental study in vivo showed that pyroptosis was likely involved in the significant suppression of tumor growth in HPV16-positive TC-1 xenograft mouse models treated with ZHPV16E7-GrB. Our findings provide mechanistic insights into a previously unrecognized anti-tumor pathway mediated by ZHPV16E7-GrB of GSDME-mediated pyroptosis via a caspase-3-independent pathway, revealing a novel approach for HPV16-specific cancer therapy.

Thyroid cancer is the most common endocrine malignancy, yet its biological underpinnings remain incompletely understood. Here we show that common risk alleles for thyroid cancer point to distinct biological pathways underlying disease susceptibility. We perform a multi-ancestry genome-wide association meta-analysis of thyroid cancer (16,167 cases and 2,430,374 controls), identifying 51 independent loci, including 21 not previously reported. By integrating these loci with genetic associations for 151 thyroid-cancer-related traits, we identify pleiotropic mechanistic clusters linked to thyroid function, oncogenic pathways, and mixed physiological function. Two thyroid-specific clusters, associated with thyroid stimulating hormone or thyroid growth and function, are enriched in thyroid tissues. Oncogenic clusters include DNA repair (ATM, CHEK2, TP53) and telomere maintenance (TERT) genes, implicating shared cancer mechanisms. Cluster-specific polygenic scores are associated with thyroid disease, cancer, and metabolic traits across ancestry groups, suggesting distinct genetic subtypes of thyroid cancer risk and supporting pleiotropy-based approaches to genetic risk stratification.

Immune checkpoint inhibitors (ICIs) have significantly improved overall survival in metastatic urothelial carcinoma (mUC). However, predictive biomarkers for therapeutic response remain insufficiently defined. Although some genomic alterations have been implicated in modulating tumor immunogenicity and ICI sensitivity in other cancers, evidence in mUC remains limited and warrants further investigation. We retrospectively analyzed 67 patients with mUC treated with ICIs and performed targeted next-generation sequencing using a 440-gene cancer panel. Tumor mutational burden (TMB), genomic alterations, and clinical outcomes were evaluated to identify biomarkers associated with ICI response. Functional studies were conducted using urothelial carcinoma cell lines co-cultured with peripheral blood mononuclear cells (PBMCs) with or without anti-programmed death-ligand 1 (PD-L1) treatment to evaluate tumor viability, cytokine responses, and antigen presentation. Gene knockdown and overexpression experiments, quantitative PCR, immunofluorescence, and western blotting were used to assess cytokine production, antigen presentation machinery components, and major histocompatibility complex (MHC) class I expression. In vivo validation was performed using CRISPR/Cas9-mediated knockout syngeneic murine models treated with anti-PD-L1 to assess tumor growth, immune infiltration, and therapeutic response through immunohistochemistry and flow cytometry. Responders to ICI monotherapy exhibited significantly higher TMB and enriched mutations in PIK3CA, ADAMTSL1, NSD1, and PRKDC. Hotspot PIK3CA mutations correlated with elevated TMB across multiple TCGA pan-cancer, breast and colorectal cohorts. Functionally, PIK3CA-mutant tumor cells demonstrated enhanced cytotoxicity when co-cultured with PBMCs and anti-PD-L1, whereas PIK3CA knockdown abolished this effect. Silencing PIK3CA reduced expression of pro-inflammatory cytokines (interferon-γ, tumor necrosis factor-α, interleukin (IL)-2, IL-6) and CD8+ effector molecules (granzyme B, perforin A), while PIK3CA E545K overexpression restored these immune mediators. Mechanistically, PIK3CA mutations enhanced antigen presentation via the IRF1-NLRC5-MHC-I axis, increasing HLA-A and B2M expression. In vivo, PIK3CA-deficient tumors displayed diminished response to anti-PD-L1 therapy, reduced CD8+ T-cell infiltration, and attenuated MHC-I expression. This study is the first to identify PIK3CA mutation as an immune-modulating driver that enhances tumor immunogenicity through activating IRF1-NLRC5-MHC-I pathway in urothelial carcinoma.

CD161+CD8+ T cells, a cytotoxic T cell subset, remain poorly defined in terms of their clinical significance and functional role in esophageal squamous cell carcinoma (ESCC), which has limited the advancement of precision immunotherapy strategies. We integrated a multicenter cohort of ESCC patients who underwent either surgery or neoadjuvant therapy (chemotherapy or chemoimmunotherapy). Multiplex immunofluorescence staining, survival analyses, and assessment of major pathological response (MPR) and pathological complete response (pCR) were performed to elucidate the clinical relevance of CD161+CD8+ T cell infiltration. The functional state and cellular interactions of this subset were characterized using single-cell RNA sequencing. A CT-based radiomics model was also developed for non-invasive prediction. High intratumoral infiltration of CD161+CD8+ T cells was identified as an independent prognostic factor for prolonged overall survival and disease-free survival (both p<0.01) and appeared to correlate with a higher MPR and pCR rate following neoadjuvant therapy. This subset was enriched in treatment responders, exhibited transcriptional features associated with cytotoxicity and activation, and showed gene expression profiles suggestive of potential interactions with tumor-associated macrophages, possibly involving the TNFRSF9/TNFSF9 (4-1BB/4-1BBL) signaling axis. A radiomics model built on the XGBoost algorithm accurately predicted the infiltration level of this subset (area under the curve=0.889), and predicted high infiltration was correlated with favorable pathological response. We identify CD161+CD8+ T cells as a pivotal prognostic and predictive biomarker in ESCC. This subset is associated with a putative 4-1BB/TNFSF9-mediated interaction network involving macrophages, correlating with a coordinated anti-tumor immune microenvironment. The CT-based radiomics model could provide a noninvasive means for assessing immune phenotypes, with potential applicability in patient stratification and treatment selection.

Post-transplant type 1 diabetes (T1D) is typically attributed to transferred donor autoimmunity. We investigated a distinct etiology in a patient who developed diabetes following haploidentical hematopoietic cell transplantation, assessing whether autoimmunity originated from donor or residual host cells. Leveraging HLA disparity between the haploidentical donor and recipient, we used HLA class II tetramers to enumerate islet-specific CD4+ T cells in peripheral blood restricted by shared versus recipient-only HLA alleles. Tetramer analysis revealed an expanded population of islet-specific T cells in the recipient. The donor showed no such expansion. Despite 99% donor T-cell chimerism, >80% of the islet-specific T cells were restricted by recipient-unique HLA alleles, suggesting they originated from the residual host fraction. T1D in this patient was most likely driven by residual recipient-derived T cells. Their survival despite myeloablative conditioning and repeated immunotherapy underscores the remarkable durability of established islet autoimmunity.

Automatic radiology report generation has gained increasing attention for its potential to assist in clinical reporting and reduce the workload of radiologists. Existing 3D radiology report generation methods employ multi-modal foundation model to encode volume-text inputs and produce diagnosis reports, while they ignore the characteristics of 3D volumes including much background regions and suffer from generating hallucinations, especially in medical domain that contains many uncommon professional terms. In this paper, we aim to efficiently adapt the pre-trained foundation model to specific 3D radiology report generation, and present a Prior-drIven Prompting with diagnosis-oriented retrieval-Augmentation (PIPA) framework. In PIPA, we design a Prior-drIven Prompting (PIP) strategy to exploit diagnostic knowledge from input volumes and a Diagnosis-oriented volume-report retrievalaugmentation Generation (DIG) module to explore beneficial knowledge from external database. Specifically, in PIP, to take full advantage of the patient's clinical information, e.g., age and symptoms, and the possible disease information, e.g., brain tumor, edema, we formulate them as the patient and disease priors to mine clinical relevant knowledge. Furthermore, we propose utilizing visual and textual embeddings as queries to retrieve similar external data by devising a diagnosis-oriented retrieval-augmentation scheme for leveraging more report resources as references for LLM to produce accuracy outcomes. With PIP and DIG, PIPA integrates clinical priors and external data to learn effective diagnostic representations for high-quality report generation. We evaluate the framework on both public and in-house 3D medical datasets with corresponding reports, demonstrating its strong performance in generating accurate diagnosis reports. Source codes have been published at https://github.com/CUHK-AIM-Group/PIPA/tree/ main.

Artificial Intelligence (AI) is transforming clinical dental practice, with most applications focusing on static images like radiographs. However, the integration of AI in analyzing video data, particularly in surgical settings rich in dynamic information, remains underexplored in oral medicine. This study introduces AI-miSMG, an intelligent surgical workflow recognition model designed for minimally invasive submandibular gland (SMG) resection. Building upon our previous work, we established a standardized annotation protocol to efficiently label endoscopic SMG resection procedures, which are divided into Creation, Position, Separation, Inspection, and Idle phases. The AI-miSMG model was trained on a dataset of 73 high-quality endoscopic SMG resection videos, comprising 386,122 labeled frames. To evaluate its performance, we used a multicenter dataset consisting of surgical videos from four different centers, totaling 85,913 images. The model achieved an overall accuracy of 0.87 on the external validation dataset. Additionally, it was explored for workflow-based analysis of surgical fluency across surgeons with different experience levels. Furthermore, our model reduced annotation time by approximately 47%, decreasing from 94.00 min to 49.90 min. Overall, we developed a deep learning-based workflow recognition model for minimally invasive SMG resection and demonstrated its feasibility for surgical phase recognition, workflow analysis, and model-assisted annotation.

In the TOPAZ-1 trial's primary analysis, durvalumab plus gemcitabine and cisplatin (GemCis) showed statistically significant improved overall survival (OS) vs placebo plus GemCis with comparable safety between treatment groups in participants with advanced biliary tract cancer (aBTC). Durvalumab plus GemCis was recently established as the first-line standard of care among patients with aBTC. To evaluate 4-year OS and safety of durvalumab plus GemCis in participants with aBTC. This post hoc analysis of the global, double-blind, placebo-controlled, phase 3 TOPAZ-1 randomized clinical trial included participants 18 years and older with histologically confirmed unresectable, locally advanced, or metastatic biliary tract adenocarcinoma. In the TOPAZ-1 trial, patients were enrolled from April 2019 to December 2020 at 105 sites in 17 countries. Data cutoff was February 28, 2025. Participants received intravenous durvalumab, 1500 mg, or placebo plus gemcitabine, 1000 mg/m2, and cisplatin, 25 mg/m2, on days 1 and 8 every 3 weeks for up to 8 cycles, followed by durvalumab or placebo monotherapy every 4 weeks. OS, duration of treatment exposure, serious adverse events, and adverse events resulting in discontinuation were assessed approximately 48 months after the last participant was randomized. Overall, 685 participants were randomized, with 341 receiving durvalumab plus GemCis (median [range] age, 64 [20-84] years; 172 [50.4%] female) and 344 receiving placebo plus GemCis (median [range] age, 64 [31-85] years; 168 [48.8%] female). Median (range) follow-up in censored participants was 56.9 (1.7-67.2) months for participants who received durvalumab plus GemCis and 50.7 (0.9-62.6) months for participants who received placebo plus GemCis. Median OS was 13.0 (95% CI, 11.6-14.1) months for durvalumab plus GemCis and 11.4 (95% CI, 10.1-12.5) months for placebo plus GemCis (hazard ratio, 0.75; 95% CI, 0.64-0.88); 48-month OS rate was 11.8% vs 4.3%, respectively. The rate of serious adverse events possibly related to treatment was similar between arms (52 of 338 participants [15.4%] in the durvalumab plus GemCis arm vs 59 of 342 participants [17.3%] in the placebo plus GemCis arm). In the durvalumab plus GemCis and placebo plus GemCis arms, 21 of 338 participants (6.2%) and 18 of 342 participants (5.3%), respectively, experienced adverse events leading to study drug discontinuation. In this post hoc analysis of the phase 3 TOPAZ-1 randomized clinical trial, durvalumab plus GemCis demonstrated long-term survival benefit and a clinically manageable safety profile, supporting its use as a first-line treatment for aBTC. ClinicalTrials.gov Identifier: NCT03875235.

TOPAZ-1 (NCT03875235) demonstrated improved overall survival (OS) with durvalumab plus gemcitabine and cisplatin vs. placebo plus gemcitabine and cisplatin with manageable safety in advanced biliary tract cancer (aBTC). The phase IIIb TOURMALINE study (NCT05771480) is evaluating the safety and efficacy of first-line durvalumab plus seven gemcitabine-based chemotherapy regimens in a close-to-real-world aBTC population, including participants with poor prognosis. Participants received 1500 mg IV durvalumab (first infusion: 60 minutes; subsequent infusions: 30 minutes) plus investigator's choice of one of seven gemcitabine-based chemotherapy regimens. The primary endpoint was incidence of Grade 3/4 adverse events (AEs) possibly related to treatment (PRAEs) within 6 months of first durvalumab dose. Secondary endpoints included progression-free survival (PFS), OS, objective response rate (ORR), and safety. As of 17 September 2025, 142 participants (median age 68.0 years) had follow-up ≥6 months and received ≥1 treatment dose; most had metastatic disease (72.5%). Participants received a median of 9 cycles of durvalumab; median (IQR) follow-up was 11.63 (6.87-17.25) months. Overall, 50.7% (95% confidence interval [CI] 42.19-59.19) of participants experienced Grade 3/4 PRAEs within 6 months of durvalumab initiation, with no serious AEs of special interest with an outcome of death. Median PFS was 7.39 months (95% CI 6.74-9.07); median OS was 13.50 months (95% CI 11.24-20.53); ORR was 33.1% (95% CI 25.44-41.48). Safety profiles of durvalumab plus seven gemcitabine-based chemotherapy regimens were manageable, and shorter durvalumab infusion duration (30 minutes vs. 60 minutes) did not increase infusion-related reactions. Efficacy data are indicative of potential clinical activity across treatment arms. These findings demonstrate the feasibility of combining durvalumab with alternative gemcitabine-based regimens in a broad aBTC population. The choice of gemcitabine-based chemotherapy backbones for the treatment of advanced biliary tract cancer (aBTC) varies globally and their use in conjunction with immunotherapy has not been fully investigated. The TOURMALINE phase IIIb study demonstrated manageable safety and efficacy data showed potential clinical activity in a close-to-real-world population of participants with aBTC receiving durvalumab plus one of seven gemcitabine-based chemotherapy regimens. These findings point towards the feasibility of combining durvalumab with different gemcitabine-based chemotherapy regimens, even in participants with less favourable disease characteristics. NCT05771480 TRIAL REGISTRATION: ClinicalTrials.gov: NCT05771480; https://clinicaltrials.gov/study/NCT05771480.

Multidrug-resistant and rifampin-resistant tuberculosis (MDR/RR-TB) remains a formidable challenge to global health. While the integration of bedaquiline (BDQ) has revolutionized the therapeutic landscape for MDR/RR-TB, the clinical necessity and safety profile of extending BDQ administration beyond the conventional 24-week regimen-especially in complex or high-risk cohorts-remain insufficiently characterized. This multi-center retrospective cohort study analyzed 189 patients with MDR/RR-TB in China between January 2019 and January 2024. Patients were stratified into a standard-duration group (≤6 months) and an extended-duration group (>6 months). Treatment efficacy, cumulative culture conversion rates, and adverse events (AEs) were comparatively evaluated. Multivariable logistic regression was employed to identify independent predictors of severe Fridericia-corrected QT (QTcF) prolongation (>500 ms). No significant differences were observed in favorable treatment outcomes between the extended and standard groups (86.8% vs. 85.8%; P = 0.845). Culture conversion rates at the end of treatment were comparable (94.7% vs. 92.0%; P = 0.457). Regarding cardiac safety, the incidence of QTcF >500 ms did not differ significantly between the two cohorts (7.9% vs. 10.6%; P = 0.528). Notably, pre-existing cardiac disease was identified as the most potent independent risk factor for severe QTcF prolongation (OR: 9.01; 95% CI: 2.53-32.12; P < 0.001), rather than the duration of BDQ exposure. Extended BDQ treatment is both efficacious and well-tolerated in patients with MDR/RR-TB. Prolonged exposure does not inherently increase the risk of cardiotoxicity, suggesting that BDQ duration can be personalized based on clinical need, provided that baseline cardiac comorbidities are rigorously managed.

Aging is a major risk factor for cardiovascular disease (CVD) and is also linked to a functional decline of the immune system. B-cells contribute to atherosclerosis progression by antibody secretion, antigen presentation and T-cell regulation. During aging, CD11c + CD21low B-cells accumulate, but it is unclear whether these age-associated B-cells (ABCs) are linked with incident cardiovascular events in humans and if they contribute to the progression of atherosclerosis. To investigate associations with first-time coronary events, circulating B-cells were phenotyped with flow cytometry in a case-control study (N = 604) nested in the Malmö Diet and Cancer Study cohort with a median of 14 years follow-up. Splenic B-cells in young (5 months) and aged (21 months) female Ldlr-/- mice were characterized with single-cell RNA-sequencing coupled with B-cell receptor (BCR)-sequencing. Atherogenicity of ABCs was determined by adoptive transfer to Ldlr-/- and Ldlr-/-Rag1-/- mice.Compared with other B-cell populations, ABCs showed a significant positive correlation with age (Spearman's rho 0.24; P < 0.0001; across 46-68 years of age) and accumulated in aged atherosclerotic mice. Coronary event cases had higher counts of ABCs than controls and regression analysis revealed an association with incident coronary events, that was independent of cardiovascular risk factors [odds ratio: 1.92 (95% CI 1.13-3.27), P = 0.016, comparing the 4th vs 1st quartile]. Adoptive transfer of ABCs to recipient mice promoted atherosclerotic lesion development. In Ldlr-/- Rag1-/- mice, ABCs also increased necrotic cores (P < 0.05). B-cells recovered from recipient mice after transfer expressed CD138 and secreted antibodies. In line with these findings, we demonstrated that ABCs expressed plasma cell differentiation genes and showed the greatest clonal expansion within the B-cell compartment, with extensive clonal overlap with plasma cells. We show that ABCs predict first-time coronary events in humans and contribute to the immunopathology of atherosclerosis in mice, indicating that clonal expansion and accumulation of ABCs contribute to the strong association between age and CVD. ABCs may serve as potential prognostic and therapeutic targets for CVD.

Timely identification of children with ileocolic intussusception likely to fail air-enema reduction is critical to avoid delays and bowel perforation. However, even expert sonographers show inter-observer variability. We developed and prospectively validated a Vision Transformer (ViT) deep learning system to predict reduction failure from static B-mode ultrasound images. This multicenter bidirectional cohort study included 5602 children (4-60 months) who underwent air-enema reduction at 14 Chinese tertiary hospitals (retrospective cohort: 2019-2024). After data augmentation, 10,151 images (8122 training, 2029 validation) were used to train a ViT model for binary classification ("success" vs. "failure"). External validation was performed on a prospective cohort of 190 patients (March-June 2025), with three junior and three senior sonographers independently predicting outcomes. The study was approved by the Ethics Committee of Yijishan Hospital of Wannan Medical University (approval No. 2025-04) and registered with ChiCTR2500098673. The model achieved high internal performance (failure: accuracy 0.880, precision 0.969; success: accuracy 0.970, precision 0.898). In the prospective cohort, the ViT model achieved 93.7% overall accuracy, significantly higher than senior (74.7%) and junior (60.7%) sonographers (p < 0.05). This study innovatively applies ViT to assess pediatric ileocolic intussusception severity, providing an objective, accurate tool to support clinical decision-making and reduce treatment risks.

Cell therapy has longstanding roots in haematopoietic stem cell transplantation and early immune cell transfers in infectious disease and transplantation, where patient- or donor-derived cells have achieved therapeutic benefit in selected contexts. The modern era has been driven largely by oncology, with engineered modalities such as tumour-infiltrating lymphocytes, CAR-T cells and TCR-engineered T cells delivering transformative responses but requiring complex, costly manufacturing. These platforms are now being adapted for autoimmune diseases to induce durable, antigen-specific immune tolerance, yet broad application is limited by safety concerns, process complexity and access. Non-engineered cell therapies for autoimmunity, including mesenchymal stem cells, polyclonal regulatory T cells and tolerogenic dendritic cells, have shown acceptable safety and proof-of-principle for immune re-education, but clinical responses have been modest and inconsistent, with limited scalability. Engineered approaches such as CAR-T cells can induce reversible B cell depletion in B cell-mediated rheumatic diseases but only addresses antibody-driven pathology and not T cell-mediated autoimmunity. TCR-engineered Tregs have emerged as a promising antigen-specific strategy, offering localized, antigen-linked suppression with bystander tolerance. Preclinical and early clinical data suggest superior potency, stability and disease control compared with polyclonal Tregs at similar or lower doses, but translation is constrained by the rarity and fragility of Tregs and by labour-intensive, CAR-T-like manufacturing. This review highlights emerging solutions for closed, automated and decentralised production, and discusses allogeneic approaches using gene-edited or banked Tregs with HLA engineering or matching. Together, these advances support the development of scalable, "off-the-shelf" TCR-Treg products with potential to provide safe, affordable tolerance-restoring therapies for autoimmune disease.

Sleep disturbances are highly prevalent among individuals with chronic non-cancer pain and are associated with worse pain severity and poorer prognosis. The comparative trade-offs between the effectiveness and safety of available pharmacotherapies for sleep outcomes in this population remain poorly defined. Ninety-eight RCTs involving 28,920 participants (mean age 53.2 years, 71.2% female) were included. Moderate-certainty evidence demonstrated that melatonin significantly improved sleep quality compared with placebo (standardized mean difference [SMD] = -0.60, 95%CI: -0.98, -0.22). Ten agents (e.g., amitriptyline, oxycodone, gabapentin, pregabalin, duloxetine) also showed statistically significant improvements in subjective sleep quality (SMD = -0.24 to -1.07), but most effects were supported by low-certainty evidence and were accompanied by an increased risk of adverse events (odds ratio [OR] = 1.90 to 37.00). Conversely, melatonin was not associated with an increased risk (OR = 0.88, 95%CI: 0.19, 3.94). Our findings indicate that melatonin shows promise as a safe, adjunctive option for improving sleep quality in this population, but larger, condition-specific trials are warranted to confirm these effects. Other pharmacological agents are limited by lower-certainty and unfavorable safety profiles. These results should be interpreted cautiously given limited direct comparisons, heterogeneous chronic pain populations, the high proportion of trials at high risk of bias, and the predominance of subjective sleep outcomes.

The psychiatric safety profile of glucagon-like peptide-1 receptor agonists (GLP-1RAs) is characterized by inconsistent findings during active treatment and remains largely unexplored after cessation. Here we show that GLP-1RA discontinuation is associated with an increased risk of psychiatric events. Using large-scale electronic health records from the Shanghai Hospital Link Database, we investigate incident depressive and anxiety disorders during GLP-1RA treatment and after its discontinuation in people with type 2 diabetes, compared with dipeptidyl peptidase-4 inhibitors (DPP4is) or sodium-glucose cotransporter-2 inhibitors (SGLT2is). During treatment, GLP-1RAs show a neutral risk compared with DPP4is and a lower risk compared with SGLT2is. In contrast, after cessation, prior use of GLP-1RAs is associated with a higher risk than prior use of DPP4is or SGLT2is. This increase is modestly mediated by elevated triglyceride levels. These findings highlight the need for clinical vigilance regarding anxiety following GLP-1RA discontinuation.

Parkinson's disease (PD) is characterized by progressive loss of nigral dopaminergic neurons, resulting in disabling motor symptoms. Intracerebral transplantation of stem cell-derived dopaminergic progenitors to replace lost endogenous dopaminergic neurons offers a new potentially restorative therapeutic approach for PD. Here we report the 12-month primary safety end point and interim efficacy outcomes from a phase 1/2, open-label, multicenter trial evaluating STEM-PD, a cryopreserved, off-the-shelf dopaminergic progenitor product derived from human pluripotent stem cells. Eight individuals with moderate PD underwent bilateral intraputaminal transplantation at two escalating doses (n = 4 per cohort), followed by 12 months of immunosuppression. Seven participants completed 12-month follow-up; one participant died from a pulmonary infection. No serious adverse events were attributed to the cell product, no graft-induced dyskinesias were observed and serial magnetic resonance imaging showed no evidence of tumor formation. These findings support the feasibility and favorable safety profile of human pluripotent stem cell-derived dopaminergic progenitor transplantation in this early-phase study, with risks primarily associated with the immunosuppression regimen. Ongoing follow-up to 36 months will further evaluate durability, clinical outcomes and graft function. ClinicalTrials.gov identifier: NCT05635409 .

Retinal vein occlusion (RVO) is a chronic retinal vascular disease that often requires repeated anti-VEGF injections and long-term follow-up. However, predicting treatment responses across different follow-up timepoints remains clinically challenging. To address this issue, we developed an AI system integrating generative adversarial networks (GANs), UNet + +, and ResNet-101 to generate post-treatment OCT and fundus images and support clinical decision-making. A total of 2304 OCT and 576 fundus images from 576 RVO patients were collected at baseline and at weeks 4, 12, and 24 after treatment. The generated images demonstrated favorable visual quality, as evaluated by mean absolute error (MAE), peak signal-to-noise ratio (PSNR), and structural similarity index measure (SSIM). The system further quantified lesion areas and predicted retreatment needs, achieving average AUCs of 0.854 and 0.744 across six models in the internal and external test datasets, respectively. In the reader study, the AI system achieved higher predictive accuracy than retinal specialists while substantially reducing image interpretation time. Clinicians' predictive performance also improved with AI assistance.

Neuroblastoma is a leading cause of childhood cancer mortality, presenting persistent management challenges due to its biological heterogeneity and the limited accessibility of molecular profiling in routine practice. Here we present NEVA (NEuroblastoma Vision-language AI), a multimodal foundation model designed to address these barriers. Unlike conventional approaches that rely on frozen encoders and multiple instance learning, NEVA implements a pathologist-inspired hierarchical workflow with end-to-end optimization. Developed and evaluated in a large multi-institutional cohort of 1,238 patients across multiple centers, NEVA outperforms ten representative foundation models, including TITAN, UNI, and Virchow, across the majority of the 11 clinical tasks evaluated. The model demonstrates diagnostic capability, achieving Area Under the Receiver Operating Characteristic curves of 0.916 for subtype classification, 0.823 for Shimada classification, and 0.806 for risk group stratification. Furthermore, NEVA predicts key molecular alterations from routinely available pathology data, reaching an AUROC of 0.924 for NMYC amplification and 0.830 for 1p36 deletion, while enabling prognostic stratification for progression-free and overall survival across multiple test cohorts. By integrating interpretable attention maps that localize histologically relevant regions, NEVA establishes a scalable framework for neuroblastoma risk stratification and clinical decision support.

Caloric restriction (CR) improves metabolic health across species, but the molecular mediators of its effects in humans remain incompletely defined. In a 24-month non-blinded randomized controlled trial (Clinicaltrial.gov: NCT00427193) of non-obese (BMI 22-27.9 kg/m2) men and premenopausal women aged 21 to 50 years, we assessed prespecified outcomes. Participants were randomized to an ad libitum or CR diet. We found that CR was associated with increased high-molecular-weight (HMW) adiponectin and reduced circulating ceramide species implicated in insulin resistance, including C16:0, C18:0, and C24:0. Mediation analysis indicated that reductions in ceramides were statistically compatible with partial mediation of the CR-associated improvements in insulin secretion, insulin sensitivity, and IGF-1 signaling markers. These effects were most pronounced at 12 months and attenuated by 24 months, suggesting partial metabolic adaptation over time. Overall, our findings are consistent with a model in which CR remodels bioactive lipid profiles and may enhance glucose metabolism in part through an adiponectin-ceramide-linked mechanism, highlighting a potential therapeutic axis for enhancing metabolic health.

Mosaic chromosomal alterations (mCAs) increase with age and are associated with many diseases, including autoimmune diseases. The associations between mCAs and systemic sclerosis (SSc) and its clinical subtypes have not been explored. We recruited study subjects from 2 independent datasets (set 1: 635 SSc, 4401 controls; set 2: 347 SSc, 2170 controls) and detected mCAs (loss, loss of heterozygosity [LOH], gain, and mosaic loss of the X chromosome [mLOX]) from their peripheral blood samples. Logistic regression analyses were conducted with covariates in each cohort, and the results were meta-analysed. We also conducted stratified analyses by age groups, the age at disease onset, clinical phenotypes based on the skin lesions, autoantibody profiles, and the presence of complications. We observed a trend of increased loss in SSc, especially in old age (P = .0063). The association of loss was strengthened in certain subtypes of SSc, including lcSSc (odds ratio [OR] = 2.22, P = .019) and SSc with vascular complications (digital ulcers, pulmonary hypertension, or renal crisis; OR = 3.30, P = .0054). The effect sizes of Loss increased in patients with high cell fractions (CFs). We also observed that mLOX was significantly associated with SSc, limited cutaneous systemic sclerosis (lcSSc), and anticentromere antibody-positive systemic sclerosis (ACA-SSc) only for subjects with high CFs. mLOX was significantly associated with lcSSc and ACA-SSc even compared with dcSSc and ATA-SSc, respectively. These associations were consistently observed in each of the 2 datasets. Finally, we identified majority of the associations of Loss were mainly driven by SSc with late age at onset. Loss and mLOX were significantly and differentially associated with SSc and its subtypes, underscoring potential phenotype-specific contributions of mCAs.

Irritable bowel syndrome (IBS) is a complex disorder of gut-brain interaction, with heterogeneous symptoms, no available biomarkers and limited pathogenetic insight. To identify genetic risk factors and actionable mechanisms for future clinical translation in IBS. We conducted a genome-wide association study (GWAS) meta-analysis of IBS in 2 775 539 individuals from 22 biobanks. IBS genetics was studied across multiple ancestries, different case definitions and symptom-related subtypes. Heritability and genetic correlations with other traits were estimated, and Mendelian randomisation was used to test causal relationships. GWAS data were functionally annotated and fine-mapped to prioritise tissues, cell types, pathways, candidate genes, specific mechanisms and druggable targets. Significant heritability was only detected in individuals of European ancestry, with near-identical genetic architecture across case definitions. Genetic correlations with GI, psychiatric and cardiometabolic traits were observed, including causal relationships with triglyceride (TG) levels. Functional annotation of IBS risk loci highlighted cell types and pathways relevant to brain, enteric neuro-glial and cardiometabolic domains, as well as actionable targets like GCKR, a regulator of TG metabolism. Druggability analyses converged on cardiometabolic mechanisms, including TG modulation. IBS polygenic risk scores were derived and showed a significant association with case status in an independent case-control dataset, supporting further evaluation in external population-based and clinically ascertained cohorts. This study provides the most comprehensive assessment of IBS genetics to date, demonstrating reproducible polygenic inheritance. We link IBS risk to convergent neurogastrointestinal and novel cardiometabolic mechanisms, highlight specific biological pathways and actionable mechanisms and outline translational opportunities emerging from integrated computational analyses.

Traditional Cox proportional hazards models show suboptimal performance for cardiovascular disease (CVD) risk prediction in Chinese populations. Transformer-based deep learning models have demonstrated promise in clinical risk prediction. In this study, sex-specific transformer-based models (China-AIHeart) for 10-year CVD risk prediction among Chinese adults were developed and validated. The derivation cohort included 156 790 participants [34.6% men; mean [SD] age, 56.7 [8.9] years) without CVD from the China Cardiometabolic Disease and Cancer Cohort. External validation was conducted in two independent Chinese cohorts (Xinjiang and CHARLS). Transformer-based time-to-event prediction models were developed, including a full model (22 predictors) and a simplified model (15 predictors). Performance was compared with Cox models using identical predictors and established risk scores (China-PAR, PREVENT-ASCVD, and SCORE2 Asia-Pacific equations). China-AIHeart demonstrated good discrimination (C-statistic [95% confidence interval, CI]: .767 [.754-.779] in men; .780 [.769-.791] in women), calibration (calibration χ2: 14.806 in men; 9.326 in women; Brier score: .104 in men; .077 in women), and net clinical benefit in predicting CVD risk. Predicted event rates closely matched observed risks across strata. Compared with Cox models with identical predictors, China-AIHeart showed improved discrimination (ΔC-statistic [95% CI]: .027 [.025-.028] in men; .031 [.029-.033] in women) and reclassification (net reclassification index [95% CI]: .478 [.466-.492] in men; .560 [.551-.572] in women), and outperformed China-PAR, PREVENT-ASCVD, and SCORE2 Asia-Pacific equations. External validation demonstrated robust performance, with C-statistics of .781/.825 (men/women) and .748/.820 for the full and simplified models in the Xinjiang cohort, and .740/.771 for the simplified model in the CHARLS cohort. The transformer-based China-AIHeart models predicted 10-year CVD risk and outperformed traditional Cox-based approaches, providing a practical tool for risk stratification in Chinese adults.

Chronological age is central to cardiovascular disease (CVD) risk estimation but poorly reflects interindividual heterogeneity in cardiovascular aging. This study developed a peri-aortic adipose tissue (PAAT) radiomic cardiovascular age (CV-Age) biomarker from routine chest CT and tested whether the PAAT age gap (ΔAge = CV-Age - chronological age) stratifies major adverse cardiovascular events (MACE) and improves risk classification. Four chest CT cohorts with different protocols were utilized to train (n = 4451) and externally validate (n = 44 214) a CV-Age model. Associations between ΔAge decile groups (resilient ≤10th; accelerated ≥90th) and incident 5-point MACE (myocardial infarction, stroke, revascularization, heart failure, and death) were assessed using survival models. Clinical utility was evaluated by substituting CV-Age for chronological age in PREVENT to quantify risk reclassification. A 31-feature radiomic signature capturing PAAT volume, attenuation, and texture heterogeneity predicted age with good accuracy (training MAE 2.2 years; primary external validation MAE 2.7 years). In a separate higher-risk cohort, predictions showed a positive ΔAge (median +5.4 years), reflecting elevated baseline cardiovascular burden. ΔAge-stratified event-free survival differed across groups (log-rank P < .001); compared with normative agers, accelerated agers had higher MACE risk (HR 1.51, 95% CI 1.37-1.66), and resilient agers had lower risk (HR 0.70, 0.63-0.77). Substituting CV-Age into PREVENT improved risk reclassification (categorical NRI +0.03, P < .01, continuous NRI +0.04, P < .05). Radiomic profiling of PAAT on routine chest CT yields a scalable imaging biomarker of cardiovascular aging. CV-Age can help improve identification of higher-risk individuals missed by age-driven risk estimation, supporting its role as a risk-enrichment tool.

Objectives. To examine the distribution of high utilizers of pediatric health care across New York State (NYS). Methods. We performed a cross-sectional study using the 2022 NYS inpatient and emergency department data sets for children younger than 18 years. We used a principal component analysis- derived composite adverse utilization index to identify high utilizers (top 5% of the distribution). We used negative binomial models to identify county-level deviation among high utilizer rates. Results. Among 663 006 children, high utilizers accounted for 34.5% of hospital days, 57.3% of admissions, 62.5% of costs, and 5.0% of emergency department visits. High utilizers were more likely to have medical complexity and ambulatory care-sensitive conditions. Bronx and St. Lawrence counties had significantly higher-than-expected rates of high utilizers after adjustment. Conclusions. Pediatric health care burden in NYS is concentrated among a subset of children within identifiable counties. A utilization index offers a pragmatic, reproducible way to target care coordination and community prevention resources, thus informing precision social medicine strategies where need and impact are greatest. (Am J Public Health. Published online ahead of print July 9, 2026:e1-e9. https://doi.org/10.2105/AJPH.2026.308486).

Chordoma is a rare malignant bone tumor with high local recurrence, metastatic spread in 40% to 60% of patients over the disease course, and significant morbidity. Because of its rarity, anatomical complexity, and prolonged natural history, high-quality evidence to guide management is limited. International consensus guidelines for localized chordoma were first published in 2015; however, advances in pathology, imaging, surgery, radiotherapy, and supportive care since then necessitate updated multidisciplinary recommendations. To update and expand the 2015 consensus recommendations on the diagnosis, treatment, and follow-up of pediatric and adult patients with primary, localized chordoma. In June 2025, a meeting of the Global Chordoma Consensus Group was held in Milan, Italy, that included experts from all relevant specialties as well as patient representatives. A comprehensive literature review guided structured discussions on the management of primary localized disease. Levels of evidence and grades of recommendation were assigned. A total of 305 articles were included in the literature review. Management strategies were stratified by anatomical site (skull base, mobile spine, and sacrum). The central principal of care was treatment at experienced, multidisciplinary centers, with maximally safe surgery followed by high-dose, highly conformal radiotherapy. Guidance was provided on diagnosis, surgical approaches, and radiotherapy planning for each anatomical site. Systemic therapy options; long-term, risk-adapted follow-up; and supportive, palliative, and rehabilitative care were also addressed. This global consensus statement provided updated multidisciplinary guidance for the management of primary, localized chordoma. It aimed to harmonize clinical practice, support shared decision-making, and identify priorities for future collaborative research in this rare and challenging disease.

Human aging is characterized by complex structural and functional decline, but quantifying its heterogeneity and assessing biological age remain challenges. We present the mCAS (multicentric Chinese aging standardized cohort) developed from 2,019 Chinese individuals aged 18-91 years. Integrating high-dimensional clinical, physiological, and molecular-level data, we constructed a three-tiered aging framework: the core capacity clock (CC-clock) to quantify clinical physiological decline, the multimodal clock (MM-clock) with extensive parameter coverage and enhanced predictive precision, and organ-associated aging clocks. Cross-layer analysis demonstrates that plasma protein clocks not only capture chronological age but also serve as efficient proxies for systemic physiological capacity. Leveraging this framework for discovery, we identified the age-dependent accumulation of coagulation factors as a driver of multi-organ senescence and systemic inflammatory activation. This study provides a foundational framework that bridges molecular signatures with functional decline, identifies new biomarkers for aging assessment, and reveals a novel translational driver of aging.

Over the past two decades, the microbiome has emerged as a central modifier of host health, whose manipulation may prevent or treat disease. Fecal microbiome transplantation (FMT) transfers stool from healthy donors to recipients to restore microbial structure and function. It is universally accepted as therapy for recurrent Clostridioides difficile infection (rCDI) and is studied across metabolic, neurological, oncological, and autoimmune disorders. However, challenges remain, including donor selection, possible transmission of infectious or non-communicable risks, and limited understanding of mechanisms driving benefits. This review summarizes FMT designs, mechanisms, indications, and obstacles. It discusses emerging strategies such as the use of microbial consortia and extra-intestinal microbiome transplantation and suggests that a better understanding of FMT functions, limitations, and off-target effects may enable safer, more generalizable modulation of microbiome-regulated diseases. Such a mechanistic understanding may manifest as refined donor screening, standardized protocols, tracked outcomes, and identified microbes and metabolites inducing durable clinical benefits.

Sickle cell disease attenuates nitric oxide signaling, limiting soluble guanylyl cyclase (sGC) stimulation needed for hemo-vascular function. Cytochrome b5 reductase 3 (CYB5R3) regulates sGC activity and expression. We show CYB5R3 T117S associates with reduced efficacy of the sGC stimulator riociguat. (NCT02633397).

There is no consensus on staging and surveillance imaging for cutaneous squamous cell carcinoma (CSCC). Although imaging has been shown to affect management, broad recommendations from the National Comprehensive Cancer Network have led to variability in clinical practice. To develop multidisciplinary consensus recommendations on the use of staging and surveillance imaging of localized CSCC. A multidisciplinary expert panel across academic and clinical practice settings convened a Delphi consensus, with 3 iterative survey rounds from January to June 2025. Data were analyzed from February 2025 to January 2026. Eligibility defined by at least 1 to 2 CSCC publications during the previous 5 years, involvement in a relevant clinical trial, experience treating at least 3 patients monthly with CSCC, and/or 5 years or longer of clinical practice. Fifty-four experts were invited and 45 (83%) completed all 3 rounds and were included in the final analysis. Structured Delphi surveys evaluated clinical scenarios, imaging modalities, and surveillance strategies for CSCC. Consensus and near-consensus recommendations were generated, defined as 80% or greater and 70% to 79% agreement, respectively. The 45 participants (21 female individuals [47%] and 24 male individuals [53%]; 14 Asian individuals [31%], 1 multiracial individual [2%], and 30 White individuals [67%]) consisted of dermatology (15 [33%]), medical oncology (7 [16%]), radiation oncology (9 [20%]), radiology (9 [20%]), surgery (3 [7%]), and otolaryngology specialists (2 [4%]). The Delphi panel recommended staging and surveillance for CSCCs with at least a 15% risk of metastasis. Consensus or near consensus was reached to recommend staging and surveillance imaging for tumors with a concern for metastasis, bone invasion, invasion beyond subcutaneous fat, large-caliber nerve invasion, or a diameter of 4 cm or larger or the combination of tumors with poorly differentiated histology and any of the following: diameter of 2 cm or larger, lymphovascular invasion and subcutaneous fat invasion, or lymphovascular invasion and small-caliber perineural invasion. Computed tomography imaging was the preferred modality for nodal staging (38 [84%]) and surveillance (35 [78%]). For surveillance duration, consensus was reached to provide imaging for at least 2 years, and near consensus was reached for at least 3 years. This study provides an expert consensus-based framework to standardize imaging in localized CSCC that may inform future guidelines.

Merkel cell carcinoma (MCC) is associated with a high recurrence rate of 40%. It is important to know which of 2 blood-based tumor biomarkers available for use in clinical practice is more accurate in detecting MCC recurrence. To compare the performance of circulating tumor DNA (ctDNA) and the MCC polyomavirus (MCPyV) oncoprotein antibody test for detecting MCC recurrence. In this multicenter cohort study across 3 US academic centers (Stanford, Dana-Farber Brigham, and University of Washington), patients with stage I-IV MCC underwent serial paired ctDNA and antibody testing for disease surveillance between April 2020 and March 2024. ctDNA was considered positive if mean tumor molecules per milliliter of plasma values were greater than 0.00, while a rising antibody test result was defined as a 30% or greater increase in titer. Inclusion criteria were clinically disease-free at baseline, detectable MCPyV antibodies at diagnosis, and both ctDNA and antibody tests performed within 45 days of each other in at least 1 time point per patient. Predictive performance for recurrence was assessed and compared between tests. The primary outcome was clinical recurrence. Predictive performance measures for each test included positive (PPV), negative predictive value (NPV), and hazard ratios (HRs). In this study among 169 patients with 703 paired tests (median testing interval, 96 days; 109 males [64%]; 60 females [36%]), there were 36 clinical recurrences from 32 patients over a median follow-up of 483 days (IQR, 274-714 days). Compared with antibody testing, ctDNA had a significantly higher PPV at 365 days (73% [95% CI, 58%-85%] vs 52% [95% CI, 32%-71%]; P = .02), higher NPV at 90 days (99% [95% CI, 98.8%-100%] vs 97% [95% CI, 95%-98%]; P = .001), and a greater HR (HR, 47.9 [95% CI, 16.5-139.0] vs 7.3 [95% CI, 3.8-13.9]; HR ratio, 6.6 [95% CI, 2.5-41.0]; P < .001). Use of both ctDNA and antibody tests together identified only 1 additional recurrence compared with the use of ctDNA testing alone. This study found that ctDNA was more accurate than MCPyV antibody testing in detecting recurrence of MCC. ctDNA appears to be a more reliable guide than the MCPyV antibody for surveillance practices and ongoing risk stratification in most patients with MCC.

Patients with phenotypically mild hypertrophic cardiomyopathy (HCM) do not require symptom management, but may be at an earlier stage in the disease course, with potential to benefit from disease-modifying therapies. However, little is known about the natural history and predictors of major adverse cardiovascular events (MACE). Using the Sarcomeric Human Cardiomyopathy Registry, we identified predictors of incident MACE and characterized disease progression in phenotypically mild HCM. Phenotypically mild HCM was defined as: having shorter disease duration (<10 years since diagnosis or age ≤30 years), no previous MACE, being NYHA functional class I, and having a left ventricular (LV) maximal wall thickness (MWT) <25 mm. These individuals were followed prospectively for the development of symptoms or MACE: atrial fibrillation (AF), malignant ventricular arrhythmia (MVA) (sudden cardiac death, resuscitated arrest, or appropriate defibrillator therapy), heart failure (HF) (cardiac transplantation, LV assist device implantation, LV ejection fraction <35%, or NYHA functional class III or IV symptoms), stroke, or all-cause mortality. Cox regression identified MACE predictors. Linear and latent class mixed models characterized LV remodeling trajectories and risk clusters. Of 2,500 participants with phenotypically mild HCM (mean age 43 years, 31% women) followed for a mean duration of 7 ± 6 years, 534 (21%) developed MACE, including 289 with AF, 69 with MVA, and 193 with HF. Individuals who progressed from NYHA functional class I to ≥ II symptoms during follow-up (n = 585, 23%) were 2.79 times (95% CI: 2.30-3.39 times) more likely to experience MACE. Age at baseline (HR: 1.24; 95% CI: 1.17-1.32 per 10-year increase), body mass index (HR: 1.10; 95% CI: 1.01-1.21 per 5-kg/m2 increase), left atrial (LA) diameter (HR: 1.16; 95% CI: 1.09-1.25 per 5-mm increase), LV MWT (HR: 1.27; 95% CI: 1.10-1.46 per 5-mm increase), and LV outflow tract (LVOT) gradient (HR: 1.08; 95% CI: 1.05-1.12 per 15-mm Hg increase) associated with higher MACE rates. LV late gadolinium enhancement presence was associated with 36% (95% CI: 5%-76%) higher hazard of MACE. Remodeling trajectories during follow-up predicted risk with each 0.5 mm/year steeper increase in LA diameter associating with doubled AF (HR: 2.24; 95% CI: 1.69-2.97) and HF rates (HR: 2.22; 95% CI: 1.62-3.04) and each 0.5 mm/year steeper LV MWT increase associating with doubled MVA rates (HR: 1.92; 95% CI: 1.38-2.69). Higher sustained values and/or steeper increases in LA diameter, LV MWT, or LVOT gradient associated with the highest MACE rates. Approximately 21% of patients with phenotypically mild HCM developed MACE over medium-term follow-up. Older age, symptoms development, and increasing LA diameter, LV hypertrophy, or LVOT gradient associated with MACE, particularly in instances of steeper rate of change. These findings can guide management strategies and inform future studies of disease-modifying therapies.

Clinical trials in IBD face difficulties of escalating complexity, high costs and challenges in recruitment. Digital twins are virtual, data-driven replicas of individual patients that model disease trajectories and treatment responses, which offer a potential innovative change in the conduct of clinical trials in IBD. Built from multimodal datasets integrating clinical, molecular, imaging and real-world data, digital twins can generate synthetic control arms, enable adaptive randomisation and predict disease relapse or treatment response. Early studies across oncology, cardiology and endocrinology demonstrate their feasibility and potential to improve statistical power while reducing patient burden. However, the integration of digital twins into clinical trials in IBD will require rigorous validation frameworks, transparent data governance and attention to algorithmic bias and consent. In this review, we explore how digital twins may transform IBD research-from in silico simulation to adaptive, patient-centred trial design-and outline the regulatory, ethical and logistical challenges to be considered in order to successfully integrate them into future trials.

Genetic studies of stool frequency (SF), an indirect proxy for gastrointestinal transit, may reveal therapeutically tractable pathways relevant to IBS and other dysmotility disorders. To identify genes and mechanisms involved in gut motility, providing a foundation for clinical translation. We performed a multiancestry genome-wide association study (GWAS) meta-analysis of SF in 268 606 European and East Asian individuals. Heritability and genetic correlations with other traits were estimated, and Mendelian randomisation was used to test causal relationships. GWAS signals were fine-mapped and functionally annotated to prioritise candidate genes and pathways. Findings implicating thiamine metabolism were followed-up with dietary interaction analyses in UK Biobank (UKB). SF heritability was comparable in Europeans (7.0%) and East Asians (5.6%). We observed strong genetic correlations with gastrointestinal and psychiatric disorders (rg=0.18-0.47), and causal effects on IBS. Novel correlations with cardiovascular traits (rg=0.12-0.14) were supported by drug signature enrichment analyses. We identified 21 independent loci, including 10 novel signals implicating bile acid synthesis (KLB) and cholinergic signalling (COLQ). Fine-mapping converged on vitamin B1 metabolism, highlighting single-variant causal effects at SLC35F3 (a thiamine transporter) and XPR1 (phosphate exporter essential for thiamine activation). In 98 449 UKB participants, thiamine intake was positively associated with SF (p<0.0001), and a combined SLC35F3/XPR1 genotype score significantly modulated this effect (p<0.0001). We identify therapeutically tractable mechanisms involved in the control of gut motility, including a previously unrecognised role for vitamin B1. These findings warrant mechanistic and clinical studies to evaluate their translational potential in IBS and other dysmotility syndromes.

IBD is characterised by recurrent flares, but evidence on whether modifiable dietary factors influence flare risk is limited. The PREdiCCt study was designed to examine demographic, clinical and dietary factors associated with disease flare among patients with IBD in self-reported remission. Multicentre, prospective cohort study conducted across 47 UK centres. Patients with Crohn's disease (CD), ulcerative colitis (UC) or IBD unclassified (IBDU) in self-reported remission were prospectively followed up. The baseline diet was assessed using a validated food frequency questionnaire. The primary outcome was time to patient-reported flare (captured by monthly IBD-Control) and objective flare (clinical flare plus C-reactive protein >5 mg/L and/or faecal calprotectin (FC) >250 µg/g with treatment escalation). Associations were evaluated using Cox frailty models adjusted for demographic, clinical and biochemical variables, including baseline FC. Between November 2016 and March 2020, 2629 participants (1370 CD; 1259 UC/IBDU) were enrolled and followed up for a median of 4.1 years (IQR 3.0-5.0). Baseline FC was strongly associated with patient-reported flares (FC ≥250 µg/g: adjusted HR (aHR) 2.22; FC 50-250 µg/g: aHR 1.52 (reference <50 µg/g)) and objective flares (FC ≥250 µg/g: aHR 3.25; FC 50-250 µg/g: aHR 1.98). In UC, higher total meat intake was associated with increased risk of objective flares (highest versus lowest quartile: aHR 1.95, 95% CI 1.07 to 3.56). No consistent associations were observed for ultraprocessed foods, fibre or polyunsaturated fatty acids and flare. Higher habitual meat intake was associated with increased risk of objective flare in UC, suggesting diet may contribute to flare susceptibility in specific patient groups. NCT03282903.

BACKGROUND: Maternal sustained smoking during pregnancy is associated with thousands of differentially methylated CpGs in newborns, but impacts of other prenatal tobacco smoking exposures remain unclear. OBJECTIVE: To identify differential DNA methylation in newborns from maternal sustained smoking and less studied prenatal smoking exposures (i.e., maternal exposure to secondhand smoke [SHS] exposure during pregnancy, maternal quitting before pregnancy, paternal smoking around conception, and paternal quitting before pregnancy). METHODS: We conducted a large meta-analysis of prenatal tobacco smoking exposures and epigenome-wide newborn blood DNA methylation through the Pregnancy And Childhood Epigenetics Consortium (PACE). Across 19 cohorts, 11,175 parent-newborn pairs contributed information on at least one prenatal smoking exposure, mostly from questionnaires. Maternal blood or urine cotinine measurements, available in a few studies, provided objective data for maternal SHS and smoking during pregnancy. Primary analyses used Illumina450 K methylation data; secondary analyses in 5 cohorts examined CpGs unique to the EPIC array. RESULTS: Maternal sustained smoking associated with differential DNA methylation (false discovery rate [FDR] < 0.05) at 8,862 CpGs on the 450 K (n = 8,148) and did not differ by infant sex. We identified over 300 novel genes not previously identified in EWAS of smoking. No differential methylation was associated with maternal SHS, maternal former smoking, or paternal smoking around conception. However, cg24805739 (MED13L) was associated with former paternal former smoking. Forty-one novel genes were identified using maternal cotinine measurements compared to questionnaire. In EPIC unique analyses (n = 3,415), differential methylation was observed with maternal sustained smoking (211 CpGs), maternal SHS (5 CpGs), and paternal former smoking (4 CpGs). Smoking-associated CpGs in blood were strongly enriched for functional elements across multiple tissues. CONCLUSIONS: Maternal sustained smoking has the largest impact on newborn DNA methylation, suggesting a strong influence of the intrauterine environment. We observed minimal impacts for less studied exposures including SHS, maternal former smoking, and paternal smoking.

Broadly neutralizing antibodies (bnAbs) are a promising tool for HIV prevention and treatment. Here we conducted a first-in-human, phase 1 trial of the bispecific 10E8.4/iMab antibody, which consists of a 10E8.4 arm binding the HIV-1 envelope glycoprotein membrane-proximal external region and an ibalizumab (iMab) arm binding the human CD4 molecule. 10E8.4/iMab was administered intravenously (IV) or subcutaneously (SC). Safety/tolerability within 2 weeks of 10E8.4/iMab administration (primary outcome) and the pharmacokinetics (PK), antiviral activity, induction of anti-10E8.4/iMab antibodies, longitudinal CD4+ and CD8+ T cell counts and long-term safety (secondary outcomes) were evaluated. 54 participants living with HIV (PLWH) or without HIV (PLWoH) received 10E8.4/iMab or placebo. In arm 1, PLWoH received 10E8.4/iMab 0.3 mg kg-1 IV, 1 mg kg-1 SC, or 1 mg kg-1 IV (n = 3 each). In arm 2, PLWoH received 10E8.4/iMab 3 mg kg-1 IV, 10 mg kg-1 IV or 30 mg kg-1 IV (n = 6 each). In arms 3/3a, PLWH received 10E8.4/iMab 10 mg kg-1 IV (n = 3) or 30 mg kg-1 IV (n = 6). In arm 4, PLWoH were randomized to receive 10E8.4/iMab or placebo 2.5 mg kg-1 SC or 10 mg kg-1 SC (n = 9 each). Participants in arms 1-3 were not randomized. No treatment-related serious adverse events (AEs) or AEs ≥ grade 3 were reported. The most common solicited AEs were tenderness (10/54, 18.5%), fatigue (18/54, 33.3%) and headache (12/54, 22.2%). Related grade 2 local and systemic solicited AEs occurred in one and six participants, respectively. Three of nine PLWH developed a generalized rash 8-12 days after infusion that resolved within 9-16 days. The primary objective of the study to evaluate the safety/tolerability of 10E8.4/iMab was met. These data support further study of 10E8.4/iMab to expand HIV treatment and prevention options. ClinicalTrials.gov: NCT03875209 .

Empirical broad-spectrum antibiotic overuse in community-onset (CO) bacteremia poses significant clinical risks. We evaluated the prevalence and risk factors of excessively broad-spectrum empirical antibiotic use and its association with clinical outcomes. This retrospective cohort study included 11,183 inpatients diagnosed with CO bacteremia. CO bacteremia was further classified as community-acquired (CA) bacteremia or healthcare-associated (HCA) bacteremia. Excessively broad-spectrum antibiotic use was defined as empirical broad-spectrum antibiotic administration without isolating a compatible resistant pathogen, and clinical characteristics and outcomes were compared according to that status using IPTW-adjusted logistic regression. Among all eligible patients, adequate empirical antibiotics were administered to 9,343 (83.5%) patients; however, 6,944 (62.1%) were administered excessively broad empirical antibiotics - 4,040 (63.5%) among HCA patients and 2,904 (60.2%) among CA patients. Despite high rate of broad-spectrum empirical antibiotics prescribing (69.9% of CA patients and 81.5% of HCA patients), resistant organisms were microbiologically confirmed in only 24.1% of CA patients and 43.1% of HCA patients. Cancer, higher Pitt bacteremia scores, and inotropics use were independently associated with excessively broad-spectrum empirical antibiotic administration. Infection sources were also associated with prescribing patterns, and broad-spectrum antibiotic use was more likely in lower respiratory tract and intra-abdominal infections. Excessive use was associated with adverse outcomes, including in-hospital mortality (aOR 1.57; 95% CI, 1.16-2.11) and Clostridioides difficile infection (aOR 1.72; 95% CI, 1.08-2.74). Excessively broad empirical antibiotic use was prevalent and associated with adverse clinical outcomes in CO bacteremia, underscoring the need for enhanced antimicrobial stewardship.

Extracorporeal life support (ECLS), including venovenous extracorporeal membrane oxygenation (VV-ECMO) and extracorporeal carbon dioxide removal (ECCO₂R), is an established treatment for selected patients with acute respiratory distress syndrome (ARDS). In this review, on the 60th anniversary of ARDS, we review the evolution of ECLS as a support strategy in ARDS, the evidence surrounding its efficacy, factors associated with prognosis, management of patients on ECLS, associated complications, and long-term outcomes among survivors. We also highlight existing areas of uncertainty, and ongoing clinical research that will define the next decade and beyond of ECLS for ARDS.

A 68-year-old man with gastric cancer underwent 18F-fluorodeoxyglucose (FDG) PET/CT, which revealed an incidental subcutaneous lesion suspicious for low-grade sarcoma or metastasis. The patient initially declined a biopsy. One month later, following a new diagnosis of prostate cancer, 68Ga-prostate-specific membrane antigen (PSMA) PET/CT was performed. The unchanged lesion demonstrated heterogeneous, moderate PSMA uptake. Subsequent histopathologic examination confirmed the lesion as a spindle cell lipoma. In conclusion, spindle cell lipomas can exhibit mild 18F-FDG and moderate PSMA uptake, serving as a potential pitfall that can mimic malignancy and cause false-positive results in oncologic PET/CT imaging.

Solitary fibrous tumor (SFT) is a mesenchymal tumor with unpredictable biological behavior that can occur in multiple anatomic sites throughout the body. This case report describes a 22-year-old man with an intracranial malignant solitary fibrous tumor. After 3 courses of 177Lu-FAP-2286 therapy, 68Ga-FAP-2286 PET/CT revealed partial tumor regression, with the patient reporting no adverse reactions.

Primary spermatic cord leiomyosarcoma is a rare malignancy. We describe FDG PET/CT findings in a case of primary high-grade leiomyosarcoma of the spermatic cord. It appeared as a soft tissue mass with heterogeneous hyperenhancement on MRI and focal activity with SUVmax of 4.8 on FDG PET/CT. This case indicates that spermatic cord leiomyosarcoma, although rare, should be included in the list of differential diagnoses of hypermetabolic spermatic cord lesions.

HIV-1 cure requires preventing viral rebound after treatment interruption, but quantitative criteria defining the rebound-competent reservoir are lacking. We studied individuals undergoing observational treatment interruption without confounding interventions to identify virologic and immunologic determinants of rebound. In 9 of 13 participants, rebound viruses were genetically identical or similar to proviruses in circulating resting CD4+ T-cells. We found no evidence of recombination among rebound sequences. Instead, resistance to autologous neutralizing antibodies (aNAbs) was a critical determinant of viral rebound. Increased suppression of viral outgrowth by contemporaneous IgG isolated from plasma was correlated with longer time to rebound. Using inhibitory potential (IP), the log reduction in single-round infection at physiologic IgG concentrations, we defined quantitative limits governing rebound-competency with respect to contemporaneous aNAbs. Contemporaneous IgG antibodies inhibited different reservoir variants with a wide range of IP values (0.4 to 8.2 logs), whereas rebound viruses were minimally inhibited (0.5 to 2.8 logs), indicating that inhibition by even up to 2.8 logs (631-fold) cannot prevent rebound. Longitudinal analyses revealed that waning aNAb potency over time on antiretroviral therapy (ART) allows previously neutralized variants to gain rebound potential, consistent with the finding that rebound can come from variants deposited in the reservoir at different pre-ART time points. Thus, rebound competency is a dynamic, immune-governed property defined by quantitative immunologic constraints, including those exerted by aNAbs.

We find that, relative to Republicans residing in more Republican neighborhoods, Republicans in neighborhoods with a higher concentration of Democratic residents report higher levels of anxiety, depression, and loneliness. While Democrats in general report worse mental health than Republicans, Democrats show no sensitivity to partisan context. Using a large-scale national survey and fine-grained data on residential political context, we establish that these patterns persist even when examining partisans who live in the same Zip Codes, and while controlling for other individual and geographic features. The correlation is strongest for the most strongly partisan individuals, suggesting that politics is a significant factor in the relationship. For Republicans, the size of the relationship between partisan context and mental health is comparable to or larger than the correlation between mental health reports and other contextual features of residential areas, such as neighborhood poverty or the way racial minorities respond to changes in the racial or ethnic composition of local geography.

Sleep health is multidimensional, yet existing research has largely examined isolated sleep characteristics, specific population groups, or only one direction of the sleep-depressive symptom relationship. As a result, the bidirectional associations between multidimensional sleep patterns and depressive symptoms remain insufficiently understood. The study recruited 75,445 adults between July 2021 and December 2024 through the smart health management digital platform for primary cancer prevention (SmartHMDP-PCP) with 34,344 participants completed the follow-up survey. Sleep was assessed across eight dimensions-sleep midpoint, duration, pre-sleep activities, insomnia, sleep problems, daytime symptoms, medication use, and subjective quality-to construct a composite sleep score (healthy vs. unhealthy). Depressive symptoms were measured using the Self-Rating Depression Scale. Multivariate logistic regression estimated odds ratios (ORs) and 95% confidence intervals (CIs). Healthy sleep was associated with reduced odds of incident depressive symptoms (OR, 95%CI: 0.62, 0.58-0.67) and increased likelihood of symptom remission (1.55, 1.41-1.69). Maintaining healthy sleep conferred the strongest protection against incident depressive symptoms (0.38, 0.35-0.42) and the greatest likelihood of remission (2.72, 2.42-3.06). In bidirectional analyses, absence of baseline depressive symptoms (0.60, 0.56-0.66), particularly when sustained over time (0.41, 0.37-0.46), was also negatively associated with subsequent deteriorations in sleep patterns, with effect sizes numerically comparable to those observed in the reverse direction. Multidimensional sleep and depressive symptoms exhibit bidirectional associations of numerically similar magnitude. These findings underscore the importance of integrating sleep health promotion into mental health strategies and highlight the need for interventional and long-term longitudinal research to clarify causal pathways.

Patients with advanced biliary tract cancers (aBTC) are in urgent need of additional/new treatment options. We aimed to assess the efficacy and safety of ivonescimab plus chemotherapy in patients with aBTC. In this multicenter, open-label, phase II study, 30 patients with treatment-naive unresectable locally advanced or metastatic BTC received ivonescimab (20 mg/kg or 30 mg/kg) combined with gemcitabine (1000 mg/m2) and cisplatin (25 mg/m2) every 3 weeks for up to eight cycles, followed by ivonescimab maintenance. The primary endpoint was the investigator-assessed objective response rate (ORR) and safety. Pretreatment tumor specimens available from the trial were subjected to a post hoc exploratory proteomic analysis. The correlation between MAP2K7 levels and ivonescimab efficacy was assessed by BTC tumor cell-T cell co-culture and BTC organoids-T cell. At data cutoff, 1 patient achieved complete response and 19 patients achieved partial response yielding an ORR of 66.7% (95% Confidence Interval [CI]: 47.2-82.7). The disease control rate was 100%. The median progression-free survival (mPFS) was 8.5 months (95% CI: 7.6-10.5) and the median overall survival (mOS) was 16.8 months (95% CI: 11.1-22.5). Treatment-related adverse events occurred in 100.0% of patients with the most common being anemia (25, 83.3%), neutrophil count decreased (23, 76.7%), white blood cell count decreased (22, 73.3%), and platelet count decreased (22, 73.3%). No treatment-related deaths occurred. Additionally, exploratory proteomic and functional analyses identified MAP2K7 as a resistance-associated biomarker. MAP2K7 was upregulated in non-responders, and MAP2K7 suppression enhanced ivonescimab-mediated antitumor activity in immune co-culture models. Ivonescimab plus chemotherapy showed potential anti-tumor activity and tolerable safety as first-line treatment of aBTC patients. Exploratory analyses suggest that MAP2K7 may serve as a candidate biomarker of resistance and a potential therapeutic target for optimizing ivonescimab-based therapy of aBTC patients. Currently, the standard of care for first-line therapy in patients with aBTC is the addition of immune checkpoint inhibitors to chemotherapy based on the TOPAZ-1 and KEYNOTE-966 trials. However, this new regimen only improved OS by less than 2 months. The ORR of 66.7%, DCR of 100% and the median overall survival of 16.8 months were observed with ivonescimab plus chemotherapy. This study provides evidence supporting the potential role of ivonescimab plus chemotherapy as a first-line therapy for patients with treatment-naive unresectable locally advanced or metastatic BTC. NCT05214482 and NCT06048289. NCT05214482 and NCT06048289.

To compare five-year systemic complication risks in non-infectious uveitis (NIU) patients treated with systemic biologic versus conventional therapy. Multicenter retrospective clinical cohort study. Adult patients (≥18 years old) with NIU from the TriNetX Collaborative Network. We identified patients with NIU using International Classification of Diseases, Ninth and Tenth Revision, Clinical Modification codes. We assembled propensity score-matched comparisons: biologic therapy versus no systemic therapy (n=6,896 each), biologic versus conventional therapy with corticosteroids matched (n=5,994 each), between January 1, 2005, and January 1, 2024. A pre-specified subgroup analysis of Analysis 2 restricted to patients receiving combination biologic + conventional therapy versus conventional therapy alone (n=3,653 each) was also conducted. We employed time-to-event analyses to identify incidence of systemic complications. Incidence of serious infections, hematologic cytopenias, heart failure, thromboembolic events, diabetes mellitus, malignancy, psychiatric illness, all-cause hospitalization, and all-cause mortality. After propensity score matching, cohorts were balanced on all measured covariates (all standardized mean differences <0.1). Compared with no systemic therapy, biologic treatment was associated with higher five-year risks of serious infections, including pneumonia (hazard ratio [HR]: 1.41, 95% confidence interval [95% CI]: 1.20-1.67), sepsis (HR: 1.43, 95% CI: 1.16-1.77), and hematologic cytopenias (HR: 1.58, 95% CI: 1.44-1.74), representing 5-year Kaplan-Meier-estimated cumulative incidences of 6.43%, 3.87%, and 30.12%, respectively, in biologic-treated patients. Risks of heart failure, thromboembolic events, psychiatric illness, diabetes, and all-cause mortality did not significantly differ between groups. When compared with conventional immunomodulatory therapy, biologic treatment demonstrated a largely comparable five-year safety profile, with the primary exceptions of higher rates of hematologic cytopenias (HR: 1.28, 95% CI: 1.16-1.41), psychiatric illness (HR: 1.13, 95% CI: 1.01-1.27), and hospitalization (HR: 1.20, 95% CI: 1.07-1.35). In patients with NIU, systemic biologic therapy was associated with higher risks of serious infections and hematologic cytopenias compared with no systemic treatment, while demonstrating a broadly comparable five-year safety profile to conventional immunomodulatory therapy. These findings support guideline-concordant stepwise immunosuppression and underscore the importance of individualized risk assessment, infection surveillance, and hematologic monitoring for patients requiring systemic therapy.

Antibiotic de-escalation (ADE) is a key antimicrobial stewardship (AMS) quality indicator. We aimed to evaluate the rate and patterns of ADE among clinically stable patients with bloodstream infection (BSI). We analyzed secondary data from two prospective multicenter cohort studies, BLOOMY and BLOOMY-PREDICT. ADE was assessed among patients eligible for safe ADE on day 5 after index blood culture. Narrowing of antibiotic spectrum was determined by a ranking based on WHO AWaRe-classification. Risk factors for not performing ADE (non-ADE) were studied using multivariable logistic regression. In total, 937 of 3824 study patients (24.50%) were eligible, of which 218 (23.27%) did not have an option for de-escalation based on antimicrobial susceptibility testing. Of 719 patients in which ADE was feasible, only 406 (56.47%) received ADE. Empiric monotherapy (OR (95% CI) = 5.68 (3.77-8.56), p<0.001), Gram-negative pathogen (OR 2.27 (1.60-3.24), p<0.001), healthcare-associated infection (OR 1.55 (1.01-2.39), p=0.046), and hospital-acquisition (OR 1.63 (1.02-2.61), p=0.042) were identified as independent factors associated with non-ADE. Conversely, ICU treatment on day 0 (OR 0.60 (0.38-0.95), p=0.029) was independently associated with de-escalation, alongside a strong study center effect (OR 0.26 (0.15-0.44), p<0.001). Further, in-hospital mortality was not associated with ADE (46/406, 11.33% vs. 29/313, 9.27%, p=0.369). Low ADE rates in patients with urogenital focus and unnecessary carbapenem use were issues of particular concern in our cohort. Available opportunities for ADE were frequently missed in our setting, especially in Gram-negative BSI. AMS efforts should therefore be strengthened to promote ADE.

Although pulmonary arterial hypertension (PAH) is a rare and fatal disease that is well-characterized, vasodilator-responsive PAH accounts for a minority of cases, with little mechanistic knowledge, but with dramatically improved survival. By assembling national cohorts, we evaluated genetic influences on acute vasodilator drug response, a key determinant of the presence of vasodilator-responsive PAH. Differences between hemodynamics at rest and after a PAH-specific vasodilator were tested in a genome-wide association study. Validated loci were functionally tested in cell culture and in a hypoxic mouse model of pulmonary hypertension. Rs8057488 in the sorting nexin 29 (SNX29) gene reached genome-wide significance in the discovery cohort (P=4.00×10-8) and was nominally replicated (P=0.027). Consistent with its predicted function, SNX29 demonstrated an endosomal distribution in PA smooth muscle cells. Silencing SNX29 redistributed stromal interaction molecule proteins to the cell membrane and enhanced store-operated calcium entry. Over-expression of SNX29, in vivo, attenuated hypoxic vasoconstriction in isolated perfused murine lung models. The data cumulatively suggest SNX29 may contribute to vasodilation partly through reduced store-operated calcium entry and endosomal trafficking of store-operated calcium entry proteins, advancing our understanding of vasodilator-responsive PAH.

Cataract is among the leading cause of vision impairment worldwide and it is essential to uncover molecule-level characteristics of different cataract subtypes for personalized treatment. Compared to existing diagnostic techniques, surface-enhanced Raman spectroscopy (SERS) shows advantages in sensitive molecular fingerprinting, tractability and compactness, making it highly suitable for clinical adoption. To further address the current lack of SERS-based techniques for cataract subtyping, this work presented an integrated methodology for robust and interpretable metabolic profiling of aqueous humor. A two-step sample pretreatment protocol has been therein developed to efficiently extract metabolites while ensuring compatibility with subsequent SERS detection. SERSome strategy was employed to enable rapid and robust metabolic profiling. Molecule-level SERSome interpretation was achieved by matching its positive intra-correlation network with metabolites' SERS barcode. Multiple analytical methods including statistical comparison, interpretable machine learning, and association analysis have been leveraged, evidencing key metabolite biomarkers such as ergothioneine, hypoxanthine and uric acid associated with cataract subtyping and diabetic retinopathy. In a word, this study has not only established a systematic methodology for metabolic analysis of aqueous humor but also provided a usable tool for both fundamental research and clinical applications in different diseases with high rapidness, cost efficiency and scalability.

BACKGROUND: The field of environmental health sciences increasingly demands comprehensive and diverse data sets, particularly in response to emerging research areas such as climate change, mixtures, and exposomics. The data needed to address the complexity of environmental health research questions often extend beyond the boundaries of a single study or data resource. Traditional data management approaches struggle to harmonize the ever-expanding and heterogeneous data sources needed for research in the environmental health sciences. Harmonization may help address this issue as it involves aligning and standardizing various elements of data to allow comprehensive analysis, data pooling, and interpretation across studies. OBJECTIVES: The primary objective is to inform researchers about the transformative potential of embracing harmonization methodologies and to motivate contributions to ongoing efforts, thereby fostering advancements. METHODS: Using the Environmental Health Language Collaborative's Data Harmonization Use Case, we provide a practical illustration of existing data harmonization approaches, identify gaps, and emphasize future research and application directions. We selected two publicly available environmental epidemiology studies on the topic of childhood asthma and three studies on the topic of biomarkers of metals exposure during pregnancy and birth outcomes and applied several existing harmonization approaches to assess interoperability. DISCUSSION: Our process revealed the potential limitations of many existing harmonization approaches, with notable failures to identify common variables across independent data sets and lack of agreement between human and computer-based approaches. This use case identified various challenges with existing approaches, including reliance on often incomplete data documentation and large amounts of manual effort. To address these challenges, we recommend the continued advancement and dissemination of community data standards, the development of software and tools to facilitate harmonization through automation, and strategic efforts to promote engagement in data harmonization within the environmental health sciences community. Collaborative science is needed to advance our understanding of environmental contributors to health, and realizing the harmonization potential of our scientific data is a step toward improved collaboration.

Extracorporeal liver cross-circulation (ELC) using gene-edited porcine liver xenografts offers a potential bridge therapy for liver failure. We previously performed five ELC procedures in four brain-dead human decedents, during which the recipients developed severe thrombocytopenia. The porcine liver xenografts maintained their parenchymal structure, with immune cell infiltration and detectable IgM deposition on endothelial cells. Here, to investigate the underlying host-xenograft interactions, we performed longitudinal proteomic, lipidomic and metabolomic profiling of 64 blood samples, alongside spatial transcriptomics and histology of 25 porcine liver xenograft and 3 native human liver biopsies. Spatial transcriptomic analysis revealed progressive infiltration of human immune cells (predominantly inflammatory macrophages and neutrophils) in the xenografts, concurrent with the loss of porcine Kupffer-like macrophages and T cells. Distinct human and porcine complement dynamics were observed, with suppressed human but elevated porcine complement levels, accompanied by increased levels of porcine acute-phase proteins and coagulation factors. Moreover, multiomics analyses identified candidate cellular and molecular factors associated with thrombocytopenia. Human platelets colocalized with activated porcine endothelial cells, which showed increasing porcine vWF expression over time, as well as with immune cells (primarily macrophages and neutrophils) and hepatocytes in the xenografts. Integrative analyses indicated that ELC procedures provided hepatic support for apolipoprotein synthesis, bilirubin clearance, energy metabolism and detoxification, although circulating lipid levels remained low under anhepatic conditions. Collectively, these findings yield insights into the complex interplay between human and xenograft systems during ELC and inform strategies to improve liver xenograft biocompatibility for clinical translation.

Atypical fibroxanthoma (AFX), pleomorphic dermal sarcoma (PDS), and undifferentiated sarcoma, pleomorphic subtype (US) are rare malignant neoplasms of mesenchymal origin with many shared clinicopathologic features. AFX are usually superficial at time of diagnosis with low risk for recurrence or metastasis following surgical extirpation. In contrast, PDS and US display greater adverse histopathological features and subcutaneous invasion which portend higher risk for local recurrence, metastasis, and death. AFX and PDS form a biological spectrum defined by shared ultraviolet (UV)-signature mutations and arise on sun-exposed skin. Malignant fibrous histiocytoma (MFH), later termed undifferentiated pleomorphic sarcoma (UPS), has been repeatedly reclassified since the 1960s, leading to ambiguity in the literature. Although histopathology may overlap with AFX and PDS, US (former MFH/UPS) lacks UV-associated mutations, shows a complex karyotype, occurs on sun-protected areas, and is a diagnosis of exclusion once lineage-defined sarcomas are ruled out by ancillary studies. Due to the rarity of these tumors, definitive diagnostic, management, and surveillance guidelines have not been established. Multidisciplinary teams are recommended in more advanced cases. For unresectable, recurrent, or metastatic disease, adjunctive therapies including radiation, chemotherapeutics, and immunotherapies have been utilized with varying efficacy.

The United States (US) and European Union (EU) have long-established orphan drug regulations to incentivise the development of medicines for rare diseases. While the numbers of orphan approvals have risen rapidly, there is increasing discordance in regulatory outcomes between the US and EU. This discordance primarily stems from two sets of cases: US Food and Drug Administration (FDA) orphan approvals not authorised by the European Medicines Agency (EMA), and FDA orphan approvals with EMA authorisation but without orphan designation. We examined factors associated with these two sets of cases to understand the growing gap in orphan approvals between the US and EU. We collected data on FDA orphan drug approvals between 2011 and 2023 from the FDA Orphan Drug Designations and Approvals Database and their corresponding EMA regulatory status from EMA medicines database. We used descriptive statistical analysis to examine trends and identify discordance in outcomes between agencies. Univariable logistic regression assessed pre-specified factors associated with discordance, including therapeutic area (cancer/non-cancer), company size (large/medium/small), company headquarters location (US/EU/others) and approval period (2011-2016/2017-2023). The main methodological limitations are that the study identified associations but does not establish causality, with unmeasured factors potentially contributing to the observed discordance. Of 814 FDA orphan approvals, only 29% received corresponding EMA marketing authorisation with orphan designation. A further 38% were authorised by the EMA but without orphan status, while the remaining 33% were not authorised by the EMA. Compared with the 2011-2016 period, cases in the 2017-2023 period were associated with lower odds (OR 0.66 (95% CI [0.48,0.92]; p = 0.013)) of EMA marketing authorisation. Compared with cancer approvals, non-cancer approvals were associated with lower odds (OR 0.53 (95% CI [0.38, 0.75]; p < 0.001)) of having EMA marketing authorisation, but when authorised, were associated with higher odds (OR 2.36 (95% CI [1.50, 3.70]; p < 0.001)) of receiving orphan designation. Compared with large companies, orphan approvals from small and medium-sized companies were associated with lower odds (OR 0.45 (95% CI [0.28, 0.74]; p = 0.001) and (OR 0.29 (95% CI [0.20, 0.43]; p < 0.001)) of EMA marketing authorisation, but among authorised products they were associated with higher odds (OR 2.95 (95% CI [1.75, 4.99]; p < 0.001) and (OR 2.14 (95% CI [1.17, 3.90]; p = 0.014)) of orphan-designated marketing authorisation, respectively. EU companies were associated with higher odds (OR 1.69 (95% CI [1.17-2.43]; p = 0.005)) of receiving EMA orphan approvals compared with US companies. Between 2011 and 2023, regulatory outcomes for orphan drug approvals increasingly diverged between the FDA and the EMA, particularly for cancer indications and approvals sponsored by small US sponsors. Among FDA orphan drugs authorised by the EMA, many were not designated as orphan products because of regulatory differences, particularly regarding requirements around significant benefit and biomarker-defined sub-populations in oncology. FDA-approved orphan drugs that lack EU marketing authorisation may be withheld by companies not because of regulatory barriers but due to insufficient commercial incentives to launch in Europe, resulting in fewer treatment options for European rare disease patients. Our findings suggest that orphan incentives are not the primary driver of commercial EU-launch decisions and that recent EU regulatory reform of these incentives may not achieve their goal of improving access to therapy for rare diseases.

In hematopoietic cell transplant (HCT) patients, intestinal Candida parapsilosis expansion and translocation can cause life-threatening candidemia, yet whether commensal intestinal bacteria prevent Candida expansion remains incompletely defined. Here, we trained a machine learning model on supernatant metabolomic profiles of Lachnospiraceae to identify bacteria-derived inhibitors of fungal growth, identifying valeric and butyric acids as top hits. Fecal samples from HCT patients supported this association, with valeric and butyric acid levels inversely correlating with C. parapsilosis growth. In cell culture and mice, valeric acid inhibited C. parapsilosis growth by increasing intracellular acidification. Administration of glycerol valerate, or free or microencapsulated valeric acid, to release valeric acid along the entire intestinal tract blunted C. parapsilosis growth at murine intestinal sites where valeric acid was detected. Thus, machine learning identified a mechanistic driver of trans-kingdom ecology limiting C. parapsilosis intestinal expansion and may inform strategies to reduce patient risk of candidiasis.

In eukaryotic cells, genomic DNA is packaged into chromatin with nucleosomes formed by core histones H2A, H2B, H3, and H4, and further stabilized by the linker histone H1. During the early stages of retroviral infection, such as with murine leukemia virus (MLV) and human immunodeficiency virus type 1 (HIV-1), host core and H1 histones are rapidly deposited onto unintegrated viral DNAs upon nuclear entry. These unintegrated viral DNAs are transcriptionally silenced through histone posttranslational modifications (PTMs), including high levels of H3K9 trimethylation and low levels of H3 acetylation. Linker histone H1 is closely associated with chromatin compaction and histone PTMs, suggesting a potential role in regulating retroviral DNA fate. In this study, we demonstrate that simultaneous knockdown of four somatic H1 variants (H1.2, H1.3, H1.4, and H1.5) in K562 cells reverses the silencing of unintegrated HIV-1 DNA, resulting in increased viral expression. Notably, this effect was specific to HIV-1, as the same H1 depletion did not alter the silencing of MLV unintegrated DNA. Furthermore, inhibition of H3K9 methylation also relieved HIV-1 silencing, and H1 depletion reduced H3K9me3 deposition on HIV-1 unintegrated DNA. These findings indicate that H1 regulates HIV-1 unintegrated DNA expression by promoting H3K9me3 deposition, a mechanism that appears distinct from that of MLV.

Homoharringtonine (HHT) is a plant-derived protein translation inhibitor used widely in ribosomal profiling and as a treatment for chronic myeloid leukemia approved by the US Food and Drug Administration. HHT is commercially semisynthesized from the alkaloid core cephalotaxine (CET) extracted from endangered Cephalotaxus species. Despite its importance, the CET/HHT biosynthetic pathway remains unresolved. Here, we use paired untargeted metabolomics (stable-isotope precursor feeding) and transcriptomics to elucidate a near-complete CET biosynthesis. We show that, while CET is biosynthesized only in growing root tips, CET and HHT accumulate throughout the plant. We identify seven pathway intermediates and six enzymes-including cytochrome P450s, an atypical short-chain dehydrogenase and a 2-oxoglutarate-dependent dioxygenase-that enable carbon excision and CET/HHT pentacyclic backbone formation to produce cephalotaxinone, the likely direct precursor of CET. This study establishes a metabolic route to the HHT core and suggests a whole-plant coordination model, in which cephalotaxinone is produced in root tips and distributed for elaboration into HHT.

Millions of phage genomes have been mined from metagenomic data recently but the genome completeness remains poor because of the limitations of existing phage detection methods, which rely on metagenomic contigs that fragment phage genomes. Here, we present PALACE, a conjugate-graph-based framework for assembling high-quality phage genomes from metagenomes. PALACE incorporates homology-based and deep-learning-based methods to detect phage signals and constructs a conjugate graph from the metagenomic sample. On simulated data, PALACE generates accurate and complete phage genomes, achieving an F1 score of 0.92-1.00 across simulation settings, outperforming the second-best method by 0.21-0.48. Applying PALACE to 914 gut metagenomic samples from healthy controls and participants with colorectal cancer (CRC) yielded 5,306 high-quality phage genomes, outperforming the second-best benchmark method by 55.98% in median genome completeness. We observed a high degree of functional organization for genes within phage genomes. Phages from participants with CRC exhibited a notable enrichment of metabolic factors, suggesting their adaptation to nutrient availability in the CRC gut environment.

Methanethiol (MeSH) is a pivotal volatile sulfur compound in plant, human, and environmental chemistry, but its study is hindered by analytical challenges. Here, we present an accessible fluorometric two-component assay toolkit for quantitative assessment of its content and enzymatic degradation by methanethiol oxidase (MTO). The first assay sensitively detects MeSH with a limit of detection of 124 nM using a novel "tube-in-tube" design for efficient MeSH capture, circumventing the need for complex chromatography and expensive instrumentation. The second assay is a real-time kinetic MTO activity assay. Using this toolkit, we (i) established an inverse correlation between MTO activity and MeSH levels in a colon cancer cell line, (ii) quantified MeSH as a key scent marker in various types of cheese, (iii) evaluated MeSH release from the selected Brassica and Allium species and (iv) determined its production in aquatic systems. The MeSH metabolism toolkit presented here will provide researchers with simple, sensitive and specific assays to elucidate multifaceted roles of MeSH and MTO in human pathophysiology and to accelerate discoveries in a broad spectrum of scientific disciplines.

The gut microbiota governs immune checkpoint blockade responses, yet actionable strategies remain scarce. Here, we report that oat-β-glucan enhances anti-PD-1 (programmed cell death protein 1) efficacy in murine models by selectively expanding Faecalibacterium prausnitzii (F. prausnitzii). Combining anti-PD-1 with either oat-β-glucan or F. prausnitzii boosts intratumoral dendritic cell and CD8+ T cell infiltration and cytotoxic activation compared with anti-PD-1 monotherapy. Metabolomics identifies F. prausnitzii-derived butyrate and indole-3-propionic acid (IPA) as key mediators. Mechanistically, butyrate activates dendritic cells via the histone deacetylase (HDAC)8/H3K27ac/nuclear factor κB (NF-κB) p65 pathway, while both butyrate and IPA potentiate CD8+ T cell effector responses. In our colorectal cancer cohort undergoing anti-PD-1 treatment, higher baseline F. prausnitzii abundance and elevated plasma butyrate and IPA correlate with improved responses, a signature corroborated in independent external cohorts. A human intervention study confirms oat-β-glucan's safety, its ability to increase butyrate and IPA, and its capacity to modulate F. prausnitzii. Collectively, our work uncovers a microbiota-metabolite-immune axis whereby oat-β-glucan primes tumors for immunotherapy sensitization.

Actuators for soft bionic robotics, utilizing disease microenvironment-responsive and mechanically flexible materials, are influenced by intra- and intermolecular structures of hydrogels. However, they lack the required endogenous stimuli responsiveness necessary for applications like gastrointestinal-targeted drug screening and liquid/mass transport. Herein, a tubular conductive, reactive oxygen species (ROS)- and an near infrared (NIR)-responsive thermo-hydrogel actuator (THA) was fabricated for viscous liquid and solid transport. The presence of diselenide-crosslinked polymer dots (PDs) loaded onto polydopamine (PDA) nanoparticles (PD@PDA) in a poly(N-isopropylacrylamide) and polyvinyl alcohol (PNIPAM/PVA) matrix provided the necessary LCST alongside ROS- and NIR-responsive behavior. In the presence of high ROS, the diselenide bonds cleaved, resulting in release of PDA, generating NIR-specific actuation. The actuation process was confirmed by the directional movement of silicone oil (MR-200) and a small glass ball, which were selective to high ROS levels. The THA also demonstrated changes in electrical resistance (decrease in ΔR: ~ 14.60 kΩ), and stretchability (~ 127% increase in breaking strain), which may be used for screening-based applications. The THA demonstrated remarkable biocompatibility with Caco-2 (colon cancer) cells, alongside ROS scavenging and downregulation of TNF-α and IL-1β. Hence, this platform provides a viable pathway for fabricating bionic and biomimetic actuators for viscous liquid and solid transport.

While cellular atlases have revealed remarkable phenotypic diversity, how cells navigate this landscape to influence tissue behavior remains poorly understood. We present an alveolosphere screening platform for investigating interactions between lung stem cells and their fibroblast niche. We assessed the role of 201 candidate genes in stem cells via imaging, then used chimeric RNAseq analysis for a transcriptome-wide understanding of cell-autonomous effects on stem cells and non-cell-autonomous effects on the niche. This phenome-transcriptome map uncovered cellular states and pathways regulating proliferation, metabolism, and immune signaling. Notably, stem cells influenced scar-forming and immune programs in fibroblasts. This injury response was dependent on stem cell identity; loss of Nkx2.1, encoding the transcription factor conferring lung epithelial identity, rewired stem cell-niche interactions and had a greater non-cell-autonomous effect than eliminating the cancer genes Trp53, Egfr, or Cdkn2b. Our study highlights how functional atlases complement the cellular diversity revealed by descriptive methods.

Inhaled pathogens, pollutants and therapeutics interact with the dynamic architecture of the alveoli, yet how individual particles move and deposit at cellular resolution remains unclear. Here, utilizing the crystal ribcage platform, we track aerosol transport in ex vivo, actively ventilated lungs using real-time fluorescence imaging with single-particle resolution, capturing droplet trajectories, free-flight motion, impact orientation and deposition timescales within functional alveoli. These measurements show that intra-alveolar transport is directional and shaped by airway-guided flow and tissue motion. At larger scales, aerosols do not disperse uniformly throughout the lung's volume but instead concentrate into geometrically constrained clusters of alveoli, forming a conserved mosaic-like compartmentalization while neighbouring alveoli remain largely unexposed. The pattern persists across particle types and species and varies with particle properties and lung age. In models of emphysema, fibrosis and metastasis, airway remodelling alters both the geometry and amount of deposition. These multiscale insights reveal how single-particle transport and airway structure together shape alveolar exposure, immune activation, development and therapeutic accessibility.

Liver fibrosis can progress to cirrhosis and hepatocellular carcinoma, yet effective antifibrotic therapies remain limited. Fibroblast activation protein α (FAPα) is a promising biomarker and therapeutic target, but existing probes are constrained by limited tissue penetration and single-modality imaging, and effective FAPα-targeted antifibrotic agents remain lacking. Here we show that Cs-FAP, a hemicyanine-based near-infrared fluorescence/photoacoustic dual-modal probe, enables real-time and noninvasive visualization of FAPα activity in fibrotic liver tissue. Using a Cs-FAP-guided screening platform integrating high-content imaging and chromatographic purification, we screened 95 medicinal herbs and identified sappanchalcone (Sap) as a potent low-toxicity FAPα inhibitor. Sap markedly attenuated fibrosis progression and improved liver function in multiple liver fibrosis models using male mice. Mechanistically, Sap suppressed hepatic stellate cell activation through inhibition of the FAPα-PI3K-AKT signaling pathway. These findings establish Cs-FAP as a versatile platform for fibrosis imaging and drug discovery and identify Sap as a promising antifibrotic candidate.

Metabolic dysfunction-associated steatotic liver disease (MASLD), especially its severe form, metabolic dysfunction-associated steatohepatitis (MASH), progresses to liver fibrosis, leading to cirrhosis and liver cancer. The cross-talk between spleen and liver in MASLD/MASH progression remains poorly understood. Here we found enlarged spleens in patients with MASLD and identified induced TRNP1hiCD8+ T cells in spleens of MASLD/MASH mouse models and patients. These cells exhibited pro-fibrotic properties through secretion of INSR-α. Mechanistically, demethylated DNA and H3K27me3, increased H3K27ac and bolstered enhancer-promoter contact synergistically reorganized chromatin topologically associating domains spatially to initiate the expression of transcription factor TRNP1 in splenic CD8+ T cells. TRNP1 then transcriptionally activated the expression of FURIN and CTSD, promoting the maturation and ectodomain shedding of INSR-α and facilitating its secretion to activate hepatic stellate cells. In vivo blockade of INSR-α using neutralizing antibodies alleviated MASLD/MASH-induced liver fibrosis. This study reveals splenic TRNP1hiCD8+ T cells with pro-fibrotic properties and suggests a potential anti-fibrotic strategy.

Industrial volatile chemical products (VCPs), particularly those used in industrial coating applications, have emerged as major sources of reactive organic carbon (ROC) emissions with implications for atmospheric chemistry and human health. However, coating-related ROC emissions remain poorly constrained, limiting assessment of their environmental and health impacts. Here, we characterized full-volatility ROC emissions from representative industrial coating processes across gas and particle phases and estimated China's national ROC emissions from downstream coating processes at approximately 5.31 Tg yr⁻¹ in 2019. Volatile organic compounds (VOCs) dominated the characterized ROC mass in the high-volatility range, with aromatics (35.6%) and cyclic alkanes (20.4%) as major hydrocarbon groups, whereas oxygenated and functionalized organics increased at lower volatility ranges. However, emission mass alone did not reflect health relevance. Species prioritized by quantitative lifetime cancer risk (LCR) and hazard index (HI) assessment represented approximately 22.6% of the characterized ROC mass but dominated the calculated risk indicators. The top two cancer-risk contributors, 1,2-dichloroethane and trichloroethylene, accounted for 96.3% of the summed LCR while representing only 5.07% of the characterized ROC mass. In addition, a recurrently detected emerging-pollutant subset, including polycyclic aromatic hydrocarbons (PAHs), phthalates, phenolic antioxidants, and cyclic siloxanes, represented only ∼0.22% of the characterized ROC mass but was prioritized by complementary toxicity screening. These findings indicate a pronounced mismatch between emission mass and health hazard in coating-related ROC emissions, suggesting that conventional mass-based VOC metrics may underestimate health-relevant risks. A hazard-prioritized, tiered management framework is therefore proposed to support risk-oriented mitigation and more comprehensive full-volatility ROC regulation.

Inorganic phosphate (Pi) is an essential macronutrient, and tight regulation of Pi import and export is required to maintain Pi homeostasis. Although Pi import mechanisms have been extensively characterized, the molecular basis of Pi export has only recently begun to emerge. Arabidopsis PHOSPHATE 1 (PHO1) and its homologue PHO1;H1 are prototypical members of the SPX [Syg1/PHO81/xenotropic and polytropic retrovirus receptor 1 (XPR1)]-EXS (ERD1/XPR1/SYG1) family of Pi exporters conserved throughout eukaryotes, including the metazoan orthologue XPR1 and fungal SYG1. We review recent structural studies redefining XPR1 and PHO1;H1 Pi transporters as channel-like proteins using a double-lock gating mechanism controlled by inositol pyrophosphates. This new knowledge enhances our ability to modulate the activity of SPX-EXS proteins involved in human diseases, such as cancer, as well as enhance phosphate-use efficiency in crops.

Anti-tumor necrosis factor (TNF) therapy is widely used for inflammatory bowel disease, yet primary non-response and secondary loss of response remain challenges in clinical practice. In this study, we demonstrate that bacterial phosphoketolase improves the primary response to anti-TNF antibodies during induction therapy by enhancing Treg-mediated immunosuppression and maintaining higher serum drug concentrations. Mechanistically, phosphoketolase acts as a microbial host enzyme in macrophages, increasing phosphoketolase pathway flux and lactate production. Elevated lactate induces histone H4K12 lactylation, leading to the upregulation of the serotonin transporter, which mediates serotonin uptake for subsequent conversion to 5-hydroxyindoleacetic acid, a potential inhibitor of TNF-α-converting enzyme. This increases surface transmembrane TNF levels, thereby enhancing TNFR2 signaling in Tregs and promoting their proliferation and differentiation. In a prospective clinical trial, phosphoketolase-producing Bifidobacterium enhanced anti-TNF antibody efficacy during induction therapy. These findings support phosphoketolase-producing probiotics as an effective adjunct to anti-TNF therapy in inflammatory bowel disease.

Seroepidemiological assays play a crucial role in tracking immunity and infectious disease exposure across populations. Here we describe an automated assay platform that robustly quantifies antibodies against up to 100 antigens in a single reaction, using Luminex suspension array technology, alongside generating ample quality control measures. This automated multiplex serology workflow is an advancement of our previously developed manual and hybrid multiplex serology platforms. It combines multiple liquid handling systems (Biomeki7, BioTek 405 LS) and bead separation technology (KingFisher Flex) to improve scalability and reproducibility and facilitate its application in large-scale population-based cohorts. Study samples undergo automated processing, and antibody levels are quantified using the Luminex Flexmap3D instrument. A custom R-based application monitors process parameters and visualizes readouts in real time. Daily quality control samples allow subsequent normalization over extended periods of time and across reagent batches. Compared with previous assay versions, the higher degree of automation requires fewer laboratory operators and results in improved robustness and a more continuous throughput. Assay robustness is excellent, with median coefficients of variations between 2.8% and 11.7%, with no relevant batch effects over a 6-month period. Reproducibility across 47 antigens and 91 duplicate samples is remarkably high (R2 = 1.00). Using the automated platform, 1,820 study samples can be processed in a typical assay week. We have successfully completed the analysis for ~38,000 cohort samples, including comprehensive quality control data, in 88 assay days. Automated multiplex serology sets the foundation to generate high-quality serolomics data for large-scale cohorts in a reproducible, scalable and cost-effective fashion.

Highly pathogenic avian influenza A H5N1 has recently expanded its mammalian host range; in 2024, genotype B3.13 emerged in U.S. dairy cattle with pronounced mammary tropism. In the past, Influenza A virus immunology has been characterized primarily in respiratory infection models, whereas this study delineates immune responses after intramammary infection. Following intramammary H5N1 challenge of Jersey cows in the early dry-off period, an integrated characterization of dose- and compartment-specific (alveoli versus teat cistern) cytokine and chemokine profiles with peripheral leukocyte dynamics and H5- or N1-specific antibody responses was performed. The results indicate that infection-induced, quarter-restricted, monophasic inflammatory networks peak at 3-7 days post-infection, accompanied by coordinated peripheral myeloid expansion and IFN-γ-competent lymphocyte activation, and rising antibody titres across quarters.

Large-scale production of artificial spider silk fibers requires heterologous expression of spider silk proteins (spidroins), yet current methods remain limited by low yields and costly purification processes. To overcome these challenges, we engineered mini-spidroins in which the poly-alanine motifs of the repetitive region were replaced with the non-natural amyloidogenic β16 peptide, significantly enhancing expression yields and solubility. Furthermore, we developed a simple, chromatography-free purification method for these constructs based on NaCl-induced liquid-liquid phase separation (LLPS). This one-step purification strategy reduced processing costs by up to 99% compared to conventional affinity chromatography while achieving yields of ~300 mg of purified protein per liter of shake flask culture and ~25 g L-1 from bioreactor cultivations. The purified engineered mini-spidroins could be spun into continuous fibers using an all-aqueous, biomimetic spinning process triggered by a pH drop. The resulting fibers exhibited mechanical properties comparable to those produced from the mini-spidroin NT2RepCT, which requires conventional chromatographic purification. Together, our protein-engineering approach and LLPS-based purification method provide a potentially scalable, sustainable, and cost-effective platform for artificial spider silk, representing a major step toward the commercial viability of recombinant silk-based materials.

Indoleamine 2,3-dioxygenase (IDO), the enzyme responsible for tryptophan catabolism, is protective in many autoimmune and inflammatory diseases. We previously developed a localized immunomodulation approach through the fusion of IDO to a carbohydrate binding protein [E. Bracho-Sanchez et al., Nat. Biomed. Eng. 7, 1156-1169 (2023)]. Here we further develop IDO as a protein therapeutic for inflammation, investigating systemic delivery via conjugation of poly(ethylene glycol) (PEG) to IDO. PEGylation extended circulation time and treatment PEG-IDO demonstrated therapeutic efficacy in five autoimmune and inflammatory models. Treatment with a single dose of PEG-IDO resulted in reversal of hind limb paralysis in experimental autoimmune encephalomyelitis as model of multiple sclerosis; protected against hepatic damage in liver ischemia-reperfusion injury; prevention of abdominal aortic aneurysm; and decreased severity of imiquimod-induced psoriasis. Treatment with two doses of PEG-IDO provided maintenance of body weight and colon length in acute ulcerative colitis. PEG-IDO treatment increased regulatory T cell populations and reduced pathogenic Th17 cell populations and inflammatory cytokine production. Systemic delivery of PEG-IDO had little-to-no off-target effects. Mice maintained immunocompetency, clearing the Listeria monocytogenes infection, and had no observed toxicity. Additionally, PEG-IDO increased serum kynurenine, consistent with a kynurenine-mediated mechanism of action. These findings demonstrate systemic immunomodulation via circulating PEG-IDO ameliorates a breadth of disparate autoinflammatory diseases, protecting against inflammation-driven tissue destruction in spinal cord, liver, abdominal aorta, skin, and colon, while presenting a positive safety profile, and expanding the scope of potential applications for this therapeutic approach.

Autoimmune uveitis is a sight-threatening inflammatory disease, with the majority of entities driven by leukocyte infiltration into the retina. A critical early step in this process is the activation of retinal vascular endothelial cells (ECs), which up-regulate adhesion molecules that mediate T cell adhesion and subsequent extravasation. Here, we identify the small terpenoid compound costunolide (COS) as a potent suppressor of retinal endothelial inflammation and disease progression in experimental autoimmune uveitis (EAU). Quantitative proteomics of primary human retinal endothelial cells stimulated with TNF-α defined a proinflammatory endothelial signature and revealed induction of adhesion molecules. Screening of a focused library of 337 terpenoids uncovered COS as a top hit that markedly attenuated TNF-α-induced endothelial activation. In vivo, COS treatment significantly reduced clinical and histopathologic EAU scores, accompanied with reduced endothelial adhesion molecule expression and decreased T cell infiltration. Mechanistically, COS directly targeted deubiquitinase USP15, inhibiting USP15-dependent deubiquitination of TRAF1 and TNF signaling in retinal ECs. These findings establish COS as a candidate therapeutic agent for autoimmune uveitis and reveal a TNF-α-USP15-TRAF1 axis in retinal endothelium that can be pharmacologically exploited to limit pathogenic leukocyte trafficking.

Learning-related synaptic plasticity is regulated by metaplasticity, which adjusts plasticity thresholds in an activity-dependent manner. We have previously described a heterodendritic form of metaplasticity whereby priming stimulation in stratum oriens (SO) inhibits subsequent long-term potentiation (LTP) in the neighboring stratum radiatum of the hippocampal CA1 region. Here, we report that this metaplasticity is transregional, in that the SO priming stimulation also inhibits later LTP induction at dentate gyrus (DG) middle molecular layer (MML) synapses, both in vitro and in vivo. This effect operates across the hippocampal fissure and occurs in the absence of CA3, highlighting a previously unappreciated reverse-direction and long-distance hippocampal crosstalk. Our findings demonstrate an essential role of astrocytes as SO priming elicited an increase in the frequency of calcium (Ca2+) events in astrocytes in the DG MML, while the metaplasticity effect was blocked by calcium-buffering in MML astrocytes. It could be triggered by either activation of M1 muscarinic acetylcholine receptors or group II metabotropic glutamate receptors, and was critically dependent on inositol 1,4,5-trisphosphate receptor type 2 signaling. The transregional inhibition of LTP was mediated by astrocytic release of tumor necrosis factor (TNF), which likely acts in an autocrine fashion on astrocytic TNF type 1 receptors (TNFR1s). Downstream of TNF-TNFR1 signaling, the inhibition of MML LTP was mediated by the activation of GluN2B-containing N-methyl-D-aspartate receptors. Thus, a complex, bidirectional neuron-glia signaling cascade orchestrates long-distance metaplasticity across hippocampal subregions, providing a framework for understanding how hippocampal neuronal networks dynamically regulate plasticity thresholds across space and time.

Vimentin intermediate filaments play essential roles in maintaining cell integrity and regulating numerous cellular functions. In particular, vimentin cooperates with the actin cytoskeleton in key cellular processes that rely on actin dynamics, such as migration, division, and mechanosensing. While there is evidence that these two cytoskeletal components interact in cells, the underlying molecular mechanisms are only partially understood. Actin and vimentin can interact through biochemical signaling pathways or via cross-linkers, but whether they engage in a direct protein-protein interaction has remained controversial, in part because such interactions are difficult to isolate and characterize in cells. Using in vitro reconstitution coupled to theoretical modeling, and total internal reflection fluorescence microscopy to monitor the elongation of single actin filaments, we show that vimentin promotes actin elongation by stabilizing actin subunits at the barbed end in a dose-dependent manner. Strikingly, this effect depends on the nucleotide state of actin, as the acceleration is only observed for the elongation from adenosine triphosphate (ATP)-actin, and not adenosine diphosphate (ADP)-actin monomers. We further establish that neither the vimentin tail nor head domains are required for this effect, and both filamentous and nonfilamentous vimentin enhance actin elongation. Finally, we find that vimentin promotes the nucleation of actin filaments. Consistently, magnetic pull-down assays demonstrate a direct interaction between vimentin and ATP-actin monomers. Altogether, these findings identify vimentin as an unexpected new actor in the regulation of actin dynamics at the barbed end and bring new insights into the functional role of vimentin through cytoskeletal crosstalk.

Postnatal uterine development requires precise coordination of epithelial differentiation, gland formation, and stromal organization, yet how transcriptional programs are spatially integrated during this process remains poorly understood. Here, we generated a spatially resolved transcriptomic map of the mouse uterus across key postnatal stages from day 3 to day 21 by combining high resolution in situ transcriptomics with histology, proteomics, genetic models, and functional assays. This approach defines the temporal emergence and spatial arrangement of all major uterine cell types and demonstrates that endometrial glands arise from luminal epithelium through progressive transcriptional reprogramming rather than from a prespecified progenitor population. Spatial analysis reveals marked compartmentalization of signaling pathways during adenogenesis. Luminal epithelium is enriched for Wnt ligands and hormone receptors, whereas glandular epithelium engages inhibitory and negative feedback programs that constrain Wnt activity while supporting proliferation and differentiation. Functional genetic studies establish that epithelial canonical Wnt signaling is essential for prepubertal gland establishment and long-term epithelial maintenance but is dispensable for postpartum gland regeneration. Retinoic acid synthesis becomes increasingly concentrated within glands, while hedgehog signaling polarizes into an epithelial to stromal axis during gland morphogenesis. Additional pathways including RTK, Notch, TGFβ, BMP, hypoxia, Hippo, and PI3K-mTOR exhibit distinct spatial biases that collectively shape epithelial identity and tissue maturation. Together, these findings provide a comprehensive spatial framework for uterine gland development and reveal how compartment specific signaling networks coordinate postnatal uterine morphogenesis.

Engineering organized microvascular networks remains a critical challenge in tissue engineering and regenerative medicine. While biochemical approaches for patterning angiogenesis via growth factor delivery have shown promise, their inability to pattern sustained growth factors with spatiotemporal control limits effectiveness. Here, we demonstrate that dynamically patterned mechanical forces enable precise spatiotemporal control over angiogenic sprouting. We developed a magnetically actuated human vessel-on-a-chip platform that integrates a perfusable endothelialized microchannel within a collagen matrix and allows noninvasive and tunable mechanical stimulation across three spatial dimensions and time (4D). Using an automated 3-axis actuator, we systematically investigated how strain magnitude, frequency, and direction modulate endothelial cell behavior and vessel morphogenesis. Dynamic mechanical stimulation at physiological strain magnitudes (5 to 15%) enhanced endothelial alignment and barrier function while promoting angiogenesis in a strain magnitude-dependent manner: lower dynamic strain (5%) maximized sprout initiation, whereas higher dynamic strain (15%) promoted elongation of sprouts. Sequential reorientation of strain direction reprogrammed sprouting trajectories along X, Y, and Z directions, generating complex sprout geometries such as L-shaped branches. RNA sequencing revealed mechanically induced transcriptional profiles distinct from unstimulated controls, characterized by upregulation of genes associated with angiogenesis, mechanotransduction, and extracellular matrix remodeling. Functional perturbation of PIEZO1 reduced strain-induced sprouting without altering barrier function, indicating that dynamic mechanical stimulation engages multiple mechanotransduction pathways to regulate angiogenesis. Collectively, these findings establish a strategy for spatiotemporally controlled angiogenesis through 4D force patterning to program vascular morphogenesis while preserving function. This approach provides a foundation for engineering hierarchically organized vascular networks for tissue regeneration.

Somatic expansion of the HTT CAG repeat is a key feature of Huntington's disease (HD) pathogenesis. Mismatch repair (MMR) enzymes drive this process through erroneous DNA repair, with variants in MMR genes modifying the onset and progression of disease features. Cell-type-specific CAG repeat sizing recently confirmed that elevated somatic expansion underlies the selective vulnerability of HD medium spiny neurons, with expansion beyond certain CAG thresholds associated with distinct stages of cellular pathogenesis. In this review, we synthesise insights from post-mortem brain tissue, cell systems, and mouse models, detailing key CAG repeat-length-dependent changes. In addition, we critically evaluate the MMR proteins MSH3, MLH3, and PMS1 as therapeutic targets for slowing somatic expansion and outline key safety considerations for emerging MMR-modulating approaches.

Endocrine tissues must continuously adjust their structure and function in response to circadian cues, metabolic state, and physiological stress. Within these dynamic systems, stem cells contribute not only to tissue maintenance and repair but also to functional plasticity. The hypothalamic-pituitary-adrenal (HPA) axis exemplifies this integration of local stem cell regulation with systemic endocrine control. In this Review, we highlight the shared regulatory principles and tissue-specific adaptations of stem cell populations across the hypothalamus, anterior pituitary and adrenal gland. We discuss tanycytes as hypothalamic stem cells, SOX2+ stem cells as key regulators of pituitary plasticity, and multipotent and lineage-restricted progenitors that sustain adrenal cortical and medullary renewal. Across HPA axis organs, conserved signaling pathways and multiple modes of cell-cell communication, including paracrine and feedback mechanisms, coordinate stem cell behavior with endocrine output. Importantly, we present growing evidence that endocrine stem cells influence organ function beyond direct cell replacement, shaping tissue responsiveness, and adaptation to physiological demand. Disruption of these processes contributes to endocrine dysfunction and tumorigenesis. Integrating insights from genetic models, human single-cell and spatial atlases, we outline how viewing the HPA axis as an integrated stem cell system provides a framework for understanding endocrine plasticity and identifies emerging opportunities and challenges for regenerative and therapeutic strategies.

Heart failure with preserved ejection fraction (HFpEF) continues to increase in prevalence and has limited treatment options. HFpEF is a systemic condition with a broad phenotype including diastolic dysfunction, pulmonary oedema, exercise intolerance, and left ventricular hypertrophy, collectively resulting in enhanced morbidity and mortality. The transcription factor RUNX1 has recently been identified as a mediator of pathological changes in multiple cardiac diseases; however, its role in HFpEF remained unknown. Here, we show that inhibition of Runx1 limits adverse cardiac remodelling in a mouse model of HFpEF. Cardiomyocyte-specific tamoxifen-inducible Runx1-deficient mice with HFpEF are protected, with preservation of diastolic function, and attenuation of pulmonary oedema, exercise intolerance, and hypertrophy. Furthermore, targeting Runx1 in HFpEF by using gene transfer or small-molecule inhibitor Ro5-3335 improves diastolic function and reduces pulmonary oedema, both in female and male mice. Overall, this work enhances our understanding of RUNX1 in cardiac disease and presents a novel translational target for the treatment of HFpEF.

Interferon (IFN) alpha (IFNα) and lambda3 (IFNλ3) constitute first-line responses of immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by increasing interferon-stimulated genes (ISGs). Prolonged IFN production may exacerbate inflammation, contributing to endotheliitis and vascular dysfunction in coronavirus disease 2019 (COVID-19). We investigated whether spike protein S1 (SP1) of SARS-CoV-2 via IFN influences inflammation in human vascular and lymphatic endothelial cells (ECs) and whether these processes contribute to vascular dysfunction in the context of hypertension. We focused on ISG15, a crucial immune protein that is also implicated in hypertension-associated vascular injury. Exposure of microvascular ECs to SP1 of SARS-CoV-2-induced expression of ISGs: ISG15, MX1, and IFIT1. These effects were potentiated by IFNs and reduced by ADAM17 and STAT1 inhibition and genetic inhibition of IFNα and beta receptor subunit 1 (IFNAR1). In microvascular ECs IFNλ3 and IFNα increased expression of ISGs, TMPRSS2, ADAM17, production of pro-inflammatory mediators (tumor necrosis factor [TNF]α, interleukin [IL]-6, plasminogen activator inhibitor [PAI]-1) and reduced phosphorylation of eNOS (Ser1177). In pulmonary, lymphatic, and aortic ECs, IFNα, but not IFNλ3, increased expression of ISGs and IL-6. To explore the relevance in intact vessels, effects of IFNs were studied in isolated micro-vessels from wildtype (WT), hypertensive and ISG15-/- mice. IFNα, IFNλ3, and SP1 reduced endothelium-dependent relaxation in WT vessels, whereas IFNα increased contraction in vessels from hypertensive mice. Vascular dysfunction induced by IFNα, IFNλ3 or spike protein was abrogated in vessels from ISG15-/- mice. SP1 and IFNs synergically increase EC expression of ISGs through ADAM17. IFNλ3 and IFNα promote endothelial inflammation and vascular dysfunction through ISG15. These processes may play a role in the endotheliopathy and vascular damage associated with SP1 and might contribute to cardiovascular sequelae, including hypertension, of SARS-CoV-2 infection.

Musculoskeletal diseases are a major health burden. Development of bone-active therapies has been hindered by limited understanding of the cells and genes that regulate the skeleton. We exploited the value of cross-species analysis and developed single-cell methodologies in skeletal tissues to define the critical endosteal compartment that regulates bone turnover. Thirty-four distinct cell types were identified, and disease-relevant cells prioritized using enrichment for rare skeletal disorder genes and bone-mineral-density-associated genes in an extended UK Biobank genome-wide association study. Functional validation was undertaken in over 1,000 genetically modified mouse models. Endothelial cells and vascular smooth muscle cells were identified as new skeletal-disease-relevant cells alongside osteoblast, chondrocyte and osteoclast cell lineages. Hundreds of cell-specific genes with unappreciated roles in skeletal pathophysiology were identified. This comprehensive cellular and molecular framework underpins skeletal physiology and disease and will help prioritize new therapeutic targets to accelerate development of therapies to treat musculoskeletal disease.

Adenine base editors (ABEs), which enable A•T-to-G•C base editing, have emerged as a powerful tool with potential therapeutic applications. However, conventional ABEs suffer from bystander nucleotide conversions, limiting their utility for precise editing. Here we present a single-nucleotide resolution ABE (snuABE) created by fusing a nickase Cas9, nCas9-H840A, with the deaminase domain of ADAR (adenosine deaminase acting on RNA), which acts on DNA:RNA hybrids, instead of TadA, which acts on single-stranded DNA in conventional ABEs. snuABE requires a target-adenine guide RNA (tagRNA) that introduces a mismatch at the target adenine, enabling highly specific A-to-G editing by ADAR. Engineering ADAR from Pediculus humanus using the in silico protein evolution algorithm EvolvePro, along with 3'-end protection of the tagRNA, enhanced snuABE activity, yielding a median efficiency of 5.4% and a maximum efficiency of 50.0% across 32 targets in HEK293T cells. snuABE exhibits no detectable DNA off-target editing at predicted off-target or orthogonal R-loop sites, highlighting its potential as a precise and safe base-editing technology.

Early embryogenesis is accompanied by dynamic epigenetic modifications. Although such dynamics are important in cell intrinsic regulation of gene expression, their extrinsic roles in mediating intercellular communication during early embryogenesis are less understood. Here, using the dTAG system, we reveal previously underappreciated stage-specific functions of PRC2 in regulating preimplantation and primordial germ cell (PGC) development. We demonstrate that PRC2 plays important roles in regulating maternal-to-zygotic transition and epiblast formation. By systematically analysing H3K27me3 and H3K4me3 dynamics, we redefine the timing of bivalency establishment and uncover a stepwise mechanism governing bivalency acquisition in early embryogenesis. Moreover, PRC2 regulates proper PGC numbers in the epiblast by controlling Esrrb expression in the extraembryonic ectoderm. Thus, our study uncovers a previously unknown cell-autonomous function of PRC2 in preimplantation development and its non-cell-autonomous impact in PGC number regulation, both through interplays between epigenetic-epigenetic and epigenetic-transcription factors networks.

Neuronal subtype-specific synaptopathy is a hallmark of many forms of neurodegeneration. We examined the cellular basis for synaptic vulnerability in the auditory system, where three subtypes of spiral ganglion neurons (SGNs)-Ia, Ib, and Ic-carry acoustic information from the cochlea to the brain. In response to noise and aging, a subset of synapses between inner hair cells and SGNs are lost, but it is unclear how this loss varies across SGN subtypes. Using genetic labeling, we showed that Ia SGNs have larger post-synaptic densities (PSDs) than Ib and Ic SGNs and are the most resilient subtype. Ia PSD volumes increase with age and are unchanged after noise exposure. By contrast, average Ib/Ic PSD volumes do not change with age but decrease with noise. Genetic reprogramming of Ib/Ic neurons to a Ia-like identity provides significant protection against noise-induced synaptopathy, linking identity to resilience and providing an entry point for therapeutics.

Toll-like receptor (TLR) signaling is critical for innate immune system. However, whether it is directly modulated by microbiota-derived metabolites remains unclear. Here, we show that the short-chain fatty acids (SCFAs) propionate and butyrate suppress TLR signaling by directly binding p38α MAP kinase, promoting its interaction with TAB1, thereby activating p38α via autophosphorylation. Activated p38α then phosphorylates TRAF3 at serine 85, inhibiting K63-linked polyubiquitylation of TRAF3 and disrupting TBK1-IRF3 activation, leading to reduced macrophage activation and intestinal inflammation. In ulcerative colitis patients, fecal levels of propionate and butyrate positively correlate with p38α activity and TRAF3 S85 phosphorylation, but inversely correlate with TBK1 activation, and cytokine levels. Notably, oral administration of propionate in three patients with ulcerative colitis markedly improved intestinal inflammation and clinical symptoms. These findings reveal p38α as a direct sensor for microbiota-derived SCFAs that suppress TLR signaling through nonmetabolic functions of propionate and butyrate, providing the first clinical evidence that propionate supplementation represents a practical dietary strategy for ulcerative colitis management.

CD46, a human-specific complement receptor, regulates gene programs essential for T helper 1 (TH1) cell differentiation, yet how it exerts direct transcriptional control remains unclear. We show that the CD46 signaling domain cytoplasmic tail 1 (CYT-1) engages the transcription factor Sp1 in human CD4 T cells to dynamically modulate Sp1-DNA interactions. Beyond promoting TH1 cell induction, CD46-Sp1-controlled programs support naive CD4 T cell survival by maintaining nutrient transporter expression and suppressing the intrinsic caspase 9-caspase 3 apoptotic pathway. The CD46-Sp1 axis also restrains HIV transcription in infected CD4 T cells in vitro. Disruption of CYT-1-Sp1-regulated programs identifies T cells from individuals with HIV who exhibit incomplete viral suppression during antiretroviral therapy. Together, these findings define a human-specific transcriptional mechanism linking complement signaling to metabolic adaptation, apoptosis regulation, and antiviral defense, highlighting an unexpected role for CD46 in coordinating T cell homeostasis and host protection.

The repair of critical-size bone defects remains clinically challenging, largely owing to the limited homing and osteogenic capacity of endogenous bone marrow mesenchymal stem cells (BMSCs). To address this, we developed a cell-free strategy that combines a decellularized fish scale (DC-FS) scaffold with stem cell sheet-derived extracellular vesicles (CS-EVs). This design leverages the scale's innate microgrooved topography for directional cell guidance while loading CS-EVs to provide sustained pro-osteogenic cues. The resulting CS-EVs/DC-FS composite retained the intrinsic micro/nanoarchitecture and mineralized composition of the biological scaffold and permitted controlled EVs release. The scaffold promoted directional migration, adhesion, and osteogenic differentiation of BMSCs in vitro, upregulating key markers including RUNX2, OPN, and BMP2. Mechanistically, EV-derived miRNAs, notably miR-146a-5p, were associated with activation of the Sirt1/Wnt/β-catenin signaling pathway. In a rat critical-size calvarial defect model, CS-EVs/DC-FS accelerated bone deposition, improved trabecular microstructure, and enhanced overall regeneration quality. This study establishes a topography-guided and EV-mediated strategy for bone regeneration, offering an integrated nature-derived solution for reconstructing challenging maxillofacial and orthopedic bone defects.

Low-cost, safe materials that absorb and emit light at near-infrared (NIR) and shortwave infrared (SWIR) wavelengths are of interest for optical communications, sensors, and diagnostics. One promising avenue to access the NIR/SWIR regions is to self-assemble chromophores into J-aggregates. However, the molecular design of two-dimensional (2D) or tubular J-aggregates with NIR and SWIR emissions is challenging, as small monomeric changes can drastically affect the resulting aggregates' morphologies and photophysical properties. Here, by synthesizing four new heptamethine benzothiazole dyes with increasing steric bulk on the polymethine chain, we rationally design SWIR-emissive 2D J-aggregates. Adding steric bulk drastically changes the self-assembled mesophases and photophysical properties, leading to an unprecedented 3-4 distinct aggregates for each dye. We isolate a total of thirteen new NIR/SWIR aggregates, out of which nine exhibit room-temperature emission. Excitingly, one of the aggregates also shows a tubular morphology─the first SWIR tubular aggregate characterized. A series of interconversion studies, structural characterizations, and modeling provide insights into the molecular packing arrangements of each mesophase. This thorough understanding of the J-aggregate mesophases allowed us to perform four-color imaging using a single chromophore scaffold by selective excitation of the monomeric fluorophore and distinct aggregate structures. Overall, this work demonstrates a rational design strategy for accessing NIR/SWIR emission via supramolecular aggregation and highlights opportunities in energy transport and multiplexed imaging derived from this fundamental understanding of long-wavelength J-aggregates.

Retinal degenerative diseases are a leading cause of irreversible blindness. Their pathogenesis is intricately linked to oxidative stress-induced dysfunction of retinal pigment epithelial (RPE) cells and subsequent retinal degeneration. Macroautophagy/autophagy, a critical cellular degradation pathway, plays a vital role in maintaining RPE homeostasis, yet its dysregulation in retinal degenerative diseases remains poorly understood. In this study, we observed that sodium iodate (NaIO3), an oxidative stress inducer, triggered lysosomal dysfunction via lysosomal membrane permeabilization (LMP), thereby impairing autophagic flux in RPE cells and exacerbating retinal degeneration. RNA sequencing identified LAMP3 (lysosomal-associated membrane protein 3) as a downregulated gene following NaIO3 treatment. Functionally, LAMP3 overexpression alleviated NaIO3-induced LMP, improved lysosomal function, and alleviated autophagic impairment. Furthermore, upregulation of LAMP3 reduced oxidative stress and apoptosis in RPE cells, while alleviating retinal degeneration in a NaIO3-induced mouse model. Mechanistically, our data suggested that NaIO3 upregulated the transcription factor SNAI1, which acts as a transcriptional repressor of LAMP3. SNAI1 knockdown increased LAMP3 expression, thereby facilitating the recovery of lysosomal function and the alleviation of autophagic impairment. Collectively, our findings indicate that the SNAI1-LAMP3 axis contributes to the regulation of the autophagy-lysosomal pathway in retinal degeneration, highlighting a potential therapeutic target for delaying disease progression.Abbreviations: AMD: age-related macular degeneration; AO: acridine orange; Baf A1: bafilomycin A1; BAX: BCL2-associated X protein; BCL2: B cell leukemia/lymphoma 2; BSA: bovine serum albumin; CCK-8: cell counting kit-8; ChIP: chromatin immunoprecipitation; CM-H2DCFDA: chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DEGs: differentially expressed genes; DHE: dihydroethidium; EdU: 5-ethynyl-2'-deoxyuridine; ERG: electroretinography; GSEA: gene set enrichment analysis; H&E: hematoxylin and eosin; HsRPE: human primary retinal pigment epithelial; JC-1: 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; LAMP3: lysosomal-associated membrane protein 3; LGALS3: lectin, galactose binding, soluble 3; LLOMe: leu-leu methyl ester; LMP: lysosomal membrane permeabilization; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NAC: N-acetyl-L-cysteine; NaIO3: sodium iodte; NC: negative control; OCT: optical coherence tomography; PCA: principal component analysis; PI: propidium iodide; qRT-PCR: quantitative real-time polymerase chain reaction; Rapa: rapamycin; ROS: reactive oxygen species; RP: retinitis pigmentosa; RPE: retinal pigment epithelium; RPE65: retinal pigment epithelium 65; siRNA: small interfering RNA; SNAI1: snail family zinc finger 1; SQSTM1/p62: sequestosome 1; TJP1/ZO-1: tight junction protein 1; ZNF135: zinc finger protein 135.

Mechanical stimulation can remodel the physiological mechanical microenvironment of cardiomyocytes and influence their maturation and injury progression. However, the dynamic process by which cardiomyocytes transition from adaptive maturation to injury remains unclear, because conventional endpoint assays cannot continuously track cellular states. Here, we developed a heart-on-a-chip platform that reconstructs physiological strain and fluidic microenvironments. The chip integrates traction force microscopy (TFM) and a cardiac troponin I (cTnI) biosensor, enabling synchronous monitoring of cardiomyocyte contractility and injury-associated phenotypes. Under physiological cyclic stretch (20% strain), the platform induced a mature cardiomyocyte phenotype, with the gap junction protein CX43 and cytoskeletal protein F-actin increasing by 1.22- and 2.03-fold, respectively. Through 14 days of dynamic mechano-chemical monitoring, we identified 25% strain as a critical turning point in cardiomyocyte mechanical responses. Below this threshold (5%-20%), mechanical stimulation promoted cellular maturation and enhanced contractile stress, whereas above this threshold (30%), it induced an injury-associated phenotype. This heart-on-a-chip provides a controllable and quantitative in vitro platform for optimizing mechanical stimulation windows, evaluating cardiomyocyte injury, and screening cardioprotective therapeutics.

Obtaining a precise genetic tuberous sclerosis complex diagnosis is a challenge as many missense TSC2 variants are variants of uncertain significance. Variants of uncertain significance in TSC2 have been resolved by one-at-a-time functional assays, but these assays cannot scale to the 3634 TSC2 missense variants of uncertain significance observed so far. To address this challenge, we use massively parallel sequencing to measure the steady-state abundance of almost 9000 TSC2 missense variants and develop an mTOR pathway activity assay using genome editing and cell sorting to generate activity scores for 391 missense variants. We observe that 1256 of 8864 (14.17%) missense variants assayed have altered TSC2 abundance, and 69 of 391 (17.65%) missense variants assayed have altered mTOR pathway activity. Calibration and integration of these data into classification of variants identified in a clinical cohort putatively reclassifies 212 of 276 (76.8%) TSC2 missense variants of uncertain significance. These datasets will lead to improved genetic diagnosis of tuberous sclerosis complex with potential positive impacts on the clinical management of patients and their families.

Chronic neuroinflammation gives rise to diverse microglial states across the brain, yet how region-specific microglial remodeling contributes to cognitive dysfunction remains unclear. Here we report that synapse-engulfing microglia in the thalamus drive cognitive impairment after cortical brain damage in mice, primarily studied in females. Region-specific manipulations of microglia during the chronic phase show that reactive microglial changes in the thalamus, but not in the hippocampus, impair recognition memory. Single-cell RNA sequencing reveals an enrichment of synapse-engulfing CD9hi microglia in the thalamus. Antibody-based CD9 blockade in the thalamus, as well as microglia-selective CD9 disruption, rescues thalamic synaptic loss, restores neuronal activity, and improves recognition memory. Further analysis shows that the blood-brain barrier disruption and subsequent γ-immunoglobulin (IgG) extravasation facilitate the generation of CD9hi microglia in an Fcγ receptor III-dependent manner. These findings demonstrate that the induction of synapse-engulfing CD9hi microglia in the thalamus by IgG/FcγRIII signaling drives recognition memory deficits following cortical damage.

In settings of persistent (self or foreign) antigen, such as autoimmunity and chronic infection, immune responses are sustained by stem-like T cells. Although TCF1 has emerged as a key transcription factor (TF) associated with stemness, the TCF1hi population is heterogeneous, raising the question of whether TCF1 exclusively defines the stem T cell (TSC) pool. Using preclinical models of autoimmune type 1 diabetes and chronic infection, we discover that a small subset of TCF1hi T cells express the TF LEF1. LEF1+ TCF1hi T cells define a true self-renewing TSC pool. TSC give rise to LEF1- TCF1hi progenitor T cells (TPRO), which lack stem functions and generate terminally differentiated TCF1lo T cells (TDIFF) (TSC→TPRO→TDIFF). We show that LEF1 is essential for T cell stemness. Autoimmune and exhausted LEF1+ TSC share a unique epigenetically encoded core program enriched for genes and pathways characteristic of embryonic and adult stem cells, including WNT/β-catenin and Notch signaling. Spatial positioning, niche signals, and migration regulate stem-cell fate; accordingly, targeting integrins or Notch signaling impairs T cell stemness and prevents disease. Our studies identify LEF1 and niche-derived factors as fundamental regulators of T cell stemness across chronic diseases.

Mammalian eyes are exposed to visible light but cannot perform photosynthesis. Here, we show that introducing a nanoscale, structurally and functionally preserved thylakoid system, LEAF (light-reaction enriched thylakoid NADPH-foundry), into corneal cells enables light-driven bona fide photosynthetic production of NADPH and ATP, similar to plant leaves, which alleviates oxidative stress and inflammation. LEAF acts in two domains. Intracellularly, it integrates with host cells to supply NADPH and ATP via intact photosynthetic electron transport, restoring redox balance. Extracellularly, photosynthesized NADPH enhances endogeneous antioxidant enzyme activity and reduces reactive oxygen species in the local environment. These results establish a strategy for using light as an energy input in mammalian metabolic systems and suggest a possible cross-kingdom, endosymbiosis-like interaction in which animal cells derive functional benefits from plant-derived photosynthetic neo-organelles.

The process of nonocclusive thrombus formation is well known, but the mechanism keeping the thrombus silent at the end stage remains unclear. The aim of this work was to evaluate the role of fibrin in limiting further growth of a thrombotic remnant. Intravital microscopy showed that attachment of platelets to a fibrin-rich thrombus stopped after partial thrombus disaggregation, indicating that the thrombus activation potential is lost, a stage we named the stillness phase. Histological analyses showed that 80% of the internal cross-section area of the thrombus remnant is bordered by fibrin, whereas 20% of the superficial thrombus area was covered only by a few platelet layers, suggesting a role of fibrin in limiting platelet recruitment. This result was confirmed in a flow-based assay in which fibrin-rich thrombi recruited circulating platelets inefficiently as compared with fibrin-poor thrombi. Moreover, we found that in vitro, lysis of fibrin with recombinant tissue plasminogen activator (rtPA) released active thrombin. This observation was confirmed in vivo because treating a thrombus with rtPA to promote fibrin breakdown during the stillness phase resulted in the release of thrombin, leading to an unexpected regrowth of the thrombus. This finding was further supported by the dynamics of thrombus formation in FgaEK mice, which displayed repeated cycles of thrombus growth and detachment after vessel injury, with an inability to reach the stillness phase, accompanied by the continuous release of active thrombin. Altogether, these findings identify a novel role of fibrin in maintaining an end-stage thrombotic remnant in an inactive state.

Intercellular mitochondrial transfer is pivotal in both healthy and pathological states. Supplementing healthy mitochondria is emerging as a promising therapeutic approach for various diseases. Non-immunogenic edible plants, which contain mitochondria, offer a novel avenue for such therapies. Mitochondria were isolated from several commonly consumed edible plants (P-Mit) using differential centrifugation followed by sucrose gradient ultracentrifugation. The distribution of P-Mit, particularly in the brain, was examined with a mitochondrial membrane-potential dye and an imaging system. As a proof of concept, the molecular interactions underlying turmeric-derived mitochondria (T-Mit) uptake by microglia were elucidated through affinity precipitation coupled with mass spectrometry. By labeling with gold-nanoparticles in a distinct triangular or spherical shape followed by electron microscopy and energy dispersive spectroscopy analysis, we demonstrated the physical fusion of T-Mit and animal mitochondria in microglia. Mitochondrial functions such as superoxide levels, ATP-linked mitochondrial respiration, glycolysis and electron transport chain activity were assessed to determine the impact of T-Mit on aging-related microglial dysfunction. Next-generation small RNA sequencing revealed the underlying mechanism by which T-Mit-derived small RNAs modulate the expression of NADH dehydrogenase (ND) genes in microglia. Orally administered T-Mit travelled from the gut to the brain in aged male mice, where they fused with microglial mitochondria (M-Mit), reprogramming M-Mit energy metabolism and reversing aging-related cognitive dysfunction. Specifically, T-Mit was taken up by microglia via the phagocytic receptor TREM2. Subsequently, T-Mit fused with M-Mit in a mitofusin 1-dependent manner. The T-Mit microRNAs Tae-miR319 and Osa-miR166a-3p then integrated into M-Mit, inhibiting the expression of complex I subunits ND4 and ND5. This inhibition alleviated reverse electron transport (RET) at complex I, reducing reactive oxygen species (ROS) production and facilitating ATP production, ultimately rescuing aging-related cognitive decline. Data from elderly human subjects also showed overactivation of the RET process and overproduction of ROS, accompanied by low ATP levels in microglia. Our findings fundamentally alter our understanding of the regulation of mammalian mitochondrial biology by P-Mit and may lead to P-Mit-based transfer therapy for preventing or treating human mitochondrial disorder-related diseases.

The skin is composed of multiple fibroblast subpopulations with different functions in homeostasis and repair, but their role in skin diseases is largely unknown. Junctional epidermolysis bullosa (JEB) is a hereditary skin disorder characterised by severe skin fragility and aberrant granulation tissue formation, caused by loss-of-function variants in basement membrane proteins, including laminin-332. We developed JEB-like organotypic (OT) cultures with distinct fibroblast subpopulations and explored their role in an inducible JEB in vivo disease model, mimicking key features of the human disease. Mechanistically, papillary fibroblasts are highly increased in the granulation tissue of blistered JEB skin, promoting pathological αvβ6 integrin and TGFβ signalling in JEB keratinocytes. Treatment with the TGFβ receptor inhibitor RepSox not only normalised aberrant cell proliferation, differentiation, and cytokine signalling in JEB OTs but also reduced aberrant granulation tissue formation and skin blistering in laminin-332-depleted mice. Collectively, our study reveals that papillary fibroblasts promote JEB pathogenesis through increasing αvβ6 integrin and TGFβ signalling and disruption of these pathological signalling interactions significantly improved skin health and regeneration in JEB.

Histone post-translational modifications are fundamental to genome regulation, yet dissecting the functions of individual histone marks in mammals remains challenging due to the presence of multiple histone gene copies. Here we develop a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) prime editing platform enabling precise, reversible and combinatorial mutagenesis of canonical and noncanonical histone H3 genes within their native genomic context. Using systematic lysine-to-arginine substitutions benchmarked against synonymous controls, we identify key residues, including H3K4, H3K9, H3K14, H3K18 and H3K79, whose mutation compromises fitness in mouse embryonic stem cells. We further show that H3K56, linked to genome stability in yeast and Drosophila, has a conserved role in mammalian cells. Through analysis of selected double mutants, we uncover functional crosstalk across residues, with combinations such as H3K27R + H3K36R impairing stem cell self-renewal and altering transcription. Altogether, this study establishes a functional map of histone H3 lysines in mammals and provides a broadly applicable platform for systematic dissection of chromatin regulation.

Nucleotides are essential for life, serving not only as the building blocks of the genome but also as cellular energy providers, metabolic cofactors and signalling molecules. To sustain cellular function and proliferation, cells must continuously generate, recycle and precisely balance nucleotide pools in response to fluctuating metabolic and environmental demands. Nucleotide metabolism is therefore not a static biosynthetic pathway, but a dynamic system tightly integrated with cell signalling and physiology. Here we highlight the regulatory logic of nucleotide metabolism, from acute post-translational regulation to transcriptional scaling, feedback control and higher-order spatial organization into multi-enzyme assemblies and filaments. Through the lens of human genetic disorders and cancer, we examine how nucleotide depletion, pool imbalance or intermediate toxicity produce striking tissue-selective pathologies. Together, these principles position nucleotide metabolism as a central regulatory axis linking cellular metabolism, signalling and fate in health and disease.

The short-term and long-term effects of genotoxic pre-transplant conditioning remain barriers to the broader application of haematopoietic stem/progenitor cell (HSPC) transplantation and gene therapies1-4. Although monoclonal antibodies targeting KIT have been proposed as alternatives to chemotherapy or radiotherapy5-7, their pharmacokinetics hinder clinical applications owing to the risk of depleting transplanted HSPCs. Here, to address this issue, we identified amino acid changes in the extracellular domain of KIT that disrupt the binding of two therapeutic monoclonal antibodies8,9, which impair stem cell factor (SCF)-mediated signalling without affecting KIT expression or functionality. We exploited adenine base editing10 or prime editing11 to efficiently introduce these mutations in HSPCs and combined them with the disruption of the BCL11A erythroid enhancer to promote expression of fetal haemoglobin (HbF)12,13, a therapeutic approach for several haemoglobinopathies. This strategy enables in vivo co-selection of gene-engineered cells to reach the threshold required to provide therapeutic benefit in patients affected by sickle cell disease and β-thalassaemia. We show progressive enrichment of KIT plus BCL11A multiplex-edited haematopoiesis under selective pressure with KIT monoclonal antibody, in vitro and in vivo. We report that extended treatment with anti-KIT regimens leads to superior in vivo enrichment while avoiding clonal selection, as assessed by a lentiviral barcoded library. Finally, by overcoming the limitations of monoclonal antibody pharmacokinetics, epitope editing enables novel haematopoietic replacement regimens that are not limited by on-target graft elimination, allowing prolonged immune-based conditioning that maximizes haematopoietic niche clearance without chemo-radiotherapy or monoclonal antibody wash-out.

Cycloaddition reactions are central to building cyclic molecular architectures for drug discovery and materials science. However, controlling single-atom-bridged cycloaddition remains elusive. Herein, we describe a selenium-atom transfer [3 + 2 + 1] heterocycloaddition of readily available arylamines, alkenes and diselenides, enabling rapid, modular access to diverse selenium- and nitrogen-containing heterocycles, including selenomorpholines and polycyclic 1,4-selenazinanes. This metal-free method operates under mild, simple conditions with scalability, showcasing broad substrate generality and functional group tolerance, is amenable to scalable synthesis, and demonstrates utility in the late-stage skeletal diversification of complex molecules. Mechanistic and computational studies reveal a Se-bridged cycloaddition pathway involving alkene aminoselenylation, followed by electrophilic selenocyclization and deprotonative rearomatization. This heteroatom-driven annulation opens opportunities for the development of reactions for the construction of multi-heteroatom cyclic scaffolds and broader application in selenium-based medicinal chemistry.

RNA modifications are fundamental to gene regulation and RNA processing, yet their diversity and transcript specificity remain incompletely defined. Here, using a genetic screen in S. pombe, we identify the RNA methyltransferase Tgs1 and Coilin-related proteins as regulators of transcripts harboring inefficiently spliced cryptic introns. These factors associate to form a protein assembly, termed TEaM, which is recruited to cryptic-intron-containing RNAs, including retrotransposon-derived transcripts, by spliceosomal components, and to gametogenic gene transcripts by a YTH-family RNA-binding protein. Upon recruitment, Tgs1 catalyzes trimethylguanosine (TMG) capping, facilitating engagement of the conserved factor Pir2/ARS2, which cooperates with the DROSHA homolog Pac1 and other factors to promote RNA processing and RNAi-mediated silencing. This pathway also targets centromeric repeat RNAs containing cryptic introns, enabling de novo production of siRNAs that specify heterochromatin nucleation. Together, these findings delineate a mechanism in which Tgs1-directed TMG capping, coupled with Pir2/ARS2, specifies RNAi substrates to broadly regulate gene expression and silence retrotransposons.

During morphogenesis and in pathological conditions, gaps can form in the plane of epithelial barriers upon cellular forces that disrupt intercellular junctions. How the size of these epithelial holes further increases over time and what sets their shape remain poorly understood. Here we analyze the formation of the olfactory orifice (the nostril) in zebrafish, which opens and grows in the skin epithelium above a rosette of olfactory placode cells, allowing the sensory neurons to directly access odor cues. Using quantitative imaging and tissue-specific perturbations, we analyze the dynamic remodeling of skin cells allowing the expansion of the orifice edge. We identify the sensory cavity located in the center of the placodal rosette as a crucial player that sets the size of the growing epithelial hole in the skin. We further show that fine-tuning of actomyosin contractility within each tissue (skin and sensory cavity) exerts non-autonomous effects on the neighboring tissue, thereby shaping the nostril structure. This study uncovers dynamic cell behaviors and reciprocal tissue-tissue interplay that control the growth and shape of an epithelial hole in vivo.

Plasma membrane damage can cause cell death and is associated with neurodegeneration. In this issue of Developmental Cell, Heffner et al. show that annexin A11 (ANXA11) first plugs membrane lesions, before ESCRT-III is recruited to extrude the damaged patch-a two-step repair mechanism compromised by ALS- and FTD-linked mutations.

Proliferative lupus nephritis (LN) is triggered by deposition of autoantibodies in glomeruli and paralleled by a T cell-rich kidney infiltrate. Although these T cells have been attributed to the propagation of tissue injury, it is unclear how they are activated and whether T cell autoreactivity drives local inflammation. Kidney-infiltrating T cells are also observed in urine, where they have high resemblance to interstitial T cells. Therefore, urinary T cells are a proxy for investigating tissue pathogenesis. Here, we analyzed urinary T cells to elucidate whether a kidney-specific T cell autoimmune reaction contributes to tubulointerstitial inflammation in LN. Using single-cell RNA sequencing, we compared transcriptomes and clonotypes of T cells from the blood and urine of patients with active LN and showed that urinary T cells were mostly activated CD8 effector memory cells recruited from a circulating CX3CR1+ subset. Several urinary CD8 T cell clones were expanded. However, upon in vitro testing of their T cell receptors, we did not observe autoreactivity against autologous tubular epithelial cells. Instead, ~20% of expanded clonotypes were Epstein-Barr virus-specific or cytomegalovirus-specific, but respective viral antigens were undetectable in kidney biopsies or urine. Conversely, kidney-infiltrating T cells had access to interleukin-15 and interferon-β (IFN-β), and stimulation with these cytokines was sufficient to trigger degranulation and production of tumor necrosis factor, IFN-γ, and granzyme K. Together, these results show that CD8+CX3CR1+ T cells are recruited into the kidney in LN, where they are activated by cytokines, enabling them to contribute to local inflammation.

Cardiovascular disease (CVD) is the leading cause of mortality in chronic kidney disease (CKD). While CKD is known to give rise to systemic inflammation, its inciting factors remain poorly defined. Oxalate, long implicated in rare genetic kidney disorders, accumulates with decreased kidney function and has emerged as a driver of inflammation and independent risk factor for CVD. Here, we investigate the immunological mechanisms linking oxalate nephropathy to systemic inflammation, cardiac damage and kidney injury. Oxalate nephropathy was induced in C57Bl6/N mice through an oxalate-enriched diet. Oxalate induced systemic immune activation, renal fibrosis, and adverse cardiac remodeling, including pulmonary congestion with systolic and diastolic dysfunction. Flow cytometry analysis identified interleukin (IL)-17A as a dominant inflammatory effector, with expansion of Th17 and Th17-like Treg in the kidney, intestine, and spleen. Bulk mRNA sequencing confirmed these findings in kidney and heart. In line, plasma IL-17A was increased in oxalate-fed mice. Confirming the oxalate-IL-17A relationship, plasma IL-17A was elevated in patients with primary hyperoxaluria. Gut microbiome analysis by 16S amplicon sequencing showed only mild oxalate-induced alterations in mice. However, soluble oxalate directly enhanced Th17 polarization and disrupted mitochondrial respiration in vitro. In vivo, antibody-mediated IL-17A blockade improved kidney function, cardiac fibrosis, reduced neutrophil infiltration, and partially restored cardiac function in oxalate-fed mice. Our study identifies oxalate as a systemic immunometabolic stressor and IL-17A as a central mediator of oxalate-induced cardiorenal injury. These findings establish the oxalate-IL-17A axis as a mechanistic link between CKD and CVD and suggest IL-17A inhibition as a potential therapeutic strategy to reduce cardiovascular damage in CKD.

DNA hydrogels are widely explored in biomedical research for their programmability and soft tissue-mimicking mechanics. However, their application in load-bearing implants is restricted by insufficient mechanical robustness, especially for mimicking the biomechanics of the nucleus pulposus, which is a naturally occurring functional tissue essential for spinal flexibility and shock absorption in the intervertebral disk. Here, we report a novel single-pot, two-step fabrication strategy in which long DNA strands produced by rolling circle amplification form an initial viscoelastic network that is subsequently reinforced through controlled thermal self-assembly of proteins, physically stapling the DNA chains into a mechanically tunable hybrid matrix. This synergistic protein reinforcement enables precise control over the morphological and mechanical properties of DNA hydrogels while improving stability under enzymatic and pH stress. The reinforced hydrogels maintain structural integrity under complex deformation and sustained compression in ex vivo nucleus pulposus models and exhibit pressure-dependent drug release. Overall, this study establishes protein reinforcement within viscoelastic gel networks as a distinct materials design strategy for creating programmable biomaterials that integrate molecular precision with mechanical resilience for use in mechanically demanding biological environments.

How distinct lineage identities are specified from pluripotent epiblast cells during gastrulation is a longstanding open question. By investigating AXIN, a negative regulator of the WNT/β-catenin pathway, we have uncovered previously unrecognized roles for WNT signaling in the specification of distinct mesoderm identities. Using complementary approaches, including a detailed analysis of Axin1;Axin2 mutant mouse embryos involving single-cell and single-embryo transcriptomics and in vitro pluripotent stem cell differentiation assays, our data revealed two critical layers of regulation. First, WNT initiates differentiation of primitive streak cells into mesoderm progenitors. Next, WNT amplifies and cooperates with bone morphogenetic protein (BMP)/pSMAD1/5/9 or NODAL/pSMAD2/3 to propel differentiating mesoderm progenitors into either posterior or anterior streak identities, respectively. We propose that Axin1 and Axin2 function to prevent precocious differentiation of pluripotent epiblast cells into mesoderm through the spatiotemporal regulation of WNT-signaling levels.

Maintenance of plasma membrane integrity is essential for compartmentalization of the cytosol and for cellular viability. Upon membrane damage, several factors including endosomal sorting complex required for transport-III (ESCRT-III) proteins, annexins, stress granules, lipids, and membrane fusion proteins are mobilized to orchestrate membrane repair. However, whether these factors operate independently or act together is unclear. Here, using human cell lines, we expose temporal differences and interdependencies in the recruitment of ESCRT-III and annexin proteins to sites of plasma membrane damage. We show that annexin proteins are recruited immediately and form a plug at the damage site, restricting membrane permeability. We find that ESCRT-III assembles later and acts to release plug-containing damaged membranes from the cell. Further, frontotemporal dementia (FTD)- and amyotrophic lateral sclerosis (ALS)-associated mutations in the ESCRT-III protein, CHMP2B, and the annexin protein, ANXA11, compromise plasma membrane repair, suggesting that defects in this process may contribute to these pathologies. These data present an integrated "sealing and healing" model of membrane repair.

A bioorthogonal click-to-release reaction employing cyclooctynes and N,N-dialkylhydroxylamines is described. The reaction is characterized by a tandem retro-Cope/Cope elimination reaction sequence in which strain-promoted hydroamination of a cyclooctyne by a N,N-dialkylhydroxylamine reagent is relayed into Cope elimination of the resulting enamine N-oxide. β-Elimination of the N-hydroxyenamine product then results in bond cleavage. The reaction is regioselective, and the cleavage is directional. The primary hydroamination reaction exhibits second order rate constants up to 2 M-1s-1, and the ensuing elimination steps are not rate limiting up to millimolar levels of hydroxylamine. The transformation enables the rapid and complete cleavage of a chemical bond in biologically relevant settings using reagents with a small molecular footprint. We demonstrate the importance of reagent size in an application involving the chemical activation of protein function using hydroxylamines that are tuned for either rapid kinetics or constrained spaces. Access limitations to enzyme active sites are a major determinant of reagent choice in bioorthogonal cleavage reaction applications.

Scholarisine A (1) represents a structurally distinct and synthetically intriguing subfamily of the echitamine/akuammiline-type alkaloids. Inspired by its postulated biogenetic logic, we developed a radical cyclization strategy that retains the key bond-forming site while replacing the aldol reaction. This reaction-altering biomimetic strategy, combined with expeditious preparation of a tetracyclic precursor, enabled a 13-step, scalable synthesis of 1. Moreover, facile access to its congeners, scholarisines I, T, and W (2-4), was achieved. Compound 2 impairs lysosomal degradative capacity and thus inhibits late-stage autophagy in cancer cells.

Bacterial infections impede wound healing and are exacerbated by antibiotic resistance. In this study, three semiconductor conjugated polymer (SCP) nanomaterials (DFPE, DTBA, and DTID) with integrated photodynamic/photothermal therapeutic functions are developed by employing different electron donor groups. Compared to DFPE and DTBA, the introduction of a benzo[e]indole unit endows DTID with a distinct A-D-A electronic configuration. DTID exhibits strong near-infrared absorption, excellent photothermal conversion efficiency (38.4%), and efficient reactive oxygen species (ROS) generation. Furthermore, the composite hydrogel HGJD is constructed by coloading DTID and curcumin into a sodium alginate/chitosan hydrogel. HGJD integrated the hydrogel's inherent moisturizing capacity and biocompatibility, DTID's dual phototherapy, and curcumin's antioxidant and anti-inflammatory activities. HGJD effectively kills Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) and exhibits antioxidant activity. In the S. aureus-infected mouse model, HGJD with light irradiation eliminates bacteria, reduces inflammation, and accelerates wound healing. This work presents a promising strategy for designing hydrogel dressings with dual phototherapy and anti-inflammatory functions.

The gut microbiota plays critical roles in constraining Candida albicans colonization of the gastrointestinal (GI) tract, which is a key precursor to disseminated fungal infection in immunocompromised hosts. Depletion of commensal microbiota, such as by antibiotic treatment, increases C. albicans burden and promotes dissemination, yet the mechanisms of colonization resistance remain unclear. Here, we show that microbiota-derived short-chain fatty acids (SCFAs) directly inhibit C. albicans growth by inducing fungal metabolic reprogramming, impairing hexose uptake, and inducing intracellular acidification. In vivo, SCFAs enhance Candida colonization resistance only in the presence of an intact gut microbiome, which drives SCFA-induced taxonomic shifts that augment resistance. A Bacteroides thetaiotaomicron mutant unable to produce SCFAs exhibits diminished capacity to restrict C. albicans colonization, while prebiotic therapy that increases luminal SCFAs enhances C. albicans clearance. These findings define a critical microbiota-metabolite mechanism underlying Candida colonization resistance and suggest strategies to modulate GI fungal burden and prevent invasive disease.

Inflammatory bowel disease is shaped by complex microbial communities, yet the contribution of fungal-bacterial interactions to disease progression remains poorly defined. Here, we identify Cladosporium tenuissimum (C. tenuissimum) as a gut fungus with potent colitis-alleviating activity. Mechanistically, C. tenuissimum restrains Candida albicans (C. albicans) overgrowth through nutrient competition, particularly via the utilization and subsequent limitation of the amino acid ornithine. C. albicans can evade this suppression and potentiate intestinal inflammation through nutrient escape by preferentially exploiting specific amino acids, such as threonine. We further reveal a bacterial-fungal metabolic axis in which threonine-producing Bacteroides fragilis facilitates C. albicans escape from gut microbiome-mediated fungal control, thereby exacerbating colitis. Notably, dietary threonine restriction markedly attenuates C. albicans-driven colitis in mice. Together, our findings uncover a cross-kingdom metabolic network that determines C. albicans homeostasis and, in turn, governs intestinal inflammatory outcomes, offering new conceptual and therapeutic avenues for IBD.

Autoimmune hepatitis (AIH) is characterised by death of hepatocytes and chronic inflammation. Patients with deleterious variants in A20 are identified with AIH presentations and immune activation. However, it remains unclear how A20 dysregulation contributes to AIH pathogenesis. This study elucidates the role and mechanism of A20 in AIH progression and its therapeutic potential. A20 expression was analysed in patient sample and AIH mouse model. A20 function was explored in mice with hepatocyte-specific A20 knockout and overactivation and wild type controls. Liver samples were assessed by histology, immunoblot and electron microscopy. Proteomics, mass spectrometry, co-immunoprecipitation and site-specific mutation experiments were used to elucidate underlying mechanisms. AdipoRon was used to identify the effects of pharmacological induction of A20 on AIH. A20 expression is reduced in livers of individuals with AIH. In experimental AIH, hepatocyte-specific A20 depletion aggravates hepatic inflammation and fibrosis, enhances ferroptosis of hepatocytes and increases sensitivity to ferroptosis inhibitors, while A20 overexpression ameliorates AIH pathology and reduces hepatocyte ferroptosis. Mechanistically, A20 is found to interact with KEAP1 and mediate its K48-linked polyubiquitination degradation, promoting NRF2 accumulation and nuclear translocation to enhance antioxidant and antiferroptosis effects. Pharmacological induction of A20 confers protective effects in AIH. The clinical correlation among A20, KEAP1, NRF2 and ferroptosis is validated in human AIH samples. Our findings elucidate a previously unrecognised A20-KEAP1-NRF2 axis that orchestrates hepatocyte ferroptosis in AIH. Targeting A20 may provide a promising therapeutic strategy for AIH.

The management of chronic diabetic wounds remains a persistent clinical challenge, characterized by prolonged healing times and high recurrence rates. These wounds typically stall in the inflammatory phase due to multiple overlapping pathological factors, including multidrug-resistant infections, poor microvascular networks, sustained oxidative stress, and impaired immune responses. Traditional single-target treatments struggle to overcome these concurrent barriers; thus, there is a pressing need for multifunctional therapeutic approaches. Metal ions (evolutionarily conserved enzymatic cofactors and redox mediators) provide this mechanistic versatility by coordinating antimicrobial defense, angiogenesis, inflammation resolution, and tissue remodeling through distinct yet complementary pathways. We systematically examine the specific mechanisms of nine functional metal ions (Ag+, Cu2+, Zn2+, Au3+, Ce3+/Ce4+, Mg2+, Ca2+, Mn2+, and Co2+), and discuss recent advances in bimetallic synergistic platforms (e.g., Fe-Cu, Cu-Ce). To connect biological potential with practical application, we compare diverse delivery architectures (such as stimuli-responsive hydrogels, metal-organic frameworks, electrospun nanofibers, and microneedle patches) focusing on their release kinetics and structural advantages. Finally, we address the primary bottlenecks limiting clinical translation, notably protein corona formation, and outline future directions for designing the next generation of safe, smart, and highly effective metal-based wound dressings.

Peripheral cannabinoid CB1 receptor antagonists that lack central nervous system effects are emerging as promising therapies for metabolic disease, yet the role of endothelial CB1 signaling in atherosclerosis remains unclear. Here, we show that endothelial CB1 is expressed in human atherosclerotic plaques, is induced by oscillatory shear stress in atheroprone flow regions, and promotes vascular inflammation, permeability and lipid uptake. Endothelial-specific Cnr1 deletion or peripheral CB1 antagonism in mice attenuates atherosclerosis, reduces endothelial caveolae-dependent low-density lipoprotein uptake by downregulating caveolin-1 and ALK1 expression, and improves metabolic parameters in brown and white adipose tissue and the liver. The anti-atherogenic and metabolic effects are more pronounced in females, which is possibly linked to estrogen signaling. These findings identify endothelial CB1 as a proatherogenic, sex-biased regulator of vascular lipid transport and plaque development and associated metabolic dysfunction.

Adult hippocampal neurogenesis is essential for learning, memory, and mood regulation, and its disruption is implicated in ageing, neurodegeneration, and mood disorders. However, the mechanisms linking inflammation to adult hippocampal neurogenesis impairment remain unclear. Here, we identify chronic tumour necrosis factor-alpha signalling as a key driver of neurogenic dysregulation via a previously unrecognised type I interferon autocrine/paracrine loop in human hippocampal progenitor cells. Using a female-derived human in vitro neurogenesis model, single-cell RNA sequencing, and functional T cell migration assays, we show that tumour necrosis factor-alpha induces a robust type I interferon response in hippocampal progenitor cells, promoting chemokine-mediated and CXC motif chemokine receptor 3-dependent T cell recruitment and suppressing neurogenesis. This inflammatory signalling cascade drives a fate switch in hippocampal progenitor cells from a neurogenic trajectory towards an immune-defensive phenotype, with critical implications for infectious and inflammatory disease pathogenesis. These findings uncover a key inflammatory checkpoint regulating human adult hippocampal neurogenesis and highlight potential therapeutic targets to restore neurogenesis in chronic inflammatory states.

Lactate was long considered a waste metabolic byproduct. Currently, lactate functions as an energy substrate, signaling molecule, and epigenetic regulator in the central nervous system (CNS). Lysine lactylation (Kla), a novel lactate-dependent PTM, acts as a core epi-metabolic mediator by directly linking cellular metabolism to epigenetic remodeling. This review summarizes lactate production, shuttling, and the regulatory mechanism of lactylation (donors, writers, erasers, readers). We outline the multifaceted functions of lactylation in neurodevelopment, synaptic plasticity, and neural energy homeostasis, alongside its pathological dysregulation in major neurological disorders, with particular emphasis on ageing-related conditions. We further discuss the interplay between aberrant Kla and core ageing hallmarks, including mitochondrial dysfunction, impaired proteostasis, and inflammageing. Kla exerts site‑specific, cell‑type‑specific, and context‑dependent effects that are either protective or pathological. We also discuss therapeutic strategies targeting lactate metabolism and lactylation-modifying enzymes, analyzing the translational challenges involved. This review suggests Kla as an emerging epi-metabolic target that merits further investigation for therapeutic interventions in the ageing nervous system.

A key feature of the intestinal immune system is balancing pathogen defense with antigen-specific tolerance to commensal bacteria. Here, using conditional deletion models, we identified the transcription factor BHLHE40 as a central regulator of group 3 innate lymphoid cell (ILC3)- and RORγt⁺ antigen-presenting cell (APC)-dependent mucosal immunity. In ILC3s, BHLHE40 drove transcription of cytokine effector programs and maintenance of mucosal immunity. Cytokine TL1A stimulation and inflammation induced Bhlhe40 expression, amplifying these programs through epigenetic modulation of chromatin accessibility at effector loci. Bhlhe40 was also highly expressed in RORγt⁺ APCs, where it was required for the generation of antigen-specific Tregs. In parallel, BHLHE40 integrated microbial cues to promote expression of the co-stimulatory molecule OX40L by ILC3s, further promoting antigen-specific Treg induction. Together, these findings define Bhlhe40 as a coordinated regulator of barrier immunity and support a model in which ILC3s and RORγt⁺ APCs act in concert to shape antigen-specific intestinal immunity.

Why is fatty liver disease associated with hyperglycemia? In this issue, Ye, Wan, Liu, Deng, Zhang et al.1 propose an unexpected mechanism: hepatic alkaline phosphatase released from diseased liver suppresses intestinal stem cell differentiation into GLP-1-secreting L-cells. This study reveals a new pathogenic route of liver-intestine communication.

Plasmonic-photonic metasurfaces enable large-area, spectrally selective field enhancement for surface-enhanced Raman scattering (SERS). However, identifying low-loss hybrid plasmon-photon modes under confocal, near-normal excitation remains elusive. Angle-resolved SERS provides direct access to their dispersion and degeneracies under stationary, ultrahigh numerical aperture excitation (NA ≈ 1.0 in air) by independently scanning polar and azimuthal incidence angles. SERS additionally demands modes that tolerate defects and periodicity fluctuations. We therefore fabricate colloidal metasurfaces across particle concentrations and lattice periods to quantify robustness. Using template-assisted self-assembly, we reveal a direct correlation between nanoparticle concentration, structural order, and the emergence of hybrid modes. At the optimized periodicity, SERS is maximized (up to 2.32× higher SERS intensity), which we can modulate through resonance detuning. Angle-resolved SERS at 633 and 785 nm under oblique incidence shows maximal enhancement when the hybrid guided-mode resonance is tuned to the laser line, yielding up to 36× higher intensity than off-resonant conditions for the same sample. An analytical model attributes these modes to phase-coupled waveguide coupling and collective plasmonic chain resonances. Thus, we yield practical design rules for robust, tunable colloidal plasmonic-photonic SERS metasurfaces via hybrid-mode dispersion engineering.

Infrared (IR) photodetectors used in advanced imaging and sensing applications rely on epitaxially grown semiconductors that are expensive to manufacture. Colloidal quantum dots (QDs) offer lower-cost production, with substantial progress using PbS and HgTe QDs. HgSe and HgCdSe QDs offer wide spectral tunability, higher absorption coefficients, and air stability, but their inherent n-type doping has hindered their use. Here, we show that n-type doping can be depleted by neutralizing nanocrystal surfaces by displacing surface metal cations acting as Z-type ligands. Heating with alkanethiols yields QDs coated with protonated thiols and with unfilled conduction bands. Surface-neutralized HgSe and HgCdSe QDs exhibit sharp excitonic absorption features at IR wavelengths up to ∼1700 nm. Photodiodes fabricated from HgCdSe QDs have a room-temperature specific detectivity of 1.4 × 1012 Jones at 1355 nm, attributable to suppressed doping and low dark current. These air-stable, broadly tunable materials with strong IR absorption may enable low-cost IR sensors.

Focal segmental glomerulosclerosis (FSGS) is a major cause of primary glomerular disease, characterized by progressive podocyte injury and loss. This study aimed to identify the RNA methylation regulator WTAP (Wilms' tumor 1-associated protein) as a novel pathogenic factor in FSGS. WTAP expression in podocytes was detected in kidney biopsies from patients with FSGS and in an adriamycin-induced FSGS mouse model. Two podocyte-specific Wtap-deficient mouse models were generated using the Cre-loxP system: a constitutive knockout (cKO) model and a tamoxifen-inducible knockout model. Loss- and gain-of-function strategies were used to investigate the functional role of WTAP in podocytes both in vivo and in vitro. WTAP expression was significantly decreased in podocytes from patients with FSGS and in the adriamycin-induced FSGS mouse model. Both cKO and tamoxifen-inducible knockout mice developed progressive glomerulosclerosis with characteristic features of FSGS. WTAP overexpression in podocytes attenuated podocyte injury both in vivo and in vitro. cKO mice exhibited alterations in the proportions of various renal cell types, including both renal intrinsic and inflammatory cells. Mechanistically, WTAP regulates SPP1 m6A methylation, thereby modulating its stability and expression. Notably, SPP1-mediated signaling emerged as a central hub coordinating pathological cell-cell interactions in cKO mouse kidneys, and neutralization of SPP1 significantly ameliorated renal injury in both cKO and adriamycin-induced FSGS mice. We successfully constructed 2 FSGS mouse models that are suitable for investigating the pathogenic mechanisms and therapeutic targets of FSGS. Our findings demonstrate that the WTAP/SPP1 (secreted phosphoprotein 1) signaling pathway contributes to the renal pathological changes in FSGS, suggesting that targeting this pathway may represent a promising therapeutic strategy.

The scientific world has lost one of its great architects with the passing of Pierre Chambon, a visionary French scientist, institution builder, and demanding yet inspiring mentor. He provided major insights into the fundamental mechanisms governing gene expression and hormonal regulation that transformed modern biology and medicine. In addition, he developed groundbreaking mouse genetic techniques, and established Strasbourg as a leading European center for life sciences research. His death on May 5, 2026, at the age of 95, marks the end of an extraordinary era in science, yet his intellectual legacy will continue to shape generations of researchers across the world.

Chemotactic receptors involved in generation of ectopic pulmonary germinal centers (GCs) within inducible bronchus-associated lymphoid tissue (iBALT) are poorly defined. Here, using CIBER Cxcr3-reporter mice, we demonstrate that the prototypical type 1 inflammatory chemokine receptor CXCR3 is highly induced in influenza A virus (IAV)-reactive B cells in the mediastinal lymph node, spleen, lung, peripheral blood, and airways following intranasal infection. Notably, elevated Cxcr3 was observed in ectopic pulmonary germinal center B (GCB) cells in iBALT relative to their contemporaneous counterparts in secondary lymphoid organs across the timecourse of the response to IAV infection. Mice with a B cell-specific deletion of Cxcr3 displayed a 50 to 60% reduction in the frequency and number of ectopic GCB cells in the lungs at the peak of the response following IAV infection, relative to controls. Furthermore, in cotransfers, Cxcr3-deficient B cells were substantially outcompeted by their Cxcr3-sufficient counterparts for ectopic pulmonary GC participation, but were not impacted with respect to GCB cell frequencies in other compartments. Thus, the data elucidate the requirement of B cell-intrinsic CXCR3 expression for efficient generation of ectopic pulmonary GCB cell responses in iBALT following respiratory viral infection with IAV, a finding that broadens understanding of the molecular cues underpinning this key component of local protective humoral immunity to IAV.

The NLRP3 inflammasome is central to host defense and sterile inflammation and forms condensates at the microtubule-organizing center (also known as the centrosome), although the mechanisms regulating this process remain unclear. Here we define a functional relationship among microtubule transport, the centrosomal kinase NEK7, priming, and NLRP3 abundance. We show that microtubule-dependent transport is required for NEK7-dependent NLRP3 activation and promotes NEK7 to the pericentriolar material (PCM). Microtubules, priming, and NEK7 synergistically converge on PCM abundance, thereby creating a permissive centrosomal environment for NLRP3 condensation and inflammasome assembly. Elevated NLRP3 expression compensates for limited PCM abundance, rendering K+ efflux-induced activation independent of both NEK7 and priming in human and mouse macrophages. By contrast, NLRP3 overexpression only partially bypasses NEK7 dependence in response to the K+ efflux-independent stimulus imiquimod, likely due to its activation of a non-trans-Golgi network pool of NLRP3 that is quantitatively limited. Together, these findings define a conserved spatial mechanism in which microtubule transport, NEK7 localization, priming, and NLRP3 abundance integrate at the PCM to establish the activation threshold and magnitude of NLRP3 inflammasome signaling across species and stimuli.

Deciphering protein function is fundamental to advancements in medicine and biotechnology. However, conventional experimental characterization remains resource-intensive. Public large language models (LLMs), though proficient in natural language processing, often fail to accurately interpret and predict the functional and structural properties of proteins, limiting their utility in bioinformatics. To address this gap, we introduce BetaDescribe, designed to generate detailed and rich textual descriptions of proteins, including their function, catalytic activity, involvement in specific metabolic pathways, subcellular localizations, and the presence of specific domains. The trained BetaDescribe model receives protein sequences as input and outputs a textual description of these properties. BetaDescribe starting point was the LLAMA2 model, which was trained on trillions of tokens. Our model was next trained on datasets containing both biological and English text, which allowed the incorporation of biological knowledge. In addition to the description generator, BetaDescribe comprises multiple validator models and a judge, which together enable accurate ranking of alternative generated descriptions. We demonstrate the utility of BetaDescribe by providing descriptions for proteins that share little to no sequence similarity to proteins with functional descriptions in public datasets. Using in silico mutagenesis, we further show that BetaDescribe relies on functionally important regions, as part of its prediction, suggesting that the model identifies regions of importance for the protein functionality without needing homologous sequence. BetaDescribe offers a powerful tool to explore protein functionality, augmenting existing approaches such as annotation transfer based on sequence or structure similarity.

The Smoothened (SMO) G protein-coupled receptor is a key part of the Hedgehog (Hh) signaling pathway, and it is an oncoprotein that is an important target for understanding cancers such as basal cell carcinoma. However, its mechanism of activation remains unknown. To this end, we investigate here the sequence of G protein and cholesterol (CHL) ligand binding to SMO on the pathway toward activation. Our results are consistent with the G protein-first activation pathway of SMO in the Hh signaling pathway. In this model, CHL first binds to the cysteine rich domain (CRD) of the inactive SMO, which remains inactive at this stage. The G protein can then spontaneously bind to this SMO, forming a precoupled complex that remains inactive while Patched (PTCH) is attached to the membrane. Upon binding of the Hh ligand to PTCH, the CHL levels in the membrane increase, enabling CHL to also bind to the transmembrane domain (TMD) of SMO. With CHL occupying both the CRD and the TMD sites, opening of the G protein becomes energetically favorable, promoting GDP release and initiating downstream G-protein signaling.

Regulated cell death (RCD) has long been conceptualized as a genetically encoded signaling process, yet its outcome is ultimately dictated by cellular metabolism. Here, we propose that cellular metabolism functions as a gatekeeper of RCD, establishing permissive or restrictive states that determine cell fate. Bioenergetic capacity, redox balance, lipid composition, and metal availability impose metabolic constraints that bias cells toward survival or distinct death modalities. At the systems level, organelle-resolved metabolism and inter-organelle communication coordinate the spatial control of death processes. We further position RCD pathways along a metabolic continuum, ranging from energy-dependent apoptosis to chemistry-driven ferroptosis. This framework explains the plasticity of death responses and suggests that metabolic reprogramming can redirect cell fate. Targeting metabolic dependencies thus offers a strategy to control cell death in disease.

Membrane receptors sense external stimuli and transduce signals by recruiting cytosolic effector proteins to initiate downstream signaling cascades. Despite precise and distinct functional outcomes, diverse receptors often engage common cytosolic effectors, implying that signaling specificity is regulated by the spatial dynamics of effector activity within membrane-proximal cytoplasmic microdomains. Here, we engineered receptor-tethered cytosolic modulators for manipulating the activity of effectors within localized cytoplasmic regions adjacent to target receptors. These modulators comprise three modules: an extracellular receptor-targeting unit, a cytosolic effector inhibitor, and an intervening transmembrane linker. By using the modulator to locally inhibit Src family kinases (SFKs, effectors) within the cytoplasmic microdomain of PD1, we observed robust suppression of PD1 phosphorylation and subsequent restoration of T-cell activity. Conversely, inhibition of SFK activity in the proximity to the T-cell receptor/CD3 complex remarkably impaired T-cell activation. Collectively, our work establishes a versatile and modular nanoplatform for dissecting signaling networks with biologically compatible spatial precision, providing opportunities for targeted bioregulation and therapeutic interventions.

Metal particle size has a significant influence on the activity of nanozymes, yet size-dependent nanozymatic catalysis under the same metal loadings in full scale (from single atom, cluster to nanoparticle) is a challenging task and has been rarely reported. Herein, porous SiO2 nanoflower surface-confined Pt-based nanozymes with the same metal loadings but different particle sizes were rationally designed and synthesized for antibacterial and cascade catalytic tumor therapy. The particle size-activity relationship is well established, presenting a volcanic curve, and the PtNC/SiO2 nanocluster (NC) nanozyme exhibits optimized peroxidase-like activity and bactericidal efficacy compared with SAzyme and nanozyme. Furthermore, a synergistic therapeutic platform (Lap@PtNC/SiO2) is constructed through the adsorption of the prodrug β-lapachone (Lap), achieving high-efficiency cascade enzymatic catalysis for tumor therapy thereby. The cluster nanozymes not only present optimized activity during the full-size engineering but also demonstrate great potential in next-generation antibacterial and tumor catalytic therapy.

Metabolic disturbances are key contributors to myocardial ischemia-reperfusion (I/R) injury, yet the underlying molecular mechanisms remain largely unclear. Rho family GTPase 3 (RND3), a cytosolic small guanosine triphosphatase (GTPase) known to antagonize ROCK1 (Rho-associated coiled-coil kinase 1), has been implicated in several cardiovascular disorders. However, its mitochondrial localization and functional role in cardiac energy metabolism and I/R injury remain unknown. A murine model of myocardial I/R injury was established through left anterior descending coronary artery ligation. Mice with cardiomyocyte-specific knockout and overexpression of Rnd3 were generated. To investigate the role of RND3 in cardiac metabolism and I/R injury, we used 13C-nuclear magnetic resonance, 18F-fluorodeoxyglucose positron emission tomography/computed tomography scanning, seahorse mitochondrial energy metabolism assays, and 13C-metabolic flux tracing. Mechanistic studies were conducted using RNA sequencing, coimmunoprecipitation, mass spectrometry, and glutathione S-transferase (GST) pulldown assays. Cardiomyocyte-specific deletion of Rnd3 (Rnd3 conditional knockout, Rnd3cKO) resulted in impaired glucose oxidation and compensatory upregulation of fatty acid oxidation, leading to pronounced cardiac dysfunction and increased mortality. Rnd3cKO hearts exhibited reduced pyruvate/malate-driven complex I respiration and marked uncoupling between glycolysis and the tricarboxylic acid cycle. Mechanistically, RND3 was identified as a novel mitochondrial matrix-localized small GTPase that directly binds to ACAT1 (acetyl-coenzyme A [CoA] acetyltransferase), disrupting its interaction with PDHA1 (pyruvate dehydrogenase E1α subunit) and thereby promoting PDHA1 acetylation and glucose oxidation. It is important to note that RND3 expression was significantly downregulated in both human and murine hearts after I/R insult. Loss of RND3 sensitized the hearts to I/R injury, as evidenced by reduced levels of phosphocreatine and ATP. Conversely, cardiac-specific overexpression of Rnd3 conferred protection against I/R injury, an effect that was abolished upon Pdha1 knockdown. Our results identify RND3 as a novel mitochondria-localized regulator of glucose oxidation that safeguards the heart against I/R injury. Therapeutic reconstitution of Rnd3 may represent a promising strategy to restore metabolic homeostasis and mitigate myocardial damage in the context of I/R.

Chronic stress (CS) exacerbates anxiety and hyperglycemia, emerging as a key risk factor for type 2 diabetes, yet the mechanism remains unclear. Here, we found that CS induces hyperglycemia and enhanced amygdaloid astrocytic senescence in mice. The amygdaloid astrocytic senescence was mediated by the reduction of hexokinase 2 (HK2) driven by pre-B cell leukemia homeobox transcription factor 1 (PBX1). The astrocytic Hk2 deletion mice and amygdala-specific astrocytic Hk2 knockdown mice both display anxiety-like behaviors and hyperglycemia. The reduction of HK2 in astrocytes reduces L-serine synthesis and decreases the supply to neurons for the generation of D-serine by disrupting the astrocyte-neuron serine shuttle. Reduced neuronal D-serine level in the amygdala impaired the balance of sympathetic and parasympathetic amygdala-pancreas projections, leading to hyperglycemia. L-serine supplementation or dasatinib/quercetin administration to eliminate senescent cells alleviates both CS-induced neurobehaviors and peripheral hyperglycemia. Together, these findings reveal that HK2 in amygdaloid astrocytes mediates CS-induced neurobehaviors and hyperglycemia.

Antibody-oligonucleotide conjugates (AOCs) have emerged as a promising therapeutic platform that integrates the targeting capability of antibodies with the gene-regulatory potential of oligonucleotide payloads. By enabling cell or tissue selective delivery, AOCs may extend oligonucleotide therapeutics beyond liver predominant distribution and broaden intervention strategies for intracellular targets that have historically been difficult to drug. However, their therapeutic performance is not determined by target binding alone, but also by productive intracellular delivery, including receptor mediated uptake, endosomal trafficking, payload release, and functional access to cytoplasmic or nuclear compartments. In this review, we summarize the key design principles governing AOCs performance, including target biology, antibody formats, oligonucleotide payload classes, chemical modifications, linker design, conjugation strategies, and critical quality attributes. We further discuss the intracellular fate of AOCs and highlight endosomal escape as a major rate limiting step that often constrains biological activity despite efficient cellular uptake. In addition, we review current translational progress, with particular emphasis on neuromuscular disorders, as well as emerging applications in oncology, central nervous system diseases, and other indications. Finally, we outline major challenges and future directions that are likely to shape the next generation of AOCs therapeutics.

Current antiretroviral therapies suppress HIV replication but fail to eliminate integrated proviral DNA in long-lived CD4⁺ cells, precluding a cure. CRISPR-Cas9 offers potential for HIV eradication but efficient and cell-specific delivery into HIV target cells remains a major hurdle. We developed CD4-directed Nanoblades (CD4-NBs), murine leukemia virus-like particles pseudotyped with anti-CD4 nanobodies and a fusogenic glycoprotein VSV Gmut, to selectively deliver Cas9-gRNA ribonucleoproteins into CD4⁺ cells. CD4-NBs selectively delivered cargo to CD4⁺ cells in vitro and in vivo, achieving efficient gene disruption in primary CD4+ cells. Dual-guide CD4-NBs targeting conserved HIV tat/rev/env regions disrupted proviral DNA, suppressing HIV infection in CD4+ cells. In HIV-infected, ART-pretreated humanized mice, CD4-NBs significantly reduced plasma viremia. While full tissue reservoir clearance was not achieved, repeated dosing did reduce viral RNA and proviral DNA in bone marrow and lungs, respectively. As such, this proof-of-concept study supports the promise of CD4-NBs as a minimally invasive, CD4⁺ cell-targeted gene editing strategy for HIV therapy.

Exhausted CD8+ T (TEX) cells undergo extensive genome reorganization during differentiation, yet the drivers of this process remain elusive. Here we show that CTCF programmed CD8+ TEX cell fates through two distinct modes of action. CTCF acquired de novo binding sites and concordantly induced open chromatin in early CD8+ TEX cells responding to chronic viral infection. The dynamic CTCF binding activated enhancers and promoted chromatin looping. Consequently, genetic ablation of CTCF diminished chromatin accessibility and interaction strength, impairing CD8+ TEX cell proliferation, effector function and bioenergetic mobilization. Conversely, invariant CTCF binding acted as essential chromatin barriers, and loss of CTCF disrupted insulation and caused aberrant chromatin self-association and undue RNA polymerase II pausing, leading to excessive activation of exhaustion- and stemness-linked genes. Thus, CTCF balanced CD8+ TEX cell differentiation by gaining dynamic binding to induce cytotoxicity and sustain metabolic fitness, while its invariant binding compartmentalized exhaustion and stemness program genes to prevent their overexuberant activation.

Within days of exposure to chronic viral infections, activated CD8+ T cells differentiate into Tcf1-Slamf6loTim3hi exhaustion-prone effector T (TEX_EFF) cells or self-renewing Tcf1+Slamf6hiTim3lo precursor exhausted T (TPEX) cells. Here we showed that early CD8+ TEX cell fates were imprinted by forming subset-specific, self-associating chromatin hubs. Chromatin hub assembly coincided with effector or stemness gene induction and identified the transcription cofactors Id2 and Id3 as key regulators that promoted CD8+ TEX_EFF and CD8+ TPEX cell fates, respectively. Id2 drove CD8+ TEX_EFF cell specification by activating effector genes, while suppressing genes involved in exhaustion and stemness. In contrast, Id3-repressed effector genes but upregulated IL-7Rα and AhR, thereby maintaining the CD8+ TPEX cell pool. Mechanistically, Id2 and Id3 exhibited a distinct impact on the chromatin accessibility landscape in early CD8+ TEX cells by engaging Runx3 and Tcf1 transcription factors along with E proteins. These findings indicated that reshaping chromatin architecture represents a critical means for specifying CD8+ TEX cell fates and ensuring lineage stability.

Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) manipulates cellular processes through the translocation of effector molecules into the host cell cytosol. Using a recently established neonatal S. Typhimurium infection model, we provide functional insights into how Salmonella outer protein B (SopB) suppresses early mucosal tissue inflammation and prolongs host survival. Mechanistically, SopB prevents a disintegrin and metalloprotease 17 (ADAM17) activation, plasma membrane translocation and the release of membrane-bound TNFα from enterocytes and reduces epithelial secretion of IL-18 via mTOR-controlled secretory autophagy. This abolishes the early epithelial transcriptional response and reduces immune cell recruitment and programmed cell death-mediated mucosal barrier disruption delaying disease progression. The immunosuppressive effect of SopB is independent of the C-terminally encoded phosphatidylinositol phosphatase and phosphotransferase activity but requires an intact N-terminal domain. Also, it is restricted to the neonatal mouse model characterised by Salmonella pathogenicity island (SPI)1 type 3 secretion system (T3SS)-dependent enterocyte invasion-driven mucosal translocation. Thus, here we demonstrate that SopB suppresses the early, post-transcriptional regulation of epithelial cytokine release in an inositol phosphatase-independent manner likely promoting pathogen transmission.

Gene expression during cellular differentiation is coordinated by combinatorial interactions between transcription factors (TFs) and cofactors. Although the TF GATA1 coordinates gene transcription in hematopoiesis, the specific cofactors required for GATA1-driven gene expression are incompletely defined. We identify the H3K4 methyltransferase KMT2D, as a cofactor of GATA1 in erythropoiesis. Loss of KMT2D in human erythroid precursors causes developmental arrest with impaired expression of numerous erythroid genes. Mechanistically, KMT2D colocalizes with GATA1 on more than one thousand erythroid enhancers, and such co-occupancy is associated with stronger transcriptional activity than occupancy by GATA1 alone. Acute depletion of KMT2D in erythroid precursors causes rapid reductions of H3K4me1 and H3K27ac on a subset of GATA1-bound enhancers and impairs their target gene expression. Moreover, acute depletion of GATA1 or KMT2D individually causes downregulation of overlapping gene sets. Our findings demonstrate how a lineage-specific TF cooperates with a ubiquitous epigenetic regulator to drive lineage-specific gene expression.

Heart failure with reduced ejection fraction (HFrEF) remains a major therapeutic challenge. B-cell lymphoma 2-associated transcription factor 1 (Bclaf1) is implicated in RNA splicing and cardiac disease, but its role in HFrEF pathogenesis is unknown. Here, we demonstrate that Bclaf1 expression is elevated in human HFrEF myocardium and in male murine pressure-overload models. Cardiac-specific Bclaf1 overexpression drives pathological hypertrophy and systolic dysfunction, whereas its genetic knockout or adeno-associated virus serotype 9 (AAV9)-mediated knockdown attenuates these phenotypes. Mechanistically, Bclaf1 interacts with the splicing factor serine/arginine-rich splicing factor 2 (Srsf2) to bind to heart and neural crest derivatives expressed 2 (Hand2) pre-mRNA and enhance its splicing efficiency, leading to increased mature Hand2 levels and maladaptive remodeling. Inhibition of either Bclaf1 or Hand2 rescues cardiac function and structure in experimental HFrEF. Our work defines a Bclaf1/Srsf2/Hand2 splicing axis as a critical driver of HFrEF and reveals a promising therapeutic target for heart failure.

Transient receptor potential vanilloid type-1 (TRPV1) channel is a polymodal receptor involved in pain perception and neuronal signalling that represents a promising target for the development of analgesics and neuroprotective agents. In this study we implement a combination of computational techniques and targeted design of chemical libraries to discover benzothiophene-substituted TRPV1 agonists with high affinity and efficacy toward TRPV1. In vitro functional experiments show that prolonged or repeated exposure to these compounds induce calcium-dependent desensitization of TRPV1, making it insensitive to noxious stimuli. We solve a cryo-electron microscopy (cryo-EM) structure of human TRPV1 (hTRPV1) in complex with the most promising benzothiophene-substituted agonist MSP20. The structure reveals molecular details of MSP20 binding to the vanilloid site and a desensitized conformation of hTRPV1, characterized by the closed ion channel pore, α-helical C-terminus and distinct behaviour of annular lipids. Our in vivo experiments demonstrate that MSP20 exhibits robust and long-lasting antinociceptive activity with ex-vivo neuroprotective effects, supporting the perspective of benzothiophene-substituted vanilloids as future analgesics.

Huntington's disease is driven by CAG repeat expansion in the mutant huntingtin gene. Nuclease FAN1 and mismatch repair protein MLH1 regulate repeat expansion through direct interaction, but the underlying structural basis remains unclear. Here, we show that the MLH1 C-terminal domain binds to FAN1-derived peptides containing either the MIP or MIM motif with comparable affinities. Crystal structures of this domain bound to each motif provide structural insights into human MLH1-FAN1 interaction, revealing a conserved mechanism for FAN1-MIP recognition and a previously unrecognized binding site on MLH1, termed the S3 site, for FAN1-MIM engagement. Co-immunoprecipitation assays confirmed that mutation of key MLH1 residues disrupts FAN1 binding in cells. These findings establish the molecular basis of MLH1-FAN1 recognition and provide a structural framework for understanding the regulation of CAG repeat expansion in Huntington's disease.

Epigenetic regulation refers to heritable changes in gene expression without altering the DNA sequence. It mainly includes DNA methylation, histone modification, RNA modification and non-coding RNAs (ncRNAs). Abnormalities in epigenetic regulation play a key role in the development of many diseases. In recent years, with the rapid development of high-throughput sequencing technology, epigenetic modification datasets have accumulated rapidly, which has facilitated the development of various database resources and computational analysis tools. At the same time, the emergence of artificial intelligence technologies such as machine learning and deep learning has further improved the speed and accuracy of epigenetic modification site prediction and functional mining. However, most existing reviews focus on databases and computational tools for single types of epigenetic modifications, whereas systematic overviews of integrated databases, multi-algorithm prediction tools, and applications of artificial intelligence technologies in this field remain scarce. Here, we systematically introduce the current major databases and prediction tools for DNA methylation, histone modification, RNA modification, ncRNAs, and disease-related epigenetic modifications, highlighting the potential of artificial intelligence technologies, including deep learning, in epigenetic modification research and pharmaceutical research. In addition, we discuss the challenges faced by existing computational methods in the field and future directions, providing references for data repositories and computational tools selected by researchers, as well as new insights into epigenetic mechanism studies.

Lysine succinylation is an emerging post-translational modification critically involved in cardiovascular pathophysiology. Malate dehydrogenase 1 (MDH1), a core enzyme of the malate-aspartate shuttle that maintains cardiomyocyte redox homeostasis, is implicated in myocardial injury, yet the regulatory role and specific mechanism of MDH1 succinylation in myocardial ischemia/reperfusion (I/R) injury remain incompletely understood. This study aims to elucidate the functional role and underlying molecular mechanism of site-specific lysine succinylation of MDH1 in myocardial I/R injury. Global succinylome profiling was performed on cardiac tissue from murine I/R models. Target succinylation was validated by immunoprecipitation and Western blot. AAV9 vectors encoding wild-type MDH1 (MDH1-WT) or succinylation-deficient mutant (MDH1-K298R) were constructed for cardiac-specific delivery. Molecular docking and co-immunoprecipitation identified the upstream succinyltransferase, and virtual screening identified Ethyl rosmarinate (ER) as a Carnitine palmitoyltransferase 1A (CPT1A)-stabilizing compound. MDH1 K298 succinylation was significantly decreased in I/R-injured hearts compared with sham-operated control hearts. In vivo cardiac overexpression of MDH1-WT suppressed ferroptosis and ameliorated myocardial I/R injury, whereas MDH1-K298R failed to confer such protection, indicating that the cardioprotective effect of MDH1 is dependent on K298 succinylation. Mechanistically, K298 succinylation enhanced MDH1 protein stability by inhibiting its ubiquitin-proteasomal degradation, thereby preserving redox homeostasis required for glutathione peroxidase 4 (GPX4) activity. Importantly, CPT1A was identified as the succinyltransferase responsible for MDH1 K298 succinylation. ER alleviated myocardial I/R injury by preventing Carnitine palmitoyltransferase 1A (CPT1A) degradation and consequently enhancing MDH1 K298 succinylation. Our findings uncover a critical cardioprotective role of the CPT1A-MDH1 succinylation axis via suppressing ferroptosis during myocardial I/R injury, and identify ER as a CPT1A-stabilizing compound with promising therapeutic potential for ischemic heart disease.

Discrete π-π stacked nanocarbons, which serve as atomically precise models for layered graphitic materials, exhibit distinct optical and electronic properties. However, most reported examples are limited to planar building blocks. In this work, two negatively curved chiral nanographenes (1 and 2) containing a hexa[7]circulene core were synthesized. Both 1 and 2 form bilayer structures in the crystalline state, adopting eclipsed and displaced conformations, respectively. The bilayer stacking is driven by interlayer π-π and C-H···π interactions. NMR studies confirmed the well-defined bilayer structure of (1)2 in solution. Cyclic voltammetry measurements demonstrated interlayer coupling in (1)2. The inversion behavior of the chiral isomers was investigated using circular dichroism spectroscopy and theoretical calculations, revealing that the bilayer stacking stabilizes the chiral structure of (1)2. Enhanced chiroptical dissymmetry factors were observed in (1)2. This work not only establishes (1)2 as a molecular model for negatively curved bilayer graphene but also highlights the modulation of chiral behavior through stacking, offering a strategy for constructing well-defined chiral assemblies based on negatively curved nanocarbons.

Diabetic wounds are characterized by persistent inflammation, impaired angiogenesis, and delayed tissue regeneration, yet effective strategies to regulate the pathological immune microenvironment remain limited. Here, integrated single-cell transcriptomics and multi-omics analyses revealed significant enrichment of endoplasmic reticulum stress (ERS)-related signaling in pro-inflammatory macrophages within diabetic wounds, suggesting a potential mechanism driving chronic inflammation. To target this process, we developed an injectable dynamic hydrogel (GPOK-2@EVs) incorporating Artemisia argyi-derived extracellular vesicles (EVs) for localized immunomodulatory therapy. The hydrogel was formed through dual dynamic crosslinking between phenylboronic acid-modified gelatin and oxidized konjac glucomannan, providing self-healing capability, injectability, tissue adhesion, and pH/ROS-responsive degradation for sustained EV release. In vitro experiments demonstrated that GPOK-2@EVs exhibited excellent biocompatibility and effectively reprogrammed macrophages toward an anti-inflammatory phenotype, accompanied by reduced inflammatory cytokine secretion, alleviated ERS activation, decreased ROS accumulation, and improved mitochondrial integrity. GPOK-2@EVs significantly enhanced endothelial angiogenic activities, including cytoskeletal remodeling, tube formation, and migration. In a diabetic mouse wound model, GPOK-2@EVs accelerated wound closure, promoted granulation tissue formation and collagen deposition, enhanced vascularization, and suppressed inflammatory and ERS responses. These findings demonstrate that EV-loaded dynamic hydrogels can modulate the inflammatory microenvironment and promote diabetic wound regeneration, providing a promising biomaterial strategy for chronic wound therapy.

Atherothrombosis, which underlies most acute coronary syndromes and is driven by intraplaque thrombosis, preferentially occurs in regions of disturbed blood flow (d-flow). Although LATS1/2 (large tumor suppressor kinases 1 and 2) are known regulators of endothelial mechanotransduction, the mechanisms by which d-flow connects endothelial senescence, proliferation, and intraplaque thrombosis remain poorly understood. We investigated endothelial cell (EC)-specific roles of Lats1/2 using inducible EC-specific knockout mice in a partial carotid ligation model. Spatial multiomics of human and mouse plaques was performed using imaging mass cytometry, COMET sequential immunofluorescence, and spatial metabolomics. Tamoxifen-induced deletion of both Lats1 and Lats2 (homozygous) in ECs caused fatal edema and increased vascular permeability. In contrast, Lats1het(±)/Lats2 homo(-/-)-EC-specific knockout mice survived and developed spontaneous atherothrombotic plaques with neovascularization. Spatial proteomics revealed that LATS1/2 loss induced a senescence-associated stemness phenotype driven by CD38 upregulation. Spatial metabolomics showed sulfite and taurine accumulation, indicating SUOX (sulfite oxidase) deficiency. CD38 suppressed SUOX, demonstrated a switch into the reverse mode of mitochondrial complex V, increased succinate dehydrogenase activity, and promoted ATP consumption. Despite ATP depletion, glutamate metabolism and the citric acid cycle flux increased, sustaining EC proliferation under energetic stress. This senescence-associated stemness state promoted both proliferation and senescence, leading to fragile, leaky neovessels and intraplaque thrombotic lesions. Pharmacological CD38 inhibition attenuated these phenotypes. Similar EC states were observed in human plaques. Loss of Lats1/2 in ECs induces a CD38-associated senescence-associated stemness-like phenotype that promotes intraplaque thrombosis through mitochondrial metabolic reprogramming, including changes consistent with reverse-mode operation of mitochondrial complex V. These findings define a mechanistic link between disturbed flow, endothelial metabolic reprogramming, and intraplaque thrombosis and hemorrhage.

Over the past two decades, omics and big data have shifted plant molecular biology from single-gene, hypothesis-driven studies to systems-level, data-driven discovery. As datasets expand in scale and diversity, bioinformatics software has become essential for routine analysis and interpretation. However, the efficiency of data exploration and evidence integration has not kept pace with data growth, leaving many datasets underutilized and only slowly translated into biological insight. A central bottleneck is the widening gap between the limited data analysis skills of many experimental biologists and the increasing complexity of biological data. TBtools was developed to narrow this gap by providing low-barrier, interactive functions for common plant omics tasks, and it has been broadly adopted. Here, we use TBtools as a decade-long case study to discuss why certain local tools achieve broad adoption in plant omics research, distill eight actionable design recommendations, and propose four capacity pillars for next-generation local workbenches: project-level data management, reproducible workflow construction, elastic remote computing, and AI-assisted navigation and automation. Together, these lessons provide a practical roadmap for accelerating the translation of omics data into biological insights.

Metal-free room temperature phosphorescence (RTP) materials in aqueous environments offer promising applications due to their long lifetimes and large Stokes shifts but face challenges in maintaining stability against water and oxygen. To address this issue, we developed a trinity strategy that integrates efficient emissive units, excited-state stabilization, and protection from quenching agents within a single polymeric carbon nitride (CN) macromolecule functionalized with imidazolium. This approach yielded homogeneous RTP materials exhibiting robust phosphorescence in water and oxygen, with lifetimes up to 80 ms and high quantum yields. Mechanistic studies revealed that incompletely condensed residues enabled efficient intersystem crossing, which was stabilized by the rigid CN skeleton and protected by the hydrophobic microenvironment from covalent imidazolium functionalization. Leveraging these properties, we fabricated a phosphorescent hydrogel for visual detection of Fe3+ in human serum. This work demonstrates the potential of engineered carbon nitride as a versatile platform for stable, water/oxygen-tolerant organic RTP materials suitable for biosensing applications.

Acetaminophen (APAP)-induced acute liver injury (AILI) is a prevalent clinical liver condition caused mostly by oxidative stress and mitochondrial damage. Dental pulp stem cells (DPSCs) possess antioxidant, anti-inflammatory, and immunomodulatory capabilities, demonstrating significant potential in liver diseases. However, during in vitro culture, they are typically maintained under normoxic conditions (21% O2), which is very different from the hypoxic oxygen level that is found in vivo. It remains unclear whether hypoxic-conditioned dental pulp stem cells (Hyp-DPSCs) exhibit superior therapeutic effects compared to normoxic-conditioned dental pulp stem cells (Nor-DPSCs). This study demonstrated that 24-h exposure to 1% O2 significantly enhanced HIF1A/HIF-1α expression in DPSCs. It promoted mitophagy through the MYC-HIF1A-BNIP3 pathway, enhancing mitochondrial shape and function while reducing oxidative stress in DPSCs. Furthermore, in vitro and in vivo experiments demonstrated that Hyp-DPSCs were far more potent than Nor-DPSCs in boosting the expression of hepatic antioxidant factors and enhancing macroautophagy/autophagy to reduce AILI. These findings revealed that hypoxia activated mitophagy in DPSCs, enhancing their therapeutic efficacy against AILI and providing a novel strategy for stem cell-based AILI treatment.Abbreviations: AILI: acetaminophen-induced acute liver injury; ANOVA: analysis of variance; APAP: acetaminophen; BAX: BCL2 associated X, apoptosis regulator; BCL2: BCL2 apoptosis regulator; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CASP3: caspase 3; CAT: catalase; CCK-8: cell counting kit-8; CM: conditioned medium; COX4I1: cytochrome c oxidase subunit 4I1; CPT1A: carnitine palmitoyltransferase 1A; CQ: chloroquine; DPSCs: dental pulp stem cells; ELISA: enzyme-linked immunosorbent assay; GO: Gene Ontology; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic - pyruvic transaminase; GPX4: glutathione peroxidase 4; GSH: glutathione; Hyp-DPSCs: hypoxic-conditioned dental pulp stem cells; H&E: hematoxylin and eosin; HIF1A/HIF-1α: hypoxia inducible factor 1 subunit alpha; HMOX1/HO-1: heme oxygenase 1; HUVECs: human umbilical vein endothelial cells; IF: immunofluorescence; IHC: immunohistochemistry; IL1B/IL-1β: interleukin 1 beta; IL6: interleukin 6; i.p.: intraperitoneally; i.v.: intravenous injection; KEGG: Kyoto Encyclopedia of Genes and Genomes; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MSCs: mesenchymal stem cells; MYC: MYC proto-oncogene, bHLH transcription factor; NAC: N-acetylcysteine; NAPQI: N-acetyl-p-benzoquinone imine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; Nor-DPSCs: normoxic-conditioned dental pulp stem cells; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; PLIN2: perilipin 2; PINK1: PTEN induced kinase 1; PPARA/PPARα: peroxisome proliferator activated receptor alpha; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; ROS: reactive oxygen species; SEM: standard error of the mean; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TNF/TNF-α: tumor necrosis factor; TOMM20: translocase of outer mitochondrial membrane 20; VDAC1: voltage dependent anion channel 1; WB: western blot.

White matter (WM) abnormalities have emerged as a critical element in Alzheimer's disease (AD) pathogenesis, shifting from their former status as a passive consequence to an active contributor to disease progression. Notably, microstructural WM alterations, detectable early via advanced neuroimaging techniques such as diffusion tensor imaging, frequently precede overt gray matter atrophy and cognitive decline, highlighting their potential as early contributors to AD pathogenesis. The origins of WM pathology are multifactorial, involving a complex interplay among β-amyloid (Aβ) and tau aggregation, energy dysmetabolism, neuroinflammation, vascular dysfunction, and cellular senescence. Importantly, we emphasize a paradigm-shifting perspective: WM degeneration acts not merely as a downstream outcome but as a key driver of AD pathogenesis, capable of accelerating protein aggregation, amplifying neuroinflammation, and compromising neural plasticity. Given its early manifestation and close association with symptom onset, WM integrity has emerged as a sensitive and reliable biomarker for early AD detection and progression monitoring. Moving beyond diagnostics, the growing understanding of WM pathophysiology has unveiled a new frontier of therapeutic interventions aimed at myelin regeneration and WM protection. Despite persisting translational challenges, targeting WM integrity represents a pivotal avenue for developing disease-modifying therapies capable of slowing disease progression and improving clinical outcomes in patients with AD.

Intragenic inversions reverse sequence orientation within genes and create non-collinear splice junctions that standard transcript assemblers miss, resulting in incomplete reconstruction and biased quantification. Here we show Invas, an inversion-aware framework operating with bulk short-read sequencing data. Invas integrates whole-genome sequencing breakpoints with transcriptomic sequencing evidence, rescues unmapped reads, and assembles isoforms using conjugate flow optimization. In silico across 42,000 events, Invas demonstrates high precision and recall and improves quantification of unaffected transcripts compared with conventional tools. We apply Invas to seven disease cohorts, identifying recurrent germline susceptibility variants and somatic drivers. Furthermore, Invas facilitates the discovery of somatic inversion-derived tumor-specific antigens with strong predicted immunogenicity. We release Invas and InvasDB as community resources to enable accurate characterization of inversion-affected genes, filling a critical gap in structural variant-aware transcriptomics.

With the surge in the number of variants of uncertain significance (VUS) reported in ClinVar in recent years, there is an imperative to resolve VUS at scale. Multiplexed assays of variant effect (MAVEs), which allow the functional consequence of 100s to 1000s of genetic variants to be measured in a single experiment, are emerging as a powerful source of evidence which can be used in clinical variant classification. Increasingly, multiple published MAVEs are available for the same gene, sometimes measuring different aspects of variant impact. When multiple functional roles of a gene need to be considered, combining data from multiple MAVEs may provide a more comprehensive measure of the consequence of a genetic variant, which could impact variant classifications. We curated published datasets from five MAVEs for the gene TP53, two MAVEs for LDLR and two MAVEs for PTEN. Statistical methods (principal component analysis), unsupervised learning (k-means clustering), and supervised learning (Naïve Bayes and random forest classifiers) were used to integrate multiple MAVE datasets. The utility of MAVE integration methods were assessed using standard metrics (sensitivity, specificity, etc) as well as evidence strength in a putative variant classification framework. Here, we provide guidance for combining such multiplexed functional data, incorporating a stepwise process from data curation and collection to model generation and validation. We also present a web applet that allows users to test various methods for combining score sets from multiple assays, calculate integrated functional scores for all variants, and assess whether combining data enables the application of stronger evidence for pathogenicity or benignity. In general, supervised learning methods such as random forest led to improved variant classification as compared to any individual MAVE dataset. By following the steps outlined herein with appropriate guardrails, researchers can maximize the value of MAVEs, strengthen the functional evidence for clinical variant classification, and potentially uncover novel mechanisms of pathogenicity for clinically relevant genes.

Transcription factor (TF) modules interact to regulate key biological processes and cell-state transitions in normal physiology and disease. Understanding these modules and how they evolve over time can be accomplished by constructing gene regulatory networks (GRNs). To identify context-specific TF subnetworks, we developed ChEA-KG, which generates enriched TF regulatory subnetworks for input gene sets. ChEA-KG is based on a GRN connecting 1559 human TFs via 131 181 signed and directed edges inferred from diverse published ChIP-seq (chromatin immunoprecipitation followed by sequencing) and mRNA (messenger RNA)-sequencing experiments. We demonstrate ChEA-KG's utility by applying it to uncover master regulators of aging, mechanisms of action (MoA) for drug classes, pan-cancer subtypes, and cell types from across 14 major human tissues. Next, we extend ChEA-KG to develop the webserver application ChEA-KG Time Series (ChEA-KG-TS), which identifies TF modules from time-series mRNA-sequencing datasets. Results from this workflow are automatically summarized as reports that include enrichment analysis, regulatory subnetworks, and UMAP projections of enriched TFs. We use ChEA-KG-TS to explain transient responses in two use cases. ChEA-KG and ChEA-KG-TS are available from https://chea-kg.maayanlab.cloud/ and https://chea-kg-ts.maayanlab.cloud/.

ProteinNetworkSight (https://proteinnetworksight.jce.ac) addresses a pervasive bottleneck in modern systems biology: the inability to simultaneously analyze multiple feature vectors generated by quantitative techniques-such as machine learning, deep learning, or statistical modeling-that provide series of patterns in a dataset. Modern computational pipelines, ranging from PCA to deep autoencoders, rarely identify a single gene list; instead, they extract a series of distinct patterns representing diverse patient subgroups or independent components. Current web servers are ill-equipped for this high-dimensional reality, forcing researchers to analyze vectors one-by-one or merge them into a static consensus, obliterating unique topological signatures. ProteinNetworkSight introduces a novel web server architecture for simultaneous multi-pattern analysis. Unlike standard tools, our server accepts multi-column tables and transforms every input vector into a discrete, interactive protein-protein interaction network in a single run. This batch vector architecture allows side-by-side visualization of distinct topologies, preserving disease heterogeneity. Furthermore, the server enables prescriptive intervention by calculating a composite perturbation score to identify key protein nodes specific to each pattern. By mapping FDA-approved anti-cancer drugs to these targets, it facilitates the rapid design of personalized combinatorial therapies.

Type 1 regulatory T (Tr1) cells are CD4+ T cells with suppressive function that are induced from conventional T cells exposed to persistent or strong antigens. Human Tr1 cells are understudied; the regulators of their antigen-driven differentiation are unknown, and identifying them in tissues, where antigen interactions occur, is challenging. Here, we conducted a multiomic profiling of human antigen-induced Tr1 cells. Using CRISPR-based functional genomics, we uncovered essential roles of transcription factors IRF4, BATF, and MAF in human Tr1 differentiation, phenotype, and function. We also derived a Tr1 transcriptional signature that detects cells with a Tr1 phenotype in single-cell datasets from patients treated with Tr1 therapy and those with solid tumors. Cross-species analysis confirmed this signature identifies bona fide Tr1 cells induced in vivo in a murine solid tumor model. These findings provide a framework for development of Tr1-based and Tr1-targeting therapies and studies of Tr1 cell biology.

N-Nitrosodimethylamine (NDMA) is a water-soluble carcinogen typically quantified using mass spectrometry coupled with chromatographic separation, which requires extensive sample preparation and sophisticated instrumentation. Here, we report a fluorescence-based detection strategy for NDMA in aqueous media that replaces column-based separation with affinity capture. Photochemical transformation of NDMA, followed by reaction with a biotinylated polymeric reagent, generates a biotin-labeled fluorescent product that can be selectively enriched through streptavidin-biotin interaction, enabling effective separation of the analytical signal from background. The assay is performed entirely in water without sample pretreatment, preconcentration, or solvent exchange. Under optimized conditions, quantitative detection is achieved in a plate-based format using only 100 μL samples, affording a limit of detection of 235 ppb. The chemical transformation is completed within 22 min, followed by rapid affinity capture via streptavidin-coated agarose beads or plates. The method is rigorously validated through spectroscopic characterization, selective capture experiments, and quantitative analysis, and demonstrates consistent performance in spiked commercial water samples. These results establish a chemically defined platform for chromatography-independent optical NDMA detection and provide a basis for further development toward scalable sensing formats built on streptavidin-functionalized solid phases.

The cytochrome P450 redox system is composed of a cytochrome P450 reductase (POR) and multiple CYP450 enzymes (CYP450). Of the CYP450 isoenzymes, CYP51A1 is essential for endogenous cholesterol biosynthesis. Elevated circulating cholesterol is a well-established risk factor for cardiovascular disease, however, the role of intracellular cholesterol synthesis in normal endothelial function remains unclear. To investigate this, we generated CRISPR/Cas9 knockouts of the cytochrome P450 reductase in primary human endothelial cells (EC) and studied an endothelial-specific, tamoxifen-inducible POR knockout mouse (ecPOR-/-). Deletion of POR led to the accumulation of lanosterol, the substrate of POR/CYP51A1, and a reduction in desmosterol. Functionally, POR deficiency promoted basal and VEGF-induced angiogenesis in spheroids and mouse aortic segments. Retinal angiogenesis was increased in ecPOR-/- mice in vivo. Mechanistically, POR deletion activated the Sterol Regulatory Element Binding Transcription Factor (SREBP2) regulatory pathway, as shown by increased nuclear translocation of cleaved SREBP2 in EC and in en face-stained mouse aortae. Overexpression of nuclear SREBP2 in endothelial cells mimicked the angiogenic phenotype observed upon POR deletion. Conversely, double deletion of POR and SREBP2 normalized angiogenesis to levels of control cells. RNAseq of POR-deficient EC revealed an upregulation of PI3K-related signaling pathways and genes involved in cholesterol homeostasis, including enhanced expression of pro-angiogenic factors. In line with these findings, knockout of POR increased cellular PIP3 levels, AKT phosphorylation, and activation of downstream targets such as p70 S6 kinase. These findings demonstrate that inhibition of the endothelial POR/CYP51A1 axis impairs endogenous cholesterol synthesis, activates SREBP2, and enhances angiogenesis via PI3K/AKT/mTOR signaling, highlighting a critical and novel link between intracellular cholesterol metabolism and vascular growth.

Image-based machine learning tools are powerful resources for analyzing medical images, with deep learning-based semantic segmentation commonly utilized to enable the spatial quantification of structures visible in images. However, dataset generation and training of segmentation algorithms requires advanced programming skills and intricate workflows, limiting their accessibility to scientists without prior coding expertise. Here we present the step-by-step instructions to carry out automatic segmentation of medical images guided by a graphical user interface using the CODAvision algorithm. This workflow simplifies the process of semantic segmentation of microanatomical structures by enabling users to train highly customizable deep learning models without extensive coding expertise. The protocol outlines best practices for creating robust training datasets, configuring model parameters and optimizing performance across diverse biomedical image modalities. CODAvision enhances the usability of the CODA algorithm by streamlining parameter configuration, model training and performance evaluation, automatically generating quantitative results and comprehensive reports. We show the use of CODA to serial histology by demonstrating robust performance across numerous medical image modalities and diverse biological questions. We provide sample results in data types, including histology, magnetic resonance imaging and computed tomography. We demonstrate the diverse use of this tool in applications, including quantification of metastatic burden in in vivo models and deconvolution of spot-based spatial transcriptomics datasets. This protocol is designed for researchers with interest in rapid design of highly customizable semantic segmentation algorithms and a basic understanding of programming and anatomy.

Endovascular embolization is one of the core techniques in minimally invasive interventional medicine. It allows catheters to be accurately delivered to target blood vessels under image guidance for the implantation of embolic materials to achieve vascular occlusion. Although this technique has become increasingly mature, it still faces major challenges in precise delivery due to the limitations of embolic-agent properties and design concepts, restricting further improvement of the embolization therapeutic efficacy. Driven by continuous breakthroughs in materials science and control engineering, as well as the inevitable trend of medical development toward miniaturization, precision, and intelligence, microrobots show broad application prospects in precision disease treatment. They present advantages in accurate embolization and remote control in endovascular embolization, which can effectively improve embolization outcomes and reduce the risk of ectopic embolism. Taking the embolic microrobot system as the research focus, this paper systematically elaborates the design strategies, validation models, and practical applications of magnetically actuated embolic microrobots, comprehensively evaluates various imaging systems, and summarizes the supporting magnetic actuation technologies. It concludes the multimodule collaboration and adaptation framework of embolic microrobot systems. Finally, it analyzes the current limitations and potential translational challenges and presents a systematic prospect of its future development trends.

Angiogenesis requires endothelial cells (ECs) to toggle between quiescence versus proliferation, migration, and invasion. While activation from quiescence is well characterized, mechanisms governing the return from proliferation to quiescence (PtoQ) remain unclear. We show here that metabolic rewiring during PtoQ renders ECs sensitive to oxidative stress, requiring nicotinamide adenine dinucleotide (NAD) turnover for protection. Limiting EC NAD does not affect proliferation or migration but prevents cell-cell contact formation and quiescence acquisition during PtoQ. In vivo and ex vivo, limiting EC NAD permits initial sprouting but impairs vascular stabilization and plexus formation. Mechanistically, NAD suppresses mitochondria-derived hydrogen peroxide (H2O2) during PtoQ. Exogenous H2O2 mimics NAD deficiency, whereas its removal rescues PtoQ. In pathological settings, inhibiting NAD synthesis limits exuberant angiogenesis of retinopathy and tumors. In summary, we unveil metabolic events critical for PtoQ, a poorly studied component of angiogenesis, and point to new ways to suppress pathological angiogenesis.

Enzymes catalyze biochemical reactions in all living systems, and their subcellular localizations can profoundly influence distinct cellular functions. Yet, the physiological roles of subcellular enzyme activity remain largely unexplored, owing to the lack of tools that allow imaging of enzymatic activity with organelle-level precision in living cells. Here, we present a class of fluorogenic reporters for spatiotemporally resolved imaging of enzyme activity within specific subcellular compartments, including mitochondria (Mito), lysosomes (Lyso), endoplasmic reticulum (ER), and Golgi apparatus (Golgi). These fluorogenic reporters comprise a rhodol fluorophore whose fluorescence is quenched by an enzyme-specific substrate and a bioorthogonal cage group, with signal restoration occurring only upon sequential uncaging by an organelle-localized bioorthogonal activator and subsequent enzymatic activation. We applied this versatile strategy to visualize the activity of enzymes from different families in live cells, such as leucyl aminopeptidases (LAP), γ-glutamyl transferases (GGT), and monoamine oxidase A (MAO-A). Application to LAPs revealed distinct, compartment-dependent functions: Mito-LAP activity maintained redox homeostasis in stressed cells, favoring tumor cell survival, whereas ER-LAP activity promoted antigen presentation and immune-mediated tumor cell killing. Notably, drug profiling showed that cisplatin (CDDP) and camptothecin (CPT) concurrently amplified both Mito-LAP and ER-LAP activities, eliciting opposing influences on tumor progression. These findings also inspire the development of a mitochondria-targeted inhibitor to improve anticancer efficacy. This platform offers a genetically independent, broadly adaptable framework for dissecting compartmentalized enzymology in living cells.

The tumor microenvironment (TME) critically shapes disease progression and therapeutic resistance. However, a comprehensive understanding of its spatial architecture remains elusive, and clinical translation is challenging. Here, we present cellular architecture and neighborhood-informed virtual AI-driven spatial profiling (CANVAS), an artificial intelligence platform that infers tumor ecological habitats from hematoxylin and eosin (H&E) histopathology. Built on an atlas of over 18 million cells profiled by 41-plex spatial proteomics across 457 patients with non-small cell lung cancer, CANVAS establishes 10 reproducible cellular neighborhoods (CNs) capturing conserved spatial organization of the TME. Through multimodal alignment and foundation-model-based morphological encoding, CANVAS predicts CN-anchored habitat structures from H&E slides and enables clinical evaluation in over 5,000 patients spanning 9 cancer types. Across patient cohorts, CANVAS supports prognostic modeling, spatial ecotype stratification, and immunotherapy outcome prediction. These results establish CANVAS as a clinically scalable platform for spatial profiling, bridging single-cell analysis to population-level insight and enabling precision oncology.

While opioids remain the primary pharmacological intervention for cancer pain management, their clinical utility is frequently compromised by dose-limiting toxicities. This study aimed to determine the comparative efficacy, opioid-sparing potential, and clinical hierarchy of non-opioid adjuvant drug classes. The study was structured around the PICO framework to evaluate the pharmacological strategies currently utilized in multimodal clinical oncology. A systematic search of electronic databases (PubMed, Embase, Cochrane) was conducted for randomized controlled trials (RCTs) published between 2000 and 2025. The primary outcome was global analgesic efficacy (standardized mean difference [SMD]), while secondary outcomes included the opioid-sparing effect, defined as the percentage reduction in morphine equivalent daily dose (MEDD) and the incidence of treatment-emergent adverse events (Harms). A Bayesian network meta-analysis (NMA) was performed to rank treatments using SUCRA values. The methodological quality was assessed using the Cochrane Risk of Bias (RoB 2.0) tool. Twenty-three RCTs (n = 1845) met the inclusion criteria. Nonsteroidal anti-inflammatory drugs (NSAIDs) (-1.10) and anticonvulsants (-1.06) demonstrated the most robust analgesic effects. The SUCRA ranking confirmed a clear hierarchy, with the combination of anticonvulsants and antidepressants showing the highest probability of efficacy. A significant opioid-sparing effect was observed for gabapentinoids and ketamine, facilitating MEDD reduction. While serious adverse events were rare, minor harms (somnolence, dizziness) were more frequent in the most effective classes. Our NMA provides a robust evidence base for a "Clinical Tier" system, ranking adjuvants by their balance of efficacy and safety. These findings support the early integration of Tier I agents (anticonvulsants and NSAIDs) to optimize pain control and reduce opioid-related toxicities in chronic cancer pain management.

Noninvasive cell-free DNA (cfDNA) fragmentation profiles are gaining popularity as diagnostic tools for a wide range of conditions contributing to the global health burden, such as cancer, autoinflammatory disorders, and adverse events in pregnancy or transplantation. Despite their diagnostic value, fragmentomics rely on DNA sequencing, resulting in significant costs and turnaround times, therefore limiting their translation to everyday clinical care. Here, we present DNAviWEB (https://dnavi.sc.hpi.de/), a freely accessible implementation of the DNAvi analysis tool for exploring cfDNA fragmentomics from DNA gel electrophoresis in research settings. DNAviWEB enables instant presequencing analysis of cfDNA while integrating into medical workflows by providing browsable European Genome-phenome Archive and European Liquid Biopsy Society metadata catalogs and delivering rich outputs of patient group-stratified cfDNA statistics, visualizations, and secure storage capacities for clinical data. The DNAviWEB platform opens the exploration, analysis, and database deposition of liquid biopsy DNA profiles to a broad community of clinicians and researchers. DNAviWEB is free and open source.

The Oral Rheumatoid Arthritis Trial Surveillance trial identified increased malignancy risk with tofacitinib vs tumour necrosis factor inhibitors (TNFis) in rheumatoid arthritis (RA), leading to class-wide regulatory warnings for Janus kinase inhibitors (JAKis). Comparative cancer risk vs other advanced therapies and across immune-mediated inflammatory diseases (IMIDs) remains uncertain. The objective was to estimate relative and absolute cancer risk associated with JAKi vs other advanced therapies across IMIDs. MEDLINE, Embase, Cochrane Library, and ClinicalTrials.gov were searched from inception to February 2026 for phases II to IV randomised trials and long-term extension studies of licensed advanced therapies in adults with RA, psoriasis (PsO)/psoriatic arthritis (PsA), or inflammatory bowel disease (IBD) reporting malignancy outcomes. Bayesian class-level network meta-analyses using Poisson models estimated incidence rate ratios (RRs) vs standard care (placebo or methotrexate monotherapy). It includes all malignancies including nonmelanoma skin cancer. A total of 305 studies (164,824 participants; 264,100 person-years exposure) were included, comprising 174 studies across IMIDs, 123 in RA, 129 in PsO/PsA, and 54 in IBD. In the combined IMID network, JAKi were associated with higher malignancy risk than TNFi (RR: 1.60; 95% credible interval [CrI]: 1.27-2.02) and standard care (RR: 1.85; 95% CrI: 1.38-2.47). Similar directional findings were observed across RA, PsO/PsA, and IBD networks. Interleukin-6 (IL-6), Interleukin-17 (IL-17), Interleukin-23 (IL-23), Interleukin-12/23 (IL-12/23), Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), B-lymphocyte antigen CD20 (CD20)-targeting therapies showed risks broadly comparable with TNFi. Absolute excess cancer risk with JAKi compared with TNFi was negligible in standard-risk populations (+0.037 cancers per 1000 person-years exposure [PYE]; ∼1 additional cancer per 27,000 PYE) but meaningful in higher-risk populations (+6.814 cancers per 1000 PYE; ∼1 additional cancer per 147 PYE). Across IMIDs, JAKi were consistently associated with higher malignancy risk relative to TNFi and standard care, extending regulatory warnings beyond RA and emphasising the need for risk-stratified treatment decisions.

Endoscopic submucosal dissection (ESD) enables en bloc resection of early gastrointestinal cancers and provides specimens suitable for precise pathological risk assessment. However, reporting remains variable for key parameters that determine curative resection and the need for additional treatment, including submucosal invasion depth and breadth, margin status, lymphovascular invasion, tumour budding, differentiation and use of ancillary stains. To develop practical international standards for pathology assessment and reporting of invasive carcinoma in ESD specimens. An international panel of 42 experts, including 28 gastrointestinal pathologists and 14 therapeutic endoscopists from 15 countries, participated in a modified Delphi consensus process. Statements addressed measurement of invasion, margin assessment, staining, specimen handling, prognostic histological features and clinically relevant reporting. 56 recommendations reached consensus across seven domains. The panel recommends using Sm1-Sm3 subclassification only when the muscularis propria is present; otherwise, submucosal invasion depth should be reported in micrometres, rounded to the nearest 100 µm. Submucosal invasion breadth should be reported in millimetres as an adjunct metric for future validation. Margin positivity should be defined as direct tumour contact with the inked surface, supported by standardised pinning, inking, complete embedding and parallel sectioning. H&E remains the baseline stain, with selective immunohistochemistry or elastic stains for equivocal lymphovascular invasion, distorted architecture or difficult margin interpretation. Tumour budding should be reported according to International Tumour Budding Consensus Conference criteria, and differentiation, histological subtype, lymphovascular invasion, perineural invasion and margin status should be integrated into composite risk assessment. These consensus standards provide immediately implementable, synoptic-ready pathology reporting criteria after ESD. By standardising measurement landmarks, margin terminology, ancillary stain use and reporting of adverse histological features, they aim to reduce interinstitutional variability, improve multidisciplinary decision-making and support future validation of risk models in early gastrointestinal cancer.

Resistance to anti-PD-1/PD-L1 therapy is a major unmet need. Growth Differentiation Factor 15 (GDF-15) has been identified as a key resistance factor for anti-PD-1/PD-L1 immunotherapy. Visugromab, a neutralizing anti-GDF-15 antibody, plus the anti-PD-1 antibody nivolumab (V+N) was evaluated in the first-in-human phase 1/2a GDFATHER-01 trial in heavily pretreated participants with locally advanced/metastatic non-squamous non-small-cell lung cancer (nsq NSCLC), urothelial carcinoma (UC), or hepatocellular carcinoma (HCC), stringently defined as anti-PD-1/PD-L1-relapsed/refractory, and showed encouraging objective responses. This analysis reports long-term follow-up of these three phase 2 expansion cohorts of the GDFATHER-01 trial. Seventy-seven participants with nsq NSCLC (N=22), UC (N=27), and HCC (N=28) received visugromab (10 mg/kg) plus nivolumab (240 mg) every two weeks until disease progression or unacceptable toxicity. Objective response rates (RECIST v1.1) were 18.2% for nsq NSCLC (4/22; 95%CI 5.2-40.3), 18.5% for UC (5/27; 95%CI 6.3-38.1), and 14.3% for HCC (4/28; 95%CI 4.0-32.7). Median duration of response (DoR) was 32.2 months (95%CI 5.5-38.0), 28.8 months (95%CI 7.4-39.4), and 19.4 months (95%CI 5.8-39.7; with protracted recruitment), respectively, with 7/13 responses (53.8%) ongoing. Confirmed complete response or complete metabolic response (CR or CMR) among responders was 61.5% (8/13), with 7/8 ongoing. In addition, 46.2% (6/13) of responders achieved a deeper response on V+N per RECIST v1.1 than with the prior anti-PD-(L)1 therapy; median DoR on V+N was 28.8 months (95%CI 7.4-38.0) versus 12.0 months (95%CI 8.0-24.0) on initial anti-PD-1/PD-L1 treatment. V+N was generally well tolerated. In heavily pretreated, advanced/metastatic participants with nsq NSCLC, UC, or HCC who were anti-PD-1/PD-L1-relapsed/refractory, V+N achieved deep and durable objective responses. The observed DoR, depth of response, and CR+CMR rate among responders exceeded those reported for their initial anti-PD-1/PD-L1 therapy. These findings suggest that GDF-15 blockade with visugromab can overcome resistance and enhance the magnitude and durability of anti-PD-1/PD-L1 responses, and warrant further exploration in randomized trials. ClinicalTrials.gov, TRN: NCT04725474, Registration date: 25 January 2021; EudraCT, TRN: 2020-002103-19, Registration date 16 Dec 2020.

Current liquid biopsy methods for multi-cancer detection using plasma cell-free RNA (cfRNA, short RNA fragments circulating in blood that can reflect disease states) typically rely on gene annotations, which can overlook signals from unannotated or repetitive genomic regions. We present GeneLLM, a Transformer-based model that directly processes the nucleotide sequences of human-mapped cfRNA reads to identify cancer-indicative signatures. By bypassing gene-level quantification, the model retains signals from transcriptomic dark matter. The model learns latent pseudo-biomarkers (prototype representations from aggregated cfRNA read embeddings) that serve as discriminative features for cancer classification, rather than corresponding to explicit genomic sequences. Here we show that, in a multi-centre cohort, GeneLLM achieves ROC-AUC values ranging from 0.9250 to 0.9962 across several cancers, while maintaining comparable performance at one-sixth of the typical sequencing depth. These results suggest that sequence-level modelling of plasma cfRNA can capture diagnostically relevant information beyond annotation-dependent approaches, enabling more cost-efficient and scalable cancer screening.

Cancer treatment can disrupt the microbiome, worsening outcomes for cancer patients. Ecology frames these changes as transitions between measurable states, enabling the prediction of microbiome trajectories to support clinical decision making. Longitudinal monitoring and microbial restoration can translate microbiome ecology into strategies that improve cancer care.

High-throughput sequencing has transformed clinical diagnostics of rare diseases (RD), cancer and infectious diseases by enabling the identification of disease-causing genetic alterations and facilitating individualised treatment and care. In response to these advances, Genomic Medicine Sweden (GMS) was established in 2017 as a national collaborative effort to accelerate implementation of genomics-based precision medicine within Sweden's regionally organized, publicly funded healthcare system. GMS brings together the seven university healthcare regions and their associated medical faculties, in collaboration with healthcare regions across Sweden, Science for Life Laboratory, patient organizations, industry and governmental agencies. Activities are coordinated through national disease-specific expert groups, supported by cross-cutting functions in bioinformatics, health economics, ethics, education and patient engagement. At the operational level, seven Genomic Medicine Centres, embedded at university hospitals, develop and deliver harmonised genomic diagnostics nationwide. The National Genomics Platform provides secure infrastructure for large-scale data storage, analysis, and national and international data sharing. Following initial project-based funding, GMS now receives long-term governmental support. This review describes the national implementation of genomic-based precision diagnostics, discusses challenges and lessons learnt, and highlights key milestones across disease areas, including whole-genome sequencing in RD and paediatric cancer, comprehensive genomic profiling of haematological malignancies and solid tumours, pathogen genomics in microbiology, pharmacogenomic testing and emerging applications of polygenic risk scores in complex diseases. Collectively, these efforts have contributed to more than 500,000 genomic tests being performed within Swedish healthcare between 2017 and 2025. Finally, we outline future diagnostic needs and priority areas to ensure sustainable, scalable and equitable access to precision medicine.

Dermatomyositis is an inflammatory muscle disease with distinctive skin changes and a strong association with cancer, but the mechanisms behind this link and predictors of malignancy remain unclear. To summarize current evidence on the relationship between dermatomyositis and cancer, including epidemiology, clinical and serologic predictors, histopathology, and prognosis. A comprehensive search of PubMed, Scopus, and Cochrane was performed according to PRISMA-ScR recommendations. Eligible studies were systematically screened, and relevant data were charted and descriptively summarized. Fifty-nine studies met the criteria. Over half of cancers occurred within one year of dermatomyositis onset, and nearly 90% within three years. Genitourinary, gastrointestinal, and lung cancers were most common. Higher risk was linked to older age, male sex, dysphagia, severe skin disease, absence of interstitial lung disease, and anti-transcription intermediary factor 1-gamma or anti-nuclear matrix protein 2, while anti-Mi-2 suggested lower risk. Prognosis was significantly worse in patients with cancer. Cancer-associated dermatomyositis is a distinct subtype, supporting early and repeated cancer screening and the need for standardized surveillance protocols.

Chemotherapy-induced thrombocytopenia (CIT) is a common complication of cancer therapy that may result in major bleeding events and reduction of chemotherapy relative dose intensity. While there remain no widely available licensed therapies to manage CIT, major studies-including global pivotal phase 3 clinical trials of the thrombopoietin receptor agonists (TPO-RAs) romiplostim and avatrombopag in CIT- have been completed, along with additional supporting evidence from other clinical trials and observational studies. Like patients with chemotherapy-induced neutropenia, not all patients with CIT require treatment-but for those who do, it can have a major impact on a patient's oncologic care and outcomes. Proper management of CIT requires an understanding of the two clinical subtypes of CIT (persistent and nadir), the limited role of platelet transfusions and antifibrinolytics, and when and how to appropriately use TPO-RA support. In this article, 4 patient cases of CIT are used to illustrate common and challenging scenarios occurring in clinical practice. Built on a framework of published studies and supported by extensive clinical experience, appropriate treatment strategies for CIT are described in detail, with emphasis on proper patient selection, dosing, titration, and monitoring for the use of TPO-RAs in CIT.

Cell-free DNA (cfDNA) in body fluids enables noninvasive cancer detection. Multifeature artificial intelligence (AI) can improve sensitivity by integrating diverse biomarkers when cancer signals are sparse. Tumor-informed assays that rely on mutations have limited practicality for early cancer detection. Emerging fragmentomic and epigenetic features underpin tumor-naive approaches to screening for individuals with low tumor burden. Here, we designed UNITE-a universal cfDNA feature ensemble framework that provides scalable cancer detection methods based on "genomic bin-fragment length" matrices derived from shallow whole-genome sequencing (sWGS) data at 0.1× depth. Using sWGS data from 2063 plasma samples (631 controls and 1432 cases from 26 cancer types), we systematically evaluated both XGBoost (UNITE-XGB) and convolutional neural networks (UNITE-CNN) across multiple feature spaces and cancer stages. In stage I-II cancer, UNITE-XGB and UNITE-CNN achieved 31 and 21% sensitivity, respectively, at 95% specificity. These findings provide roadmaps for developing multifeature AI beyond plasma biopsies.

Medical artificial intelligence research is shifting from single-task models toward multimodal large language model-based agents for complex clinical workflows, requiring benchmarks that assess clinical reasoning, process safety, and resource stewardship rather than final outputs alone.

A discredited trial has fractured trust in one landmark result-the first randomized evidence on checkpoint inhibitor dosing timing-but not the broader hypothesis it was meant to prove: that circadian biology shapes immunotherapy efficacy. Researchers on both sides of the debate are now recalculating what the surviving evidence can actually support.

Chimeric antigen receptor (CAR) T cell therapy faces challenges, such as tumor relapse due to antigen loss and poor CAR T cell expansion in vivo. Optimal tonic signaling (spontaneous CAR signaling) is crucial for CAR T cell fitness and antitumor function, requiring precise calibration to avoid dysfunction or exhaustion. Although CAR phase separation can enhance CAR clustering and tonic signaling to improve cytotoxicity and antigen sensitivity, constitutively high signaling could lead to potential side effects, including cytokine storm and neuronal toxicity. To overcome this limitation, we developed a drug-inducible phase-separation CAR (iPhase-CAR) system utilizing FDA-approved fulvestrant and an engineered estrogen receptor α (ERα) mutant as the drug-inducible phase-separation (DiPS) module. Incorporating the DiPS module into CARs enables drug-inducible clustering of CARs on T cell membranes. iPhase-CAR T cells demonstrate superior antitumor function. This system provides quantitative, reversible promotion of CAR T effector functions, enabling precise control over therapeutic efficacy only upon administration of the FDA-approved drug.

Peptidylprolyl isomerase A (PPIA) catalyzes cis-trans isomerization of proline residues, a key process regulating protein folding and signal transduction. Its aberrant secretion is closely associated with tumor metastasis and progression, making it a promising biomarker. However, currently available immunoassays often suffer from insufficient sensitivity for low-abundance PPIA detection. Herein, we present an electrochemical biosensor that integrates self-propelled nanocarriers with DNA cascade amplification strategy for ultrasensitive PPIA detection. The biosensor employs PtNPs@COF particles that catalyze H2O2 decomposition to generate O2. This autonomous propulsion accelerates target binding in homogeneous solution and helps mitigate diffusion-limited binding kinetics. After PPIA binding, the liberated DNAzyme catalytically cleaves the co-immobilized substrate strands, generating numerous triggers for the catalytic hairpin assembly (CHA) reaction. The biosensor achieves a wide linear range from 1 pg/mL to 10 μg/mL with a detection limit of 0.330 pg/mL. It also exhibits excellent specificity and performs reliably in clinical lung adenocarcinoma serum specimens, demonstrating its promising applicability for early diagnosis of this malignancy.

Accurate deconvolution of cell states from bulk tumor RNA-seq is hindered by heterogeneous malignant cells specifically in cancer applications. We present Statescope, a Bayesian framework that incorporates DNA-derived malignant cell purity to overcome this heterogeneity and explicitly models inter-sample variation to accurately identify cell states. Comprehensive benchmarking shows Statescope outperforms existing methods in both cell fraction and state estimation, and is unique in its ability to identify states entirely absent from single-cell references. In real-data applications, Statescope successfully recapitulates established cell states, including multiple states in neutrophils, a cell type often missed by single-cell methods in lung cancer. Critically, in the POPLAR/OAK clinical trials, Statescope identifies a combinatorial signature of effector CD8 + T cells and conventional dendritic cell states that together predict a striking survival benefit from immunotherapy. Collectively, Statescope transforms deconvolution into a versatile discovery platform, enabling deeper biological and clinical insights from widely available bulk multi-omics data.

The conformational dynamics of METTL3-METTL14 (M3/M14) heterodimer, the catalytic core for N6-methyladenosine (m6A) deposition, remain largely unexplored, limiting insight into dynamic regulation of catalysis and opportunities for therapeutic targeting in cancer. Here, we report the first single-molecule visualization of M3/M14 dynamics by high-speed atomic force microscopy (HS-AFM). Our measurements show that substrate RNA binding induces a conformational transition from a rigid apo heterodimer with an open interlobe groove to a compact, catalytically competent state. Guided by these dynamic insights, we identify two potent DNA aptamer inhibitors of M3/M14, M3B, and M3L, using a competitive in vitro selection strategy. HS-AFM integrated with molecular docking reveals that both aptamers insert into the M3/M14 interface, forming a sandwich-like complex that stabilizes a distorted, open conformation and prevents RNA-induced compaction. This conformational trapping inhibits methyltransferase activity, reduces global m6A levels, and suppresses A549 lung cancer cell growth. These findings define a dynamically regulated, interlobe targetable state of the m6A writer complex and demonstrate the utility of HS-AFM for uncovering dynamic regulatory mechanisms and guiding the development of conformationally targeted therapeutics in epitranscriptomic biology.

Despite compelling evidence that tumor-derived extracellular vesicles (TEVs) exhibit either pro- or antitumorigenic phenotypes, pharmacological efforts have focused primarily on their indiscriminate suppression. Here, we propose a strategy of "switching TEVs off and on" to redirect them toward an immunogenic phenotype. Designed as a nanoproteolysis-targeting chimera (Nano-PROTAC) for TEV reprogramming, EVOTAC is composed of tripartite building blocks that integrate a PROTAC and a photosensitizer via a cancer biomarker-responsive cleavable linker and spontaneously self-assemble into supramolecular nanostructures. Upon biomarker-guided activation preferentially in tumors over normal tissues, EVOTAC initially eliminates TEVs by selectively degrading intracellular proteins involved in extracellular vesicle (EV) biogenesis. Subsequent localized laser irradiation reactivates EV generation, prompting tumor cells to predominantly produce immunogenic TEVs in response to photodynamic therapy (PDT). TEVs generated through this switching-off-and-on strategy independently exert pleiotropic effects by inhibiting tumor growth, migration, and metastasis while increasing mature dendritic cells and cytotoxic T lymphocytes in lymphoid organs and tumor tissues. This TEV-toggling process, therefore, significantly enhances both innate and adaptive immune responses to photoimmunotherapy, which leads to a complete regression of triple-negative breast cancer (TNBC) and prevents metastasis and recurrence. Our study highlights the potential of this therapeutic approach for precise TEV modulation and encourages further exploration, adding new breadth to the growing list of EV-targeting cancer immunotherapy concepts.

Silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (SiNc) possesses intense near-infrared absorption, promising NIR-II emission, and a high reactive oxygen species quantum yield in its monomeric state. However, its potential as a NIR-II fluorescent photosensitizer has been long hindered by aggregation-caused quenching in aqueous environments. Herein, we report a supramolecular engineering strategy to overcome this limitation by precisely tuning the molecular packing of SiNc within a biocompatible DSPE-PEG2000 matrix. Through systematic optimization of the dye-to-matrix ratio, initial dye concentration, and solvent composition, we obtain optimized nanoparticles (SiNc NPs) that effectively disrupt tight π-π stacking. Compared to severely quenched control nanoparticles (Ctrl-NPs), SiNc NPs exhibit a 3-fold enhancement in fluorescence quantum yield (4.2% vs 1.4%), alongside 17.7-fold and 8.7-fold increases in 1O2 (ΦΔ = 10.6% vs 0.6%) and O2•- generation, respectively. Consequently, SiNc NPs deliver potent NIR-II fluorescence-guided photodynamic efficacy in vivo. Following a single low-dose intravenous injection and one session of 781 nm laser irradiation, SiNc NPs achieve near-complete 4T1 tumor eradication, showing improved efficacy compared to both a clinical photosensitizer (Ce6) and Ctrl-NPs under the tested conditions. This work establishes a programmable supramolecular paradigm for reactivating highly quenched, aggregation-prone chromophores, offering a practical alternative to complex de novo synthesis in the pursuit of high-performance NIR-II fluorescent photosensitizers capable of effective photodynamic monotherapy.

Nanoparticle mechanical properties, as critical factors in tumor targeting drug delivery, have recently been revealed to regulate cellular biological functions. While macroscopic mechanical stimuli are demonstrated to reprogram tumor-associated macrophages (TAMs), it is unclear how TAMs respond to microscopic stimuli from nanoparticles. Herein, we demonstrated that the stiff 15%NGs presented stronger interactions with integrin than the soft 2%NGs, promoting the polymerization of actin and the activation of yes-associated protein 1. Therefore, the pro-inflammatory phenotype of TAMs was enhanced and the secretion of tumor necrosis factor α was increased. In synergy with high liver enrichment, 15%NGs exhibited superior tumor suppression by reprogramming TAMs in orthotopic liver tumors. Crucially, the pro-inflammatory phenotype of macrophages reprogrammed by 15%NGs significantly induced the apoptosis of tumor cells in ex vivo hepatocellular carcinoma tissues from patients. This study proposes a mechanical signaling pathway to reprogram TAMs by manipulating nanogel stiffness and provides new insights for rational design of cancer nanomedicines.

Intratumoral signaling via the cytosolic double-stranded RNA (dsRNA) receptors RIG-I and MDA-5 enhance antitumor immunity and can overcome resistance to cancer immunotherapy. Although BET proteins are established epigenetic drivers of immune escape mechanisms, their role in regulating RIG-I-like receptor (RLR) signaling remains undefined. Therefore, we investigated the role of BET proteins in modulating innate antiviral responses to cytosolic dsRNA in tumor cells and how BET inhibition combined with dsRNA stimulation influences tumor immunogenicity and recognition by T cells. To address this, human tumor cell lines were treated with the BET inhibitor JQ1 and transfected with the synthetic viral dsRNA analog poly(I:C) to stimulate innate antiviral signaling. We found that BET inhibition synergistically amplifies dsRNA-induced immunogenicity, as indicated by enhanced cytokine and chemokine production, JAK-STAT signaling, antigen presentation, and immunogenic cell death. Combined treatment of melanoma cells with JQ1 and poly(I:C) resulted in a marked, synergistic enhancement of recognition by autologous tumor-specific CD8+ TIL, immunogenic cell death and maturation of dendritic cells. Furthermore, an antiviral gene signature induced by poly(I:C) and enhanced by JQ1 correlated with immune infiltration and improved survival in larger patient cohorts across different types of cancer. Overall, our findings demonstrate that targeting BET proteins in tumor cells strongly potentiates dsRNA-induced antiviral responses and suggests that combining BET inhibitors with RLR agonists offers a pharmacological strategy to enhance antitumor T cells responses and improve the clinical efficacy of cancer immunotherapies.

Δ1-Pyrroline-5-carboxylate synthase (P5CS), therate-limiting enzyme in the proline biosynthesis, has been implicated in diverse physiology and pathology, including cancer. However, whether P5CS exerts functions beyond its enzymatic activity has remained unclear. Here, we identify P5CS as a non-canonical RNA-binding protein that inhibits cancer cell growth and metastasis by inhibiting translation initiation in an enzyme-activity-independent manner in human cancer cells and cell-derived xenograft mouse models. Mechanistically, P5CS binds to the 5' untranslated region (UTR) of oncogenic mRNAs and disrupts the recruitment of eukaryotic translation initiation factor (eIF) 3a/3d-containing 43S preinitiation complex to cap-binding complex eIF4F, thereby blocking 48S assembly and subsequent global protein synthesis. Loss of P5CS accelerates the translational efficiency of IGF1R and promotes tumor progression. Collectively, our study highlights a non-canonical function of P5CS in translational regulation and the emerging non-metabolic functions of metabolic enzymes in tumorigenesis.

Targeted delivery of chemodrugs to tumor sites is highly desirable for efficient chemotherapy. Extracellular vesicles (EVs)-based carrier is an ideal candidate for drug delivery in cancer therapy. However, the insufficient tumor-targeting capability remains to be solved. Herein, we designed an aptamer circuit with cascade recognition capacity to guide the vesicles for tumor targeting. The sequential signal transduction triggered by the specific binding between aptamers and their targets leads to the preferential binding of vesicles with cancerous cells rather than noncancerous cells with fewer targets. Moreover, a self-promoted tumor-targeting loop induced by Doxorubicin Hydrochloride (DOX) released from vesicles further amplified the targeting capability of the vesicle-based drug carrier to tumor sites through up-regulating PDL1 marker on the surface of tumor cells. Ultimately, the accumulation of DOX in tumor cells resulted in the immunogenic cell death (ICD) for evoking anti-tumor immune response. Overall, this work presents a self-promoted tumor-targeting strategy for vesicle-based carrier by coupling the cascade recognition and tumor-targeting loop, which improves the therapeutic effect of immunogenic chemotherapy.

ConspectusImmune checkpoint blockade therapies have revolutionized cancer treatment. However, their clinical efficacy remains limited by dysfunctional T-cell states within the tumor microenvironment. These limitations are particularly evident in aged hosts, where metabolic and signaling impairments compromise immune fitness and reduce responsiveness to PD-1/PD-L1-directed therapies. Emerging evidence suggests that chemical modulation of immune cell function represents a promising strategy to overcome these barriers. Our work explores how small molecules can be leveraged to restore T-cell activity and potentiate antitumor immunity through complementary chemical mechanisms. By integrating cell-based screening with electrophile-focused chemoproteomics, we identified a covalent small-molecule activator, arvenin I, which engages a ligandable cysteine in MKK3, promoting signaling programs that revive exhausted T-cells and synergize with immune checkpoint blockade. In parallel, previous studies revealed that age-associated depletion of the endogenous polyamine spermidine contributes to impaired T-cell metabolism and diminished responses to checkpoint blockade. Using chemoproteomic tools, we profiled spermidine-interacting proteins and found that the majority were mitochondrial proteins, including lipid-metabolism factors. This chemoproteomic platform also enabled the identification of a biostable spermidine mimetic that restores mitochondrial fitness and enhances antitumor immune responses in vivo. Together, these studies establish a unified chemical biology framework in which covalent signaling activation and metabolite-inspired energy support converge to restore T-cell fitness. This Account highlights how chemoproteomic discovery can guide the development of immune-activating small molecules and underscores the potential of chemical approaches to complement and extend the impact of cancer immunotherapy.

Cancer cells avert apoptosis to sustain growth, evade immunity, and resist therapy. However, how they escape specific extrinsic death signals remains unclear. We show that certain epithelial carcinomas co-opt the death-receptor ligand Apo2L/TRAIL to suppress apoptosis while acquiring killing capacity. Analysis of CRISPR knockout screens from the Cancer Dependency Map uniquely identified TRAIL as an essential tumor necrosis factor superfamily member in epithelial malignancies. Renal cell carcinomas (RCC) and squamous cell carcinomas (SCC) expressed TRAIL within both malignant and nonmalignant compartments. In RCC, loss of the tumor suppressor VHL induced HIF2α-dependent TRAIL transcription, whereas in SCC, 3q26 amplification increased TRAIL abundance. Fractional TRAIL silencing revealed killing of TRAIL-deficient cells by TRAIL-expressing counterparts, while full silencing sensitized cells to extrinsic TRAIL. Mechanistically, endogenous TRAIL suppressed apoptosis through lysosomal degradation of its cognate death receptors. Thus, cancer cells can repurpose TRAIL via a surprising apoptotic "cloak-and-dagger" mechanism with significant therapeutic potential.

Elevated intracellular glutathione, intratumoral hypoxia, and excessive polyamines severely compromise the efficacy of radiotherapy (RT) by attenuating radiation damage and maintaining an immunosuppressive tumor microenvironment (ITME). Strategies that concurrently address these obstacles are urgently needed to enhance RT outcomes. Herein, we fabricated a biomimetic nanoplatform (PCuP) via the self-assembly of copper ions and piceatannol into CuP nanometal-polyphenol cores, followed by surface camouflage with platelet membranes (PM). Owing to the platelet membrane coating, PCuP preferentially accumulates in tumor tissues with disrupted vasculature. At the tumor site, PCuP catalyzes hydrogen peroxide to produce oxygen, relieving hypoxia and sensitizing the tumors to RT. Simultaneously, it generates reactive oxygen species (ROS) that cooperate with RT to induce immunogenic cell death (ICD). Furthermore, PCuP inhibits arginase 2 (Arg2) to deplete polyamines, suppressing tumor DNA repair and remodeling the ITME. In vivo experiments using oral squamous cell carcinoma (OSCC) models demonstrated that PCuP promotes dendritic cell maturation, activates systemic antitumor immunity, and drastically reduces post-RT tumor recurrence. This study presents the first nanoplatform integrating polyamine depletion with radioimmunotherapy, offering a promising strategy to design advanced radiosensitizers for improving RT efficacy and lowering tumor recurrence.

Conventional localized drug delivery systems (DDS) and chemodynamic therapy (CDT) platforms often lack structural integrity, hydration-mediated transport control, and architectures capable of sustaining redox activity at tumor sites. Here, we report a hybrid nanofibrous hydrogel that integrates CoMn layered double hydroxide (CoMn-LDH) within electrospun polycaprolactone (PCL) fibers, followed by a polyacrylamide (PAAm) hydrogel coating to construct a localized, ECM-mimetic therapeutic platform with dual functionality. Unlike Conventional CDT systems that treat drug delivery and catalytic activity independently, this design integrates a hydrated diffusion network with redox-active centers within a single architecture. The CoMn@PCL/PAAm HNF exhibits enhanced wettability of 26.4° and pronounced swelling-assisted transport, enabling sustained and non-Fickian release with cumulative release of 96.99%, 86.77%, and 97.83% at pH 6.2, 7.4, and 8.8, respectively. Furthermore, the system promotes peroxide-activated ROS generation, enhances intracellular oxidative stress, and induces apoptosis-mediated cytotoxicity against HuH7 and SiHa cells, with inhibition rates of 64.73% and 52.03%, respectively, while remaining nonhemolytic. These results indicate that interface-engineered transport reaction coupling governs both drug diffusion and ROS generation, highlighting the potential of CoMn@PCL/PAAm HNF for CDT integrated implantable anticancer applications with reduced system toxicity.

Epithelial-to-mesenchymal transition (EMT) is a dynamic process during which cells lose their epithelial characteristics and acquire mesenchymal traits. In cancer, EMT is closely associated with tumor initiation, progression, invasion, metastasis, and therapy resistance. Rather than being a binary state switch, EMT encompasses a spectrum of tumor states with distinct functional properties. However, the transcription factors (TFs) that govern transitions between these EMT states remain poorly defined. Here, using multi-omic approaches combining single-cell RNA-seq and single-cell ATAC-seq, we delineate the transcriptomic and chromatin landscapes of distinct EMT states in a mouse model of skin squamous cell carcinoma (SCC). Through CRISPR/Cas9-mediated loss-of-function studies coupled with in vitro and in vivo functional assays, we identify TFs regulating specific EMT states. Klf5 and Pitx1 control the early stages of EMT and are essential for metastasis formation. In contrast, Nfatc1 and Creb3l1 act at later stages of EMT. Similar EMT states and regulatory patterns are found in mouse pancreatic adenocarcinoma and human cancers. Altogether, our study defines the transcriptional and chromatin landscape controlling EMT progression in mouse skin SCC, identifies EMT state-specific TFs and highlights their essential roles in regulating metastasis.

PROTACs are commonly developed by linking E3 ligase-recruiting ligands to established inhibitors of a protein target, often resulting in degraders that retain enzymatic inhibition. Type II inhibition of cyclin-dependent kinases (CDKs) has been challenging, as reported compounds generally exhibit weak biochemical potency and limited cellular activity. Consistent with these limitations, most reported CDK degraders have been derived from type I ATP-competitive inhibitors. Here, we explored whether targeted protein degradation could enable functional CDK targeting from a type II kinase scaffold. Using the multikinase inhibitor regorafenib as a starting scaffold, we generated a focused library of CRL4CRBN-recruiting bifunctional molecules and profiled their degradation activity using quantitative mass spectrometry-based proteomics. This analysis unexpectedly revealed CDK5 and CDK6, kinases not inhibited by the parent scaffold, as degradation targets. Optimization of this series led to JHK-02-108-2, a selective CDK6 degrader that does not display a hook effect and promotes potent CDK6 degradation despite weak CDK6 binding and negligible CDK6 inhibition. In cellular models of acute myeloid leukemia (AML) and glioblastoma, JHK-02-108-2 induced sustained G1 arrest and reduced phosphorylation of the retinoblastoma protein. Interestingly, subtle modifications in PROTAC architecture redirected degradation selectivity, yielding JHK-02-102-1 as a selective type II CDK5 degrader derived from the same scaffold. Together, these findings establish the first type II inhibitor-derived selective CDK6 degrader and demonstrate that targeted protein degradation can enable functional CDK targeting from type II kinase scaffolds.

In mammalian cells, gene-distal regulatory elements enable long-range gene regulation and support cell-type-specific transcriptional programs. This regulatory architecture is frequently perturbed in cancer, particularly when oncogenic transcription factors are targeted therapeutically. However, how cancer cells adapt under such selective pressure has remained poorly understood. Here we show that mesothelioma cells dependent on the oncogenic TEAD family of transcription factors acquire resistance to a pan-TEAD inhibitor. Such resistance is accompanied by a promoter-centric regulatory mechanism, a process we term promoter reinforcement, to sustain gene expression following TEAD inhibition. Using base-pair-resolution Micro Capture-C on a set of TEAD target genes, we find that regulatory element-promoter interactions are weakened or lost in resistant cells, even as promoter activity and gene expression recover in the context of partial epigenetic restoration. Mechanistically, resistance-induced transcription factors show promoter-biased localization and can increase promoter activity, whereas distal regulatory element function can become dispensable. Together, these findings identify promoter reinforcement as a locus-specific compensatory response that supports transcriptional resilience under TEAD inhibition, indicating promoter-associated vulnerabilities in drug-resistant cancer.

Multispecific antibodies (MsAbs), capable of targeting multiple antigens simultaneously, are regarded as the 2.0 version of monoclonal antibodies (mAbs), providing therapeutic benefits that significantly exceed those of mAbs. This enhanced functionality makes MsAbs a particularly promising strategy for treating solid tumors. In this comprehensive review, we summarize diverse targeting strategies, clinical trial outcomes, and existing hurdles, while proposing novel directions to enhance the development of MsAbs for overcoming the immunosuppressive tumor microenvironment (TME). Based on their therapeutic mechanisms, we categorize their actions into six principles: 1) blocking various oncogenic signaling pathways; 2) activating and redirecting cytotoxic immune cells toward tumor cells; 3) enhancing macrophage-mediated phagocytosis; 4) targeting immune checkpoints to alleviate immunosuppression; 5) modulating matrix components within the immunosuppressive TME; and 6) promoting immune cell infiltration and localized cytokine signaling. In addition, the integration of MsAbs with sophisticated technologies has opened new avenues for enhancing therapeutic efficacy. These approaches include antibody-degrader conjugates, chimeric antigen receptor T cell (CAR-T) therapies, antibody-oncolytic virus combinations, nanomaterial-based delivery systems, and messenger RNA (mRNA)-based technologies. Such innovative strategies highlight the transformative potential of MsAbs, positioning them at the forefront of next-generation cancer immunotherapies.

Emerging data suggest fecal microbiota transplantation (FMT) may improve cancer patients' responses to immune checkpoint blockade not only by enriching beneficial bacteria but also by depleting harmful taxa. Here, we discuss the "supplementation" and new "depletion" FMT paradigms in cancer management and highlight key knowledge gaps to be addressed to move this field forward.

mRNA-based cancer vaccines offer a modular and safe platform to elicit antitumor immunity, yet their efficacy is often limited by inefficient mRNA delivery and inadequate dendritic cell (DC) activation, both of which are essential for initiating robust cytotoxic T cell responses. Inadequate innate immune activation coupled with poor antigen presentation further diminishes their effectiveness, particularly in immunologically "cold" tumors. While stimulator of interferon genes (STING) agonists can enhance DC maturation and cross-presentation, their therapeutic utility is constrained by poor intracellular delivery and limited colocalization with tumor antigens. In this study, we developed a lipid nanoparticle (LNP) platform via high-throughput screening of ionizable lipids for potent mRNA delivery to DCs both in vitro and in vivo. To amplify immune activation, we coencapsulated the STING agonists c-di-AMP (AMP) and manganese (Mn2+) together with tumor antigen-encoding mRNA into the lead LNP formulation. This codelivery strategy synergistically activated type I interferon signaling, upregulated costimulatory molecules, enhanced antigen presentation, and elicited potent tumor-specific T cell responses and superior antitumor efficacy. Our results demonstrate that integrating innate immune stimulation with mRNA-LNP delivery provides a promising strategy to overcome current limitations in mRNA vaccine efficacy and to improve cancer immunotherapy outcomes.

Cancer cells confronting oxidative stress must coordinate their extracellular vesicle (EV) secretion to balance intercellular signaling with the intracellular programs required for survival, yet how these decisions are integrated remains poorly understood. Here, we identify a stress-adaptive mechanism in which stress granules (SGs) selectively suppress CD63+ EV release. Using a bioluminescent EV-reporter screen, we found that the clinical compound YM155 selectively inhibits CD63+ EV secretion across diverse tumor cells. Mechanistically, YM155 rapidly inactivates the antioxidant transcription factor FOXO3a, diminishing expression of key detoxifying enzymes and leading to delayed but sustained accumulation of reactive oxygen species (ROS). Elevated ROS drives SG formation, and these SGs function not as passive storage sites but as RNA triage hubs that exclude and destabilize a subset of transcripts. Among them, Rab27A mRNA-encoding a GTPase essential for multivesicular-body docking to the plasma membrane-is selectively excluded and degraded, resulting in loss of Rab27A protein and suppression of CD63+ EV secretion. Forced Rab27A expression restores EV release but paradoxically reduces proliferation under oxidative stress, indicating that EV suppression is prosurvival. The same FOXO3a-ROS-SG-Rab27A axis operates during physiological glucose deprivation and is evident in vivo, where SGs form in xenograft tumors and circulating CD63+ EVs decline. Pancancer transcriptomic analyses further show that Rab27A expression correlates with FOXO3a-dependent antioxidant programs, underscoring clinical relevance. These findings reveal that SGs actively reprogram RNA fate to tune vesicle output, establishing a redox-responsive mechanism by which cancer cells transiently suppress EV secretion to enhance survival.

Transposable elements (TEs) represent an abundant and important source of HLA-presented antigens, but their immunopeptidomic characterization remains challenging due to the inflated search space. We present TIPs (TE-derived Immunopeptidomic Search), a deep learning-guided proteogenomic framework that integrates de novo sequencing, database refinement, multiple search engines and stringent FDR controls. Across various cell lines and cancer types, TIPs identified 20-fold more TE-derived peptides on average than conventional approaches. It further revealed many recurrent, tumor-specific antigens from TEs, including candidates induced by epigenetic therapy. These findings highlight the potential of TIPs to expand the antigenic landscape beyond canonical sources.

Cancer multi-omics faces challenges in handling the scale, complexity, and heterogeneity of multi-omics data, limiting progress in variant interpretation, tumor classification, and modeling cancer evolution. Quantum computing offers a new paradigm using superposition, entanglement, and quantum interference to efficiently explore vast solution spaces. This Perspective highlights how quantum algorithms-such as Quantum Support Vector Machines, Quantum Principal Component Analysis, and quantum generative models-could enhance key tasks in precision oncology, including multi-omics integration, spatial transcriptomics, and neoantigen prediction. Current technical barriers, like qubit noise and limited quantum memory, are discussed alongside strategies to connect quantum computing with biomedical research. Interdisciplinary collaboration will be essential to realizing quantum advantage in cancer multi-omics.

ConspectusPlatinum (Pt)-based anticancer drugs have been a cornerstone of chemotherapy for decades, yet their clinical application remains constrained by dose-limiting systemic toxicity and drug resistance. In this Account, we summarize our systematic efforts to address these challenges through two complementary strategies: 1) functionalization of Pt(IV) prodrugs and 2) spatially controlled targeted delivery. The kinetic inertness and octahedral geometry of Pt(IV) complexes offer a versatile platform for axial functionalization, allowing the integration of diverse bioactive ligands that are released upon intracellular reduction. Exploiting this feature, we have developed multifunctional Pt(IV) prodrugs that co-target DNA damage repair and apoptotic pathways, rewire cholesterol and energy metabolism, induce nonapoptotic cell death including PANoptosis and autophagy-associated death, and epigenetically silence resistance-associated gene networks via chromatin compaction. To engage the tumor immune microenvironment, we have incorporated immunomodulators─including STING agonists, TREM2/CD33 inhibitors, and STAT3 blockers─to amplify innate and adaptive antitumor immunity. Furthermore, we have developed radiotherapy-responsive Pt(IV) prodrugs that undergo rapid, X-ray-triggered reduction mediated by hydrated electrons, enabling spatiotemporally precise drug activation with markedly attenuated systemic toxicity. This strategy is currently advancing toward clinical translation through IND-enabling studies. In parallel, we have established targeted delivery platforms to improve the spatial precision of Pt agents. Mitochondria-targeted complexes redirect cytotoxicity to an organelle lacking efficient DNA repair, disrupting bioenergetics and triggering intrinsic apoptosis. At the tissue level, biotin-mediated targeting exploits overexpressed vitamin transporters for tumor-selective accumulation, while Pt(IV)-antibody conjugates (Pt-ADCs) achieve antigen-specific delivery, upregulate tumor MHC-I expression, expand TCR clonotypes, and synergize with PD-1 blockade. Additionally, a stimuli-responsive in situ self-assembly strategy enables enzyme-triggered nanostructure formation and intracellular disassembly for enhanced tumor accumulation and burst drug release. An immunocompetent patient-derived organoid platform has been established to screen these agents in a clinically relevant setting. The integration of multifunctional modulation, targeted delivery, and externally controlled activation within single Pt-based systems creates a synergistic framework that simultaneously addresses resistance and toxicity. Moving forward, our research will focus on optimizing pharmaceutical properties, advancing radiotherapy-responsive Pt(IV) prodrugs and Pt-ADCs toward clinical evaluation, and refining predictive screening platforms. These programmable Pt therapeutics hold considerable promise for delivering safer and more effective precision chemotherapy to cancer patients.

The human acetyltransferase complex HBO1 is implicated in cancer and developmental diseases. Here, we report the chromatin association mechanisms for scaffolding subunits of the complex, the JADE and BRPF paralogs. Structural and biochemical studies reveal a distinct paralog-specific engagement of the PZP domains of JADE1/2/3 and BRPF1 with the nucleosome. We show that only JADE1's PZP domain possesses a strong intrinsic DNA binding activity and binds to an almost entire histone H3 tail, forming a tight complex with the nucleosome. In contrast, the BRPF1 PZP domain's association with the nucleosome is a thousand-fold weaker due to its inability to engage with the large portion of H3 tail and a weak binding to DNA. These results suggest that the JADE1 PZP domain can protect the H3 tail from being modified, but the BRPF1 PZP domain cannot, which could contribute to functional differences observed in the HBO1 complexes. We demonstrate non-redundant functions of the paralogs throughout embryonic development and embryonic stem cell pluripotency, during cardiac cell differentiation, and in differentiated liver cells. Our findings provide insights into the diverse mechanisms of action of the HBO1 complex and help to understand how the scaffolding subunits mediate its different cellular functions.

Accurate identification of immunogenic cancer epitopes remains a central challenge in immuno-oncology. The Cancer Epitope Database and Analysis Resource (CEDAR, https://cedar.iedb.org/) was developed to provide comprehensive curation of experimentally validated epitopes and to foster the development of computational tools tailored to the cancer context. Recently, we released a suite of cancer-specific tools and analysis pipelines as part of the https://nextgen-tools.iedb.org/ platform, enabling users to generate, evaluate, and prioritize candidate T cell epitopes in a modular framework. Here, we present the design and functionality of these tools, describe their core methodologies, provide guidance for their use, and illustrate how they can be integrated into end-to-end pipelines. We highlight applications in cancer immunology and personalized immunotherapy by presenting practical use cases.

SNPnexus is a long-standing web-based platform for the functional annotation and prioritization of genetic variants. Since its previous release, SNPnexus has undergone substantial backend re-engineering resulting in major performance improvements and a complete restructuring of the underlying datasets. A redesigned interface now enables larger queries and multi-sample analysis enabling comparative workflows such as identifying shared pathogenic variants in disease cohorts and divergent mutations in cancer evolution studies. SNPnexus increased its capacity to 150 000 variants per query and introduced pre-annotation filters that allow users to restrict analyses to selected genes or genomic regions, improving efficiency while enabling targeted interrogation of high-priority targets. SNPnexus integrates updated and expanded annotations across both GRCh37 and GRCh38, covering genomic consequences, in silico pathogenicity predictions, population allele frequencies, evolutionary conservation, regulatory elements, biological pathways, and clinical associations. The refreshed result interface provides interactive visualizations for single-sample and cohort-level outputs, together with advanced filtering and export options. Optional user accounts now support query history and real-time job monitoring while preserving full, unregistered access for all users. SNPnexus remains free and open to all users without login requirements at https://snpnexus.org/.

DAVID is a widely used bioinformatics resource that provides functional annotation and functional enrichment analysis for gene and protein lists derived from high-throughput studies. It integrates a comprehensive gene-centered knowledgebase with a suite of web-accessible analytical tools. Since its initial release in 2003, DAVID developments have been published in 12 papers and cited >80 000 times. Here, we report updates made since the previous NAR Web Server Issue publication in 2022. This update introduces two new tools: DAVID Ortholog for cross-species functional analysis and DAVID Gene Search for identifier-agnostic gene exploration, modernizes the web interface, and implements a new backend architecture that decouples the frontend from the legacy Java processing engine. A new Servlet layer and REST APIs enable asynchronous processing and support integration of a Neo4j graph database for relationship-based queries. Major existing tools have been redesigned with modern, interactive interfaces, and multiformat result export. The pathway viewer has been redesigned with interactive drag-and-zoom navigation, animated user gene highlighting, and publication-quality downloads. Collectively, these updates enhance performance and usability, and the new backend architecture enables independent evolution of frontend and backend components while maintaining continuity with legacy analyses. DAVID remains freely available at https://davidbioinformatics.nih.gov without login.

RNA secondary structure plays a critical role in gene regulation, yet existing computational and experimental tools for structure analysis are often fragmented across prediction, ensemble modeling, and functional interpretation workflows. Here, we present ShapeRNA, a user-friendly web server for integrated RNA secondary structure prediction, ensemble inference, and structure-aware regulatory annotation. ShapeRNA supports three complementary analytical workflows, including sequence-based structure prediction, reactivity-guided modeling using SHAPE or DMS data, and sequencing-guided ensemble inference from high-throughput probing experiments. The platform integrates multiple established prediction algorithms and provides standardized data processing, ensemble clustering, and visualization. In addition, ShapeRNA enables mapping of RNA modification sites, microRNA target regions, and RNA-binding protein interaction motifs onto predicted RNA structures and representative ensemble conformations. We demonstrate the utility of ShapeRNA through applications including analysis of mutation-associated structural changes in MAPT exon 10, characterization of conformational heterogeneity in the HIV-1 Rev Response Element, and regulatory annotation of the oncogenic long non-coding RNA HULC. ShapeRNA provides an accessible and extensible platform for investigating RNA structural heterogeneity and regulatory mechanisms. This website is free and open to all users, and there is no login requirement. The server is accessible at https://shaperna.com.

Radiotherapy remains a cornerstone in the clinical management of malignancies, leveraging DNA damage and oxidative stress to eradicate tumor cells. Nonetheless, the emergence of intrinsic and acquired radioresistance significantly compromises its therapeutic efficacy. While nanomedicine has substantially advanced radiosensitization strategies, the existing literature has largely focused on physical dose enhancement or conventional apoptosis, and the systematic reprogramming of diverse cell death modes beyond conventional apoptosis in the radiotherapy context has received less systematic attention. The present review provides a cross‑pathway synthesis of how engineered nanomaterials redirect tumor cell fate beyond apoptosis to achieve next‑generation radiosensitization, while also identifying the specific limitations and knowledge gaps that currently impede progress in this rapidly evolving field. We first delineate the hierarchical sensitization mechanisms, beginning with physical energy deposition via high-Z elements, followed by chemical amplification of reactive oxygen species through nanozyme catalysis, and biological intervention in the "6R" principles of radiobiology. Crucially, we evaluate the potential capacity of advanced nanomaterials to bypass conventional apoptotic resistance by triggering ferroptosis, pyroptosis, cuproptosis, disulfidptosis, and other emerging programmed death pathways, with a focus on the current evidence base and remaining preclinical and translational challenges. Beyond localized cytotoxicity, we highlight the mechanistic potential of nanomedicine to induce immunogenic cell death and activate the cGAS-STING pathway, suggesting a possible framework for transforming RT into an "in situ vaccine" that could reshape the immunosuppressive TME. Furthermore, we discuss the clinical translation of landmark nano-radiosensitizers, such as NBTXR3 and AGuIX, while critically addressing fundamental bottlenecks in targeting efficiency, biodistribution, and biosafety. By synthesizing current trends and future perspectives, this review contributes a strategic roadmap for advancing the design of next-generation, intelligent nanoplatforms toward more precise and systemic radiosensitization.

Targeted protein degradation repurposes endogenous E3 ubiquitin ligases to eliminate disease-driving proteins, yet the ligase toolkit deployed clinically remains narrow and largely tissue-agnostic. To support rational expansion of this toolkit, we built a harmonized pan-tissue proteomic atlas of the ubiquitin-proteasome system (UPS) by integrating four major resources: (1) CPTAC tumor and normal-adjacent tissues, (2) PRIDE healthy tissues, (3) the Pan-Cancer Proteome Atlas (TPCPA), and (4) the Cancer Cell Line Encyclopedia (CCLE). The resulting atlas spans 20 distinct tissue contexts and quantifies 5998 proteins, including 473 UPS components and 181 E3 ligases. Cross-resource validation confirmed successful harmonization while maintaining biological signal. We then derived a sample-level relative rank score (RRS) for every quantified UPS protein and identified 139 E3 ligases (of 181 detected) as being significantly tissue- or tumor-specific, including XIAP in lung cancer, KLHL7 in female-specific malignancies, and FBXL18 in head-and-neck and brain tumors. To enable broad accessibility, we developed UbiDash ( https://ruggleslab.shinyapps.io/UbiDash/ ), an interactive R Shiny platform that supports queries of UPS expression, mutation effects, protein co-regulation, and clinical associations. Together, the atlas and UbiDash provide a tissue-aware framework for ligase prioritization and rational degrader design that complements the mechanistic mutation- and lineage-driven UPS analyses described in our companion manuscript [1].

Immunogenic cell death (ICD) is a type of cell death that can enhance anti-tumour immune responses of chemotherapies and targeted therapies by releasing DAMPs and cytokines that activate dendritic cells and T cells, thereby engaging the patient's immune system to combat the cancer. Pyroptosis and necroptosis are strongly immunogenic because they release DAMPs and inflammatory signals through pore-forming proteins, whereas apoptosis can be tolerogenic. This immunogenic response is contingent on a functional immune system. Unfortunately, most conventional chemotherapies and many targeted therapies also impair the immune system. Here, we investigated the mechanism by which the tumour-selective treatment of Checkpoint kinase 1 inhibitor (CHK1i) combined with low-dose hydroxyurea (LDHU) promotes ICD and anti-tumour immunity. We show that CHK1i+LDHU induces S-phase arrest and caspase-dependent lytic cell death with features of pyroptosis, including gasdermin E cleavage, but cell death was not dependent solely on gasdermin cleavage. Inhibiting caspases was sufficient to block both tumour cell killing and treatment immunogenicity. The mechanism does not rely on any single caspase or gasdermin, consistent with the contributions from multiple caspase-dependent processes. By contrast, doxorubicin that predominantly triggers apoptosis was less effective at stimulating anti-tumour immune responses despite triggering similar levels of cell death. These findings demonstrate that caspase-dependent lytic cell death with pyroptotic features promotes a more effective stimulus for anti-tumour immunity.

Phenotypically healthy cells frequently harbor somatic variants at cancer-associated genes, indicating that malignant transformation requires the selection of several alterations. Predicting which combinations of mutations, or co-mutations, exhibit oncogenic capacity requires identifying co-mutations that occur more or less frequently than expected. However, statistical frameworks to solve this problem are hampered by tumor heterogeneity and data availability. Here we curated putative oncogenic mutations in >70,000 human tumors from 119 subtypes, and designed a strategy to search for co-mutations based on in silico simulation of mutagenesis (SelectSim). Using this dataset and tool, we discovered and validated co-mutations across independent human cohorts, compared co-mutations across different tumor types and identified potential risk factors of metastatic progression. Notably, across several cohorts of phenotypically normal tissue samples, we show that, unlike individual oncogenic variants, significantly co-occurring mutations are largely cancer-specific and are observed rarely in healthy tissues, providing clues about the paths to tumorigenesis.

Autophagy underlies several immunosuppressive mechanisms that collectively endow cancer cells with prominent immunoevasive features. Recent data from Hoslett et al. demonstrate that a specialized variant of autophagy commonly known as nucleophagy actively removes PARP1 molecules trapped on DNA upon pharmacological inhibition, mediating robust cytoprotective and possibly immunosuppressive effects.

Protein therapeutics have garnered significant enthusiasm for oncological applications, owing to their exquisite specificity and intrinsic bioactivity. Yet, proteins remain constrained by limited transcellular permeability and proteolytic susceptibility during delivery. To circumvent these impediments, we engineered a redox-responsive protein prodrug, RP-ss-PK, via covalent conjugation of cytotoxic proteinase K (PK) to a multifunctional polymer, RGD-PEG-ss-NPC. This linear hetero-difunctional PEG scaffold contains a cyclic RGD peptide at the α-terminus for integrin-mediated tumor targeting and a 4-nitrophenyl ethyl carbonate (NPC) group at the ω-terminus for site-specific conjugation to amine-rich lysine residues on PK. An internal disulfide linker (-ss-) renders the prodrug cleavable in the presence of intracellular glutathione (GSH). Upon systemic administration, RP-ss-PK selectively accumulates within neoplastic lesions via RGD-integrin (αvβ3/αvβ5) recognition. Endocytosis exposes the disulfide tether to the cytosolic glutathione milieu, triggering reductive cleavage and subsequent liberation of native, fully active PK. The unleashed protease initiates indiscriminate proteolysis that precipitates rapid tumor cell death, accompanied by plasma-membrane permeabilization. This membrane disruption facilitates the extracellular release of damage-associated molecular patterns (DAMPs), thereby igniting anti-tumor immunity and remodeling the tumor microenvironment. Collectively, this prodrug paradigm not only amplifies the tumor-selective delivery of protein therapeutics but also exemplifies a facile, broadly applicable strategy for intracellular activation and functional restoration of protein cargos. Our findings establish RP-ss-PK as a versatile platform poised for the targeted protein treatment of diverse pathologies.

Intracellular chromophores {e.g., NADH [reduced form of nicotinamide adenine dinucleotide (oxidized form)] and FAD (flavin adenine dinucleotide)} play a central role in regulation of cellular metabolism. Although autofluorescence has been extensively used for label-free mapping of chromophores inside a cell, its sensitivity and molecular specificity are constrained by the low quantum yield and the fluorescence spectral overlap. Here, we address these challenges by using a photothermal approach to measure the optical absorption of chromophores rather than its autofluorescence. Our two-photon photothermal (2PPT) microscope exploits localized thermal transients generated through two-photon absorption, enabling detection of chromophore-specific signatures beyond the reach of autofluorescence. We demonstrate submicromolar limits of detection for the metabolic coenzymes NADH and FAD of 0.87 and 0.99 μM, respectively. Such high sensitivity enables differentiating the influence of mitochondrial shapes on metabolism. 2PPT can identify the biomolecular source of contrast from cellular mitochondria in a label-free manner on the basis of spectroscopy. 2PPT microscopy is used to study metabolic alterations of mitochondria in cancer under chemotherapy at the single-organelle level.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, plays a central role in glutamine, proline, and polyamine metabolism and is increasingly recognized as a metabolic vulnerability in multiple cancers. Previously, we established a second deprotonation strategy to achieve efficient mechanism-based inactivation of hOAT over closely related aminotransferases. Building on this concept, we report the rational design, synthesis, and mechanistic investigation of cyclopentene-based γ-aminobutyric acid analogues bearing alkyne or nitrile warheads as potent hOAT inactivators. These compounds undergo enzyme-catalyzed γ-deprotonation to form ketimine intermediates, priming for a subsequent tautomerization event that leads to irreversible inhibition. Inhibitory activity evaluation revealed pronounced stereochemical effects on binding affinity and partition ratio, with one nitrile analogue (4b) exhibiting an exceptional inactivation efficiency (kinact/KI = 111.8 mM-1·min-1) and ∼400-fold selectivity for hOAT over γ-aminobutyric acid aminotransferase. Intact protein mass spectrometry and X-ray crystallography demonstrated that alkyne-containing analogues form covalent adducts with hOAT, whereas nitrile-containing analogues generate noncovalent but tight-binding species. Kinetic isotope effect studies identified γ-deprotonation as the rate-determining step, and a complementary small-molecule mass and computational study elucidated the inactivation and turnover pathways. Collectively, these results expand the mechanistic repertoire of PLP-dependent enzyme inactivation and provide a generalizable framework for designing highly selective mechanism-based inactivators.

Cancer stem cells (CSCs) represent functionally defined and phenotypically plastic tumor cell populations implicated in therapeutic resistance, relapse, and metastasis. CSC plasticity is regulated through coordinated stemness signaling and epigenetic mechanisms. Canonical stemness-associated pathways, including Wnt, Notch, and Hedgehog, interact with epigenetic programs to maintain dynamic stem-like states and facilitate cellular adaptation to environmental and therapeutic stress. These regulatory networks are also associated with metabolic reprogramming and may support CSC survival under therapeutic and immune pressure. This review summarizes recent advances in our understanding of CSC plasticity, with a particular focus on the bidirectional interplay between CSCs and the tumor immune microenvironment. From a translational perspective, it further summarizes emerging strategies for targeting CSC plasticity in combination with immunotherapy, and discusses the present limitations and challenges of combination strategies. These observations may provide a conceptual framework for the development of more rational combination strategies, although their clinical benefit remains to be validated.

The immunosuppressive tumor microenvironment (TME) poses a significant challenge to effective cancer immunotherapy, as it enables tumor escape through redundant checkpoint pathways, metabolic constraints, and direct inhibition of effector cells. To overcome these barriers, we developed a next-generation NK cell platform using a tri-cistronic retroviral vector that enhances NK cell activation, recruitment, survival, and metabolic fitness. Pan-cancer transcriptomic analyses reveal consistent co-expression of PD-L1 and HLA-E across tumors, which correlates with immune infiltration accompanied by strong immunosuppression, highlighting these molecules as key targets for immune evasion. To improve NK cell recruitment, activation, cytolytic function, survival, and metabolic fitness in the TME, we developed a next-generation NK cell platform using a tri-cistronic retroviral vector that encodes an extracellular PD-1 domain (exPD1) fused to the intracellular portion of NKG2D with the costimulatory molecule 4-1BB, and expressing soluble IL15 and NKG2A single-chain variable fragments (scFv). Thus, the exPD1 allows the recognition of cells expressing PD-L1, while the intracellular costimulatory signaling transforms the inhibitory interaction PD-1/PD-L1 into an activating one. This strategy effectively targets PD-L1-positive tumor cells and induces de novo PD-L1 expression in otherwise negative tumors. To further enhance anti-tumor activity, we incorporated a module encoding soluble NKG2A-scFv to mask the NKG2A receptor on NK cells and hinder NKG2A/HLA-E inhibitory interaction. Additionally, we improved NK cell survival and expansion by delivering controlled low doses of IL15, which prevents NK cell exhaustion and extends their presence in vivo. This integrated strategy may provide a novel, ready-to-use allogenic mature NK cell therapy that could overcome checkpoint-mediated inhibition, metabolic suppression, and immune escape, offering a promising model for treating high-risk and treatment-resistant tumors.

Cancer vaccines offer a promising strategy to initiate de novo T cell responses or enhance existing ones, either functioning independently or synergizing with T cell-modulating therapeutics to reduce tumor burden. The clinical development of cancer vaccines faces challenges such as limited antigen coverage, insufficient antigen presentation, immune suppressive microenvironment, and the availability of personalized vaccines. In this study, we developed a universal "all-in-one" cancer cell-derived vaccine (UniCVac) with comprehensive antigen spectrum coverage by programming tumor cells into antigen-presenting cells (APCs) through the codelivery of CIITA, NLRC5, CD80, and IL-2. This reprogramming mimics the professional APC phenotype, providing simultaneous HLA-I and HLA-II antigen presentation, costimulation, and T cell proliferation signals. These tumor-derived UniCVac can directly activate both CD4+ and CD8+ T cells in vitro, independent of APCs. In addition, their costimulation and T cell growth-stimulating capabilities result in superior CD4+ and CD8+ T cell activation and proliferation comparable to traditional APCs, with enhanced PI3K-AKT pathways activation. Single-cell transcriptome analysis confirmed the similarity in cellular subtypes between UniCVac-activated and traditional APC-activated T cells. In mouse models, the UniCVac vaccination reprogramed the tumor microenvironment from immunosuppressive to immune-permissive, induced robust CD4+ and CD8+ T cell expansion in both preventive and therapeutic tumor models, and achieved complete tumor regression in vivo. Our approach provides a platform for the development of universal cancer vaccines with full antigen spectrum coverage and the ability to directly activate both CD4+ and CD8+ T cells, offering potential combinatorial opportunities with existing T cell-based immunotherapies against cancer.

Taxane chemotherapeutics, including paclitaxel, docetaxel, and cabazitaxel, are commonly used in cancer therapy because of their cytotoxic effects against malignant cells. Although their primary mechanisms involve microtubule stabilization and induction of apoptosis, emerging evidence suggests that taxanes also modulate antitumor immunity. Drug delivery systems (DDSs) have been used to enhance taxane therapeutic efficacy by improving tumor targeting and immune modulation. This review discusses the immunomodulatory effects of taxanes and their effects on spatially distinct immune processes within tumors and draining lymph nodes. We highlight recent advances in taxane DDSs that facilitate tumor- and lymphatic-targeted delivery, thereby enhancing antigen presentation, adaptive immune activation, and systemic antitumor immunity. Furthermore, we explore synergistic strategies that combined taxane-based DDSs with immune checkpoint inhibitors and other immunotherapeutic agents. By integrating these approaches, taxane-based chemoimmunotherapy presents potential for advancing next-generation cancer immunotherapy.

Virtual screening (VS) is a powerful approach to exploring a vast chemical space, encompassing libraries of millions to billions of compounds. However, the low hit rates of VS require testing numerous candidates to validate true binders, followed by iterative optimization cycles, which makes experimental validation costly and time-consuming. Here, we report COMBINAUT, an automated parallel synthesis platform that generates diverse chemical scaffolds to accelerate hit validation and refinement. Using a faculty-wide collection of in-house building blocks, the system enables enumeration of over 22.9 million compounds, each designed for parallelized synthesis within 32 h using repurposed solid-phase peptide synthesis equipment. Using this platform, we performed large-scale VS targeting the allosteric pocket of the immuno-oncology target, C-C chemokine receptor 2 (CCR2). Our approach facilitated the rapid synthesis and testing of 100 VS hits spanning diverse molecular architectures. In radioligand binding assays, we successfully validated nine hits with distinct scaffolds, including completely novel CCR2 ligand chemotypes. Iterative hit-to-lead optimization using the automated workflow produced cell-active CCR2 antagonists. This work demonstrates the synergy of automated synthesis and VS, enabling the efficient exploration of chemical space and the rapid discovery of novel ligands.

Bimodal protein expression, defined as the distribution of protein expression with two modes, is linked to phenotypic variation across various biological systems. To advance the identification of cancer biomarkers and targets for precision oncology beyond RNA-based studies, we developed a proteomics-specific bimodality model. By analyzing proteomics data from various cancer types, 2401 tumor-associated bimodal proteins significantly linked to critical cancer pathways were identified, including amino acid metabolism, extracellular matrix-receptor interaction, and central carbon metabolism. Utilizing an AI-enhanced knowledge graph, we further delineated common patterns among pan-cancer tumor-associated bimodal proteins. A case study on TROP2 in colon adenocarcinoma highlights up-regulation of MYC and WNT/β-catenin pathways and down-regulation of inflammatory pathways in TROP2-high groups. This research highlights the biological differences impacting cancer heterogeneity and vulnerability, ultimately aiding treatment decisions. Our findings illustrate the value of proteomics in uncovering novel biomarkers and advancing precision medicine, paving the way for multi-omics integration and clinical validation.

Immune checkpoint blockade has achieved remarkable success in cancer treatment; however, enhancing its efficacy remains a challenge. Here we identified an immunoregulatory micropeptide encoded by the long noncoding RNA USP30-AS1 gene, highly expressed in tumor-associated macrophages. The so-designated UEIS (USP30-AS1-encoded immune suppressor) drives macrophages toward a protumorigenic phenotype, thereby inhibiting antitumor T cell immunity. Mechanistically, UEIS is induced in macrophages by cGAS-STING-type I interferon signaling at a relatively late stage following tumoral DNA stimulation, and exerts a negative feedback regulation on the type I interferon signaling by forming biomolecular condensates with TBK1, thereby inhibiting its interaction with STING. Both an intrinsically disordered region and an alpha helix at the extreme N terminus of UEIS were essential for its function. A peptide designed to disrupt UEIS-TBK1 condensation successfully inhibited UEIS function in tumor-associated macrophages, leading to reduced tumor growth and increased response to immune checkpoint blockade. Thus, these findings highlight UEIS as a promising therapeutic target for cancer treatment.

Neuron-tumor communication is emerging as a distinct layer of tumor-host interaction beyond conventional stromal, vascular, and immune regulation. Recent studies show that malignant cells can detect neuronal activity and convert neural signals into growth-promoting cellular responses. In the brain, glioma cells can become electrically integrated into neuronal circuits, where glutamatergic input drives depolarization, calcium influx, and downstream signaling. In extracranial tumors, neural influence appears more heterogeneous, involving spatially organized neurochemical niches, receptor-enriched cancer-nerve contacts ('pseudo-synapses'), autonomic pathways, and injury-associated neuroimmune remodeling. These findings raise important questions about how neural input regulates tumor cell state, metabolism, immune tone, and therapeutic adaptation. This review evaluates the evidence linking neural activity to cancer progression across anatomical contexts and outlines the experimental standards needed to distinguish structured neuron-tumor interfaces from broader neural effects within the tumor microenvironment.

Redox-responsive lipid nanoparticles (LNPs) are emerging as a powerful platform for precision nanomedicine by exploiting disease-associated redox imbalances, such as elevated glutathione and reactive oxygen species, to trigger controlled cargo release and structural activation. This strategy is of great importance for developing gene-based therapeutics, where efficient cytosolic delivery is essential. In addition to nucleic acids, redox-responsive LNPs have been also explored in delivering small molecules, proteins, and theranostic agents, broadening their potential in both cancer and non-cancer diseases. This review summarizes the biological basis of redox responsiveness, key design principles for responsive chemical structures, and major advances in payload delivery and targeting capability of LNP carriers. Moreover, some major bottlenecks, including redox heterogeneity, stability, responsiveness trade-offs, and translational complexity, are critically discussed. Future directions are also highlighted, particularly for organ-selective delivery, multifunctional theranostics, and clinically translatable LNP-based medicines.

In this issue of Cancer Cell, Hernández-Verdin et al. reveal why some tumors with tertiary lymphoid structures (TLSs) resist immunotherapy: their findings identify cancer-derived gamma-aminobutyric acid (GABA) as an immunosuppressant factor resulting in dysfunctional TLS and immunotherapy resistance. Targeting GABA with immunotherapy reshapes the tumor and TLS microenvironment and ultimately improves therapeutic efficacy.

Neutrophils are acute responders to bacterial and fungal infection; however, their roles in disease extend far beyond their critical roles in countering pathogens. In this review, we seek to provide some broader context based on recent literature that highlights the contributions of the neutrophil lineage to cancer and chronic inflammation. Their importance for immunomodulation is underscored by their capacity to generate cytokines and chemokines, reactive oxygen species, granule secretory proteins, and use regulated inflammatory cell death pathways for the release of neutrophil extracellular traps. The potential to alter the balance between circumscribed inflammation and collateral tissue damage is most notable when considering the diverse contents and functions of secretory vesicles, and tertiary, specific, and azurophilic granules. We still have much to learn about the generation of granules in neutrophil progenitors, the factors controlling their release from mature neutrophils, and the cellular consequences for misfolding, misplacement, and premature release of granule contents from neutrophils. This review discusses recent advances in our understanding of neutrophil biology, roles in disease, and opportunities for depletion, inhibition, or supplementation and restoration of function.

Spatial transcriptomics (ST) enables spatially resolved gene expression profiling within intact tissue sections. However, its widespread adoption is constrained by the high cost and low throughput of current sequencing-based protocols. This has motivated growing interest in computationally predicting gene expression directly from routinely acquired histology images. Existing methods are largely restricted to isolated 2D tissue slices and fail to capture richer spatial relationships or structured dependencies among spot-level gene expression profiles. In this paper, we propose STAG, a dual-branch framework for gene-aware expression prediction and spatial context modeling. A Query branch predicts ST expression for an individual target spot, while a Neighbor branch acts as an auxiliary branch to model structured relationships among multiple spots. By leveraging hypergraph learning, the Neighbor branch captures higher-order spatial and molecular dependencies, enabling unified modeling of both intra-slice and inter-slice relationships. This design supports standard 2D settings (a single slice) and naturally extends to 3D scenarios when adjacent tissue sections are available. Moreover, STAG leverages gene semantic information as biological guidance by encoding gene names with a foundation model, enabling coordinated gene-aware interactions beyond independent gene prediction. STAG achieves an average gain of 5.16% in PCC@250 across six datasets. Under highly variable gene selection, STAG maintains the lowest RMSE and highest PCC@50 across three datasets. The effectiveness of the learned representations is further demonstrated in pseudo-3D prediction and downstream cancer classification tasks. Code is available at https://github.com/MCPathology/STAG.

Immune cells in the tumor microenvironment are not only powerful regulators of immunosuppression and tumorigenesis but also a dominant cell population, with tumor-associated macrophages (TAMs) comprising up to 50% solid tumor mass. Immunotherapies such as immune checkpoint inhibitors derive efficacy from this cancer-immune interface; however, immune-related adverse events from systemic blockade remain a major challenge. To address this need for potent, tumor-specific immunotherapies, we developed tumor immune cell targeting chimeras (TICTACs) that selectively deplete immune checkpoint receptors such as SIRPα from TAM surfaces. These chimeras consist of a synthetic ligand targeting CD206, a TAM marker, conjugated to a nonblocking antibody that binds to the checkpoint receptor without inhibiting it. By engaging CD206, which constitutively recycles between the plasma membrane and early endosomes, TICTACs drive robust checkpoint degradation in CD206high macrophages, with no effect on CD206low cells. This decoupling of antibody selectivity from blocking function presents a new paradigm for tumor-specific immunotherapies.

Physiological host factors, such as the gut microbiome and obesity, independently influence anti-tumour immunity and responses to immune checkpoint inhibitors (ICIs)1, with high body mass index (BMI) having an unexpected link with greater ICI efficacy2-6. However, how these factors interact across diverse dietary contexts remains unclear. Here, using 12 mouse diet models that reflect a spectrum of obesity biology, we characterize diet-driven metabolic, immune and gut microbiota features associated with ICI sensitivity. We find that obesity-associated ICI responses are poorly correlated with metabolic dysfunction and are instead dependent on the diet-gut axis. Obesogenic diets promote a robust and persistent gut microbial ecosystem that is capable of restoring ICI sensitivity following a short-term diet switch or fecal microbiota transplants (FMTs) from non-responder models. Monocolonization of germ-free mice with favourable bacteria such as Lactobacillus johnsonii, together with an obesogenic diet, synergistically promotes tumour regression through an enrichment of microbiota-derived aromatic amino acid metabolites. Moreover, human-to-mouse FMT from donors with a high BMI enhanced ICI efficacy compared with donors with a normal BMI, and an obesogenic diet restored sensitivity following FMT from a non-responder patient. Our study provides insight on epidemiological associations between BMI and ICI efficacy, and suggests that immunomodulatory synergy between diet and the gut microbiota could be leveraged to improve ICI outcomes and FMT interventions.

Tumor-seeking fluorescent dyes enable precise lesion localization by recognizing overexpressed receptors, providing a critical adjunctive technology for cancer histopathology. However, tumor heterogeneity and the poor understanding of targeting mechanisms limit their efficacy. Here we engineer CETIF6a, a click chemistry-compatible heptamethine cyanine dye, for multi-cancer targeting, intraoperative histopathology, and proteome-wide target identification. CETIF6a demonstrates margin delineation across multiple cancer types ( > 90% concordance with H&E staining). Quantitative proteomics reveals that the dye targets 5-15 times more tumor-specific proteins than in paracancerous tissues. Synergistic pan-cancer targeting is achieved through 491 conserved tumor-enriched proteins involved in ribosomal, proteasomal, and metabolic pathways, effectively overcoming heterogeneity. Mechanistic studies confirm that CETIF6a emits bright fluorescence upon covalent binding to targets via nucleophilic substitution at cysteine thiol residues within hydrophobic cavities. The modifiable scaffold of CETIF6a supports both intraoperative tumor diagnosis and functional targets profiling, providing a foundation for systematic probe optimization.

Bulky tumors remain challenging to treat, and immune checkpoint inhibitors (ICIs), alone or combined with conventional radiotherapy (RT), yield limited efficacy. We present EclipseRT (ERT), an RT technique that delivers low-dose RT (LDRT) to the gross tumor volume (GTV) and stereotactic body RT (SBRT) to selected subvolume(s) within the GTV. Combined with ICIs (iERT), this approach achieves marked control of bulky tumors through the coordinated activity of NK and CD8⁺ T cells. Single-cell RNA sequencing and validation experiments show that the SBRT component robustly induces type I interferon (IFN-I), which activates NK cells to secrete XCL1, thereby recruiting cross-presenting XCR1⁺ dendritic cells (DCs). SBRT also promotes the release of extracellular vesicles carrying neoantigens, enhancing DC cross-presentation and CD8⁺ T-cell responses. The LDRT component further promotes NK and CD8⁺ T-cell recruitment. iERT also induces precursor exhausted CD8⁺ T cells in tumors and tumor-draining lymph nodes. Collectively, iERT activates the IFN-I/NK/DC/CD8⁺ T-cell axis, driving potent antitumor immunity against bulky tumors.

Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS) is a dynamics-sensitive structural method that has rapidly achieved widespread adoption in the biopharmaceuticals industry, owing to its ability to quickly identify binding sites and to provide a molecular mechanism of action for drug candidates. However, a central limitation of conventional HDX-MS is that it has substantially lower spatial resolution than other structural techniques, typically providing information averaged over "segments" of five amino acids or more. Here, we demonstrate a sensitive, broadly applicable method for single amino acid resolution, i.e., site-specific HDX-MS measurements. Using a set of five therapeutic candidates targeting the highly druggable cancer target WDR5, we explore the greatly enhanced analytical power that arises from site specificity, including binding mode characterization, affinity ranking, and the detection of features that are "silent" in conventional peptide-level HDX-MS experiments.

Cancer immunotherapies have significantly improved treatment efficacy and patient survival by exploiting antigen-specific T cells to eliminate cancer cells. However, current approaches for identifying and isolating antigen-specific T cells typically require prior knowledge of target antigens, limiting discovery, and reducing the ability to consistently detect rare tumor-reactive T cells. We therefore sought to develop an unbiased platform for the identification and enrichment of antigen-specific T cells using naturally processed and presented tumor antigens. We developed ATTACH (Assessment of T cells Tethered to Antigen Class I Histocompatibility), a microfluidic platform that applies controlled shear stress and leverages tumor cells as a natural source of endogenous major histocompatibility complex (MHC)-peptide complexes to isolate antigen-specific T cells based on MHC/peptide binding avidity. ATTACH was evaluated in both human and mouse systems for its ability to enrich rare tumor-reactive T-cell populations and deplete bystander virus-specific T cells. ATTACH resulted in up to a 10-fold enrichment of antigen-specific T cells across both human and mouse systems, enabling the isolation of clonotypes present at frequencies as low as 0.1%. In addition to enriching rare tumor-reactive T cells, ATTACH efficiently depleted virus-specific bystander T cells. ATTACH provides a streamlined and unbiased approach for the rapid identification and isolation of antigen-specific T cells, and may facilitate the optimization of cellular therapies for the treatment of solid tumors.

As exosomes serve as pivotal mediators of intercellular communication, research on exosomes has expanded markedly from its conventional focus on mammalian sources to the burgeoning field of nature-derived exosome-like nanoparticles (NELNs). Endowed with favorable biocompatibility, low immunogenicity, and wide, readily accessible sources, NELNs have emerged as promising delivery vehicles to address long-standing bottlenecks in RNA-based therapeutic delivery. However, their clinical translation is significantly impeded by the lack of a consensus definition and standardized manufacturing workflows. This review focuses on NELNs-mediated therapeutic RNA delivery; systematic delineation of NELNs' characteristics and biogenic pathways remains insufficient, and further investigation is warranted for details of NELNs' properties, including biochemical composition, inherent biological functions, in vivo biodistribution regulated by distinct administration routes, cellular uptake mechanisms, and regulatory pathways of targeted delivery, as well as state-of-the-art strategies for the construction and optimization of RNA-loaded NELNs. This review systematically characterizes the intracellular trafficking, cargo release kinetics, and metabolic degradation profiles of NELNs-RNA complexes, summarizes their therapeutic applications in brain disorders, intestinal diseases, and tumors, analyzes critical translational challenges including scalable production, batch-to-batch consistency, long-term safety, and targeting precision, and proposes rational optimization strategies to advance mechanistic research and clinical translation of NELNs-based RNA delivery systems.

Cancers are complex cellular communities comprising tumor cells and their microenvironment. Recent advances in cancer biology have emerged from efforts to analyze tumors as tissues, in which emergent properties arise as tumor cells interact with one another, their microenvironment, and the host tissue. In this review, we consider interactions involving mechanical forces and mechanosignaling pathways that detect changes in physical inputs to regulate cellular behavior. This rapidly developing field provides perspectives for understanding cancer biology and tumor interactions with the host ecosystem.

In a recent Immunity paper, Lobel and colleagues integrate cross-cohort human microbiome meta-analyses with mechanistic studies in mice to uncover a dietary sulfur amino acid-microbiota-immune axis that enhances anti-tumor immunity. Sulfur amino acids expand the mucus-associated bacterium Mucispirillum schaedleri and trigger an NKT-cDC1 immune circuit.

The human gut microbiota is now established as a vital contributor to drug metabolism and therapeutic efficacy. Nevertheless, the interaction between gut microbes and pharmaceutical agents is inherently complex, posing significant challenges to the use of the microbiota to reduce toxicity and improve efficacy. Gaining a deeper understanding of the complex role of the gut microbiota in regulating drug metabolism and influencing treatment outcomes is essential for enhancing diagnostic accuracy, prognostic stratification, and therapeutic approaches. This review systematically summarizes recent advances in gut microbiota-mediated drug metabolism and effectiveness and assesses the potential of targeting microbial communities to improve drug performance. The insights provided here are set to advance personalized medicine and promote the development of microbiota-targeted therapies.

Engineered live biotherapeutic products (eLBPs) represent an emerging class of programmable microbial therapies capable of sensing and responding to host physiology. Advances in microbiome science and synthetic biology have driven the development of engineered bacteria that deliver therapeutic molecules, modulate host metabolism, or detect disease-associated signals. In this review, we summarize recent progress in the development of eLBPs across diverse disease indications, including inflammatory diseases, metabolic disorders, cancer, and infectious diseases. We highlight key factors that drive successful eLBP design, including chassis selection, methods for DNA delivery, approaches for tuning therapeutic expression, and genetic systems for biocontainment. Although early clinical studies demonstrate promising safety profiles, challenges remain in achieving predictable colonization, durable therapeutic activity, and robust biocontainment in vivo. By synthesizing advances across these areas, we propose a framework for the rational design of next-generation eLBPs that can more reliably translate from experimental systems to clinical application.

Focal radiotherapy can provide an in situ vaccine (ISV) effect that may prime adaptive antitumor immunity, which can be augmented by combination with intratumoral immune adjuvants and systemic immune checkpoint inhibition. However, propagation of an antitumor immune response is not consistently achieved in metastatic disease. Delivery of low-dose targeted radionuclide therapy (TRT) can promote clonal expansion and propagation of antitumor immune responses. Therefore, in this study, we combined dual immune checkpoint blockade (DCP; anti-PD-L1 and anti-CTLA-4) with ISV (focal radiotherapy with intratumoral injection of tumor-specific monoclonal antibody and IL2) to prime and low-dose TRT to propagate antitumor immunity. C57BL/6 mice were engrafted with a primary and secondary tumor, and intravenously injected with tumor cells to model advanced metastatic disease. Mice with poorly immunogenic, syngeneic MOC2 head and neck squamous cell carcinomas or B78 melanomas received monotherapy or double or triple combinations of: low-dose TRT; DCP; and primary tumor-targeted ISV. When compared to dual or monotherapies, TRT+DCP+ISV eradicated bulky well-established tumors, prevented development of lung metastases from circulating tumor cells, and extended survival (p< 0.01). TRT+DCP+ISV conferred tumor-specific immune memory, enabling uniform rejection of tumor re-engraftment. TRT+DCP+ISV activated innate and adaptive immune response in the primary and secondary tumors. Overall, these data highlight TRT+DCP+ISV as a promising approach to prime and propagate antitumor immunity and promote response to checkpoint inhibition.

Membrane proteins participate in most cellular processes from sensing the cellular environment to regulating gene expression. The fundamental unit in eukaryotic membrane proteins is the transmembrane (TM) helix, which in addition to anchoring the protein to the lipid bilayer, often plays a central functional role. We review here advances in our understanding of the factors underpinning the thermodynamic stability of TM helices in cellular membranes. Interestingly, a class of TM sequences exists that has the ability to, when isolated as a peptide, be stable both in solution and in the membrane. This Review covers such conditional TM peptides, including their discovery in the form of the pHLIP peptide, the factors that determine their membrane insertion, and the basis for their use in cancer theranostics. We also discuss how this knowledge has been the conceptual basis for the design of sequences that partition into lipid bilayers and target membrane proteins. These peptides act as both positive and negative allosteric regulators of their membrane targets. Such TM allosteric conditional peptides (TMACs) additionally constitute tools that can advance our understanding of the activity and regulation of membrane proteins.

Reversing the immunosuppressive tumor microenvironment (ITME) is a crucial obstacle in the immunotherapy of cold tumor. The STING signaling pathway has shown promise in overcoming the ITME issue. However, reversing ITME via precise activation of the STING signaling pathway remains a significant challenge. In this work, photoresponsive nanophotocages with photoimmunogenic performance were developed for ITME-reversed synergistic photoimmunotherapy of cold tumor. In the photocage design, photoresponsive boron dipyrromethene (BODIPY) was used to link a STING agonist (DMXAA) and then prepared into water-dispersible nanoparticles. The iodinated nanophotocage showed the best photoreaction efficiency and spatiotemporal controllability among the four nanophotocages. Under light irradiation, the iodinated nanophotocages precisely released the STING agonist and triggered immunogenic cell death (ICD) via the photodynamic therapy (PDT) effect, by which the ITME was reversed to enhance the immune checkpoint inhibitor (aCTLA4) immunotherapy. The robust immune response not only achieved primary tumor eradication in 80% of the mice but also significantly inhibited the distant tumor. This work offers a photoimmunogenic photocage for light-controlled STING activation for ITME-reversed cancer photoimmunotherapy.

Autophagy is an evolutionarily conserved lysosomal degradation pathway. In cancer, its role is paradoxical: it functions as a tumor-suppressive gatekeeper during initiation in part by preserving genomic stability, but it is frequently co-opted by established tumors to maintain metabolic fitness and therapeutic resistance. Early clinical efforts using broad, non-selective lysosomal inhibition (e.g., chloroquine) produced mixed outcomes and toxicities, prompting a paradigm shift toward modular, context-specific modulation.This review synthesizes the dynamic spatiotemporal evolution of autophagy in tumorigenesis, and characterizes it as an adaptable evolutionary trajectory governed by stress, tumor genotype, and microenvironmental context. We outline four conceptual pillars: genotype-defined modular networks, dynamic spatiotemporal adaptation, autophagy as an immunometabolic rheostat, and rational therapeutic modulation. Importantly, autophagy exerts cell-type-specific effects-promoting immune evasion in tumor cells while remaining indispensable for lymphocyte fitness. To address this paradox, we evaluate the transition from empirical global blockade to precision-guided intervention, including pathway-selective modulators, exploitation of selective vulnerabilities, and advanced targeted degradation technologies. Autophagy in cancer is a highly dynamic, context-dependent variable. Therapeutic control requires movement beyond universal flux inhibition toward pathway-specific, biomarker-guided interventions that match a tumor's distinct autophagic dependencies. Integration of dynamic monitoring with precise delivery systems may allow active modulation of the tumor microenvironment, transforming autophagy from a tumor resilience mechanism into an exploitable therapeutic vulnerability.

Deciphering the mechanistic links between aging and cancer remains a central challenge in biomedical research. This review begins by presenting recent epidemiological data on cancer incidence across age groups, underscoring the complex and paradoxical interplay between aging and cancer. A systematic analysis follows four core signaling pathways that underpin both aging and cancer: (i) nutrient sensing and energy metabolism pathways, (ii) stress response and genome maintenance pathways, (iii) inflammatory and microenvironmental pathways, and (iv) sirtuin-mediated epigenetic and transcriptional modulation pathways. Each pathway is analyzed regarding its distinct and overlapping roles in aging and cancer, with exploration of strategies for comodulating these shared mechanisms to simultaneously mitigate both conditions. Established and emerging druggable targets within these pathways are highlighted, offering new perspectives for future therapeutic development. Current integrative strategies to achieve concurrent antiaging and anticancer effects include targeting cellular nutrient sensing and metabolism, enhancing genomic integrity, suppressing chronic inflammation, clearing senescent cells, and modulating epigenetic programs. Finally, the impact of aging on cancer therapy, particularly immunotherapy, is discussed, along with a review of representative aging-related pathways and genes and their roles in modulating immunotherapy efficacy. Analysis of agents targeting each pathway spanning preclinical studies, clinical trials, and clinically approved drugs reveals disparities in the research progress between anti-aging and anticancer applications. Consequently, a promising future strategy for drug discovery lies in repurposing well-established anticancer agents for antiaging interventions, as well as translating insights from aging research into novel antitumor therapies, thereby achieving synergistic benefits in both fields.

CD8+ T cell dysfunction, characterized by impaired effector function, proliferative capacity, and sustained inhibitory receptor expression, limits immune control in both cancer and chronic viral infections. Despite arising from distinct disease processes, these conditions induce a shared state of CD8+ T cell dysfunction, suggesting convergence on common regulatory pathways. Adenosine (ADO), an immunosuppressive purine metabolite generated through extracellular ATP catabolism, has emerged as a context-integrating metabolic checkpoint that regulates immune responses in response to tissue stress and inflammation. Across tumors and HIV, dysregulated ADO signaling reinforces checkpoint pathways and stabilizes dysfunctional CD8+ T cell states. In this review, we examine how the ADO-adenosine deaminase-1 axis shapes CD8+ T cell dysfunction across disease contexts and discuss its potential as a broadly applicable target for immune restoration.

Three decades after the approval of the first cancer nanomedicine, low (<1%) tumor delivery remains the central unsolved challenge in nanoparticle (NP)-based therapy. This barrier has prompted a research shift toward biologically derived delivery systems able to reduce immune clearance while preserving tumor-homing capabilities. In particular, extracellular vesicles (EVs) seem obvious candidates on account of their intrinsic biocompatibility, cell-specific tropism, and biological functionality. In this mini-review, we critically analyze EVs as nanoparticle delivery vectors in cancer therapy. We describe current EV engineering approaches, including loading methodologies, surface modification strategies, and the development of artificial or biomimetic EVs, highlighting technical, scalability, and characterization challenges. We also summarize key in vitro and in vivo results, addressing encapsulation strategy, biodistribution, and therapeutic outcomes. From this discussion, we outline research needs that must be addressed to develop EV-NP hybrids as tools to overcome the delivery challenge in cancer.

Nanomedicine translation is limited by inefficient delivery into solid tissues of multilayered cells such as tumors. Recently, several studies highlighted the importance of transcellular transport in nanoparticle (NP) extravasation into solid tumors. However, the underlying mechanism, especially the process from NP export from one cell and re-entry into another (termed "intercellular exchange" collectively), remains poorly understood. This review summarizes the progress in eliciting the NP transport across multilayered cells, and the efforts in studying the intercellular exchange of NPs. Particularly, our studies have revealed extracellular vesicles (EVs) as a novel conduit of NP transfer between cells in vitro and NP delivery into solid tumors in vivo. We further overviewed the efforts on how to chemically regulate this EV route for boosting the NP delivery efficiency. Overall, this review highlights the importance of EVs in NP delivery and provides new insights on enhancing nanomedicine efficacy through regulating EV machinery.

Glutathione (GSH) serves as a core mediator in diverse life activities. Its abnormal expression or function is also closely linked to the pathogenesis of numerous human diseases. However, the current GSH detection generally has only diagnostic capabilities and is rarely linked to therapeutic functions, leading to a disconnect between basic detection and clinical intervention. Herein, we developed a AuNRs@ZIF-8/AuNPs core-shell nanocomposite as a "three-in-one" substrate, endowed with superior POD-like activity, strong SERS amplification performance, and mild photothermal properties. Its mimetic POD-like activity can facilitate the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB (ox-TMB), generating a strong SERS signal. In the presence of GSH, this oxidation is inhibited, leading to color fading and diminished SERS signals. Based on the above characteristics, a versatile platform was developed for SERS-colorimetric dual-mode detection of GSH, with detection limits as low as 0.019 and 1.28 μM for SERS and colorimetric modes, respectively. Furthermore, the developed dual-modal biosensor has been successfully applied for the determination of intracellular GSH levels, providing a potent and straightforward tool for intracellular GSH analysis. Significantly, the experiments also showed this material possesses photothermal sterilization and tumor ablation effects, thereby endowing the nanocomposite with a "diagnosis-therapy integration" potential application. This study presents a promising example for precision detection and synergistic therapy.

Display technologies discover high-affinity peptides from vast combinatorial libraries. In mRNA display, flexizyme-enabled genetic code reprogramming has become the dominant approach to extend building block diversity and improve drug-relevant properties, yet post-translational modifications (PTMs) capable of installing more uniquely constrained backbone topologies remain underexplored. Here, we introduce a PTM strategy that broadens this scope by installing an aspartyl aldehyde (X), which undergoes spontaneous Pictet-Spengler-type cyclization with proximal nucleophilic side chains to yield polycyclic α-amino-γ-lactam (pcAgl) motifs. Systematic studies define the equilibria, stereochemistry, and side-chain requirements governing pcAgl formation under biocompatible conditions. Incorporation of this chemistry into reprogrammed peptide libraries enabled direct in vitro selection of pcAgl-containing ligands against the oncology target MAT2A. The selected peptides contained structurally critical pcAgl motifs, inhibited the enzyme (best IC50 = 9 μM), and showed improved stability in human serum. This work establishes aldehyde-mediated peptide backbone alkylation in mRNA display and showcases the value of applying chemistries traditionally not considered biocompatible to expand the chemical space of genetically encoded libraries.

Precise measurements of small molecule-protein interactions are critical for drug discovery. However, most biochemical profiling platforms measure binding using recombinant kinase domains or proteins in cell lysates, which can miss conformational regulation present in intact living cells. Here, we used flow cytometry-based fluorescent probe cellular binding assays (FPCBA) to demonstrate that the anticancer drug dasatinib binds native, untagged ABL1 kinase with 3-6-fold higher affinity than NanoLuc- or mVenus-tagged constructs in living cells. We further used this method for in-cell profiling of 25 native kinases, revealing conformational regulatory mechanisms, including SRC autoinhibition and membrane-dependent conformational states of DDR1, DDR2, and EPHA4 that are absent or attenuated in biochemical assays. For these studies, coumarin-dasatinib probes spanning a range of fluorophore acidity (pKa 4.1-7.3) were optimized for intracellular target engagement. To enhance sensitivity of detection, we found that uptake of acidic probes can be promoted by expression of the organic anion transporter OATP1B3. Quantitative flow cytometry with NIST-standardized beads established that intracellular concentrations of an intermediate-acidity 6FC-dasatinib probe approximated extracellular concentrations in HEK293T cells at equilibrium. Cellular Ki values of dasatinib and imatinib for 25 kinases by FPCBA were broadly concordant with kinobead LC/MS measurements in cancer cell lysates but diverged substantially from recombinant KINOMEscan values, with divergences attributable to competition with ATP, autoinhibition, and membrane-dependent conformational states in living cells. FPCBA enables profiling of native protein-small molecule interactions in a physiologically relevant cellular context.

Protein corona formation is widely viewed as an inevitable but poorly controlled event when nanoparticles encounter biological environments. Converting this interfacial layer into a programmable functional entityremains a major challenge. Here, we report an adaptive protein corona nanoassembly strategy that integrates cytokine signaling with endogenous antigen transport. Coordination-engineered manganese nanoscaffolds were assembled with interleukin-12 (IL-12) and protected by an enzyme-responsive hyaluronic acid shell to form nanoshuttles (NSs) capable of dynamic protein recruitment in the tumor microenvironment (TME). Following enzymatic activation, exposure of the cationic manganese surface promotes adaptive corona formation and efficient capture of tumor-derived antigens. The resulting antigen-associated nanoassemblies traffic to tumor-draining lymph nodes, where they facilitate dendritic cell cross-presentation and tumor-specific T-cell priming. This coordinated process couples intratumoral cytokine signaling with antigen delivery, generating potent systemic antitumor immunity at significantly reduced IL-12 doses. These findings establish adaptive protein corona nanoassembly as a chemical strategy for programming nano-bio interfaces to coordinate cytokine signaling and antigen transport.

Redox metabolism in tumor cells is considered a vulnerable target for cancer therapy. Benefiting from the elevated copper-ion levels characteristic of tumorigenesis and progression, copper-mediated chemodynamic therapy (Cu-CDT) demonstrates remarkable efficacy in inducing oxidative stress-mediated cancer cell death. However, Cu-CDT encounters challenges related to the inherently low hydrogen peroxide (H2O2) level and the high glutathione (GSH) level in cancer cells to counteract oxidative stress-mediated therapeutic efficacy. Although strategies for elevating H2O2 levels, depleting GSH levels, and synergizing with other tumor therapies to enhance Cu-CDT have been increasingly reported, their underlying redox metabolism mechanisms have yet to be systematically summarized and thoroughly elucidated. In this regard, the latest progress in combating oxidative stress resistance in Cu-CDT through redox metabolism regulation has been comprehensively reviewed following the "chemical principles-biological mechanisms-regulatory approaches" logical flow. Firstly, the opportunity for Cu-CDT in tumor therapy and the related GSH-mediated oxidative stress resistance mechanism was discussed. Secondly, the existing redox metabolism regulation via H2O2 elevation and GSH depletion in Cu-CDT were classified. Thirdly, the underlying biological regulation mechanisms along the molecular route of "stress response-execution pathway-cell death" were elucidated. Fourthly, mechanism-modulated and engineered delivery strategies for enhancing Cu-CDT through redox metabolism regulation were further proposed. Finally, several foreseeable challenges and opportunities for advancing Cu-CDT were outlined. Generally, this review seeks to provide novel perspectives on enhancing the Cu-CDT therapeutic efficacy, with the broader aspiration of contributing to its future bench-to-bedside research.

The concept of bacterial therapy dates back over a century to clinical observations that incidental infections could induce tumor regression. Recent advances in genetic engineering and synthetic biology have since transformed bacteria into versatile living therapeutics with significant preclinical potential against diseases such as cancer, inflammatory disorders, and metabolic conditions. However, clinical translation faces considerable hurdles. Here, we provide a clinically oriented perspective on the translational gap in bacterial therapy. Drawing inspiration from the success of antibody-drug conjugates in achieving precise payload delivery, we highlight an emerging paradigm of "precision living therapeutics" enabled by bacterial surface engineering. we propose the concept of "Tumor accessibility" for the first time, and identify its insufficiency as a critical bottleneck in current therapeutic applications. We then systematically summarize recent advances in bacterial surface engineering, encompassing physical, chemical, and biological strategies, with a focus on their capacity to evade immune clearance, enhance tumor colonization, and improve therapeutic performance. Chemical approaches primarily involve covalent conjugation, including the SpyTag/SpyCatcher system and bioorthogonal click chemistry-based metabolic labeling. Physical strategies center on cell membrane encapsulation and surface coatings such as layer-by-layer encapsulation. Biological strategies include cell camouflage and genetic modulation of surface structures, display of functional biomolecules, and affinity-based systems such as biotin-streptavidin interactions. Finally, we discuss integrative strategies that combine surface-engineered bacteria with conventional treatment modalities, including physical therapy, chemotherapy, and immunotherapy. We propose that future clinical translation of bacterial therapy should shift from localized modification design to a holistic consideration of systemic accessibility.

Recombinant in vitro-transcribed mRNA is broadly used for vaccination and is evaluated in numerous clinical studies for multiple indications. Typical features of mRNA are the 5' cap, 5' untranslated region, start and stop codons, 3' untranslated region and 3' poly(A) tail. Here, contrary to current dogma, we show that short, chemically synthesized RNA oligonucleotides lacking some or all of these features are efficiently translated when they encode epitopes recognized by CD8+ T cells. In particular, one design that we termed ChemRNA with the structure 5'-OH-AUG-coding sequence-3'-OH strongly stimulates antigen-specific CD8+ T cells both in vitro and in vivo. Our results challenge the current understanding of canonical mRNA structure and introduce the possibility that defective or supposedly non-coding RNA may encode human and murine major histocompatibility complex class I-associated peptides. Moreover, ChemRNA could help overcome challenges associated with the design and purification of individualized anti-cancer vaccines.

Despite advances in IgG-based cancer immunotherapy, challenges remain in effectively engaging innate immune responses against solid tumors. Here, IgA antibodies hold promise due to their ability to activate neutrophils and macrophages. We present a novel retargeted adenovirus-mediated approach that transforms cancer cells into "biofactories" for localized production of monomeric or dimeric IgA antibodies and a CD47 blocker to potentiate the effect of IgA antibodies. With our approach, tumor cells effectively produced IgA antibodies against tumor antigens such as EGFR or EpCAM and a soluble SIRPα-Fc fusion protein, which blocks the CD47-SIRPα axis. In a perfused tumor-on-a-chip model, locally produced IgA triggered neutrophil- and macrophage-mediated tumor cell killing, further potentiated by SIRPα-Fc co-production. In FcαRI-transgenic, tumor-bearing mice, intratumoral adenoviral injection induced strong local IgA and SIRPα-Fc expression, immune cell infiltration, and more than 50% tumor volume reduction after a single treatment. We found that dimeric IgA exerts stronger effects than monomeric IgA. Together, these results demonstrate that adenovirus-mediated, tumor-restricted delivery of IgA antibodies and CD47 blockade effectively engages innate immune mechanisms and has therapeutic promise.

DNA-based molecular computation enables targeting of cells via multiple surface receptors. However, existing platforms are often limited to single-receptor detection per operation or rely on freely diffusing components, a common source of off-target interference. Here we show an intelligent DNA nanodevice (DND) that integrates multivalent recognition, logic‑gated computation, and spatially precise drug delivery in a single nanostructure. The DND comprises a doxorubicin-loaded tetrahedral DNA framework (tFNA@Dox) connected via three allosteric aptamer arms to a cholesterol-modified membrane anchor. Simultaneous binding of all three aptamers triggers an AND‑logic gate, releasing tFNA@Dox specifically at the target cell membrane. This localized sense‑and‑act mechanism maximizes therapeutic specificity and minimizes systemic exposure. In tumor-bearing mice, DND@Dox effectively accumulated in tumors, significantly inhibited tumor growth under local and systemic administration, and reduced systemic toxicity. This work establishes a versatile platform for logic‑controlled, multimarker‑guided cancer theranostics.

Paclitaxel exemplifies one of the key taxanes, a group of versatile and effective drugs commonly used in chemotherapy for several major cancer types. These drugs work by stabilizing cellular microtubules, a unique mechanism to account for their impressive success in oncology. Hair loss (alopecia) is a well-known adverse side effect and poses a significant quality of life issue for many patients. We discovered that a brief exposure to low intensity and low frequency ultrasound at a defined timing was able to break cellular microtubules and mitotic spindles transiently, and thus annulled the consequent hair follicle damage and suppression of hair growth following paclitaxel administrations. These experimental findings herald a practical method that is within reach to prevent hair loss in cancer chemotherapy using taxanes.

Ionizable lipids, widely employed as pharmaceutical excipients for gene delivery, exhibit acidity-responsive membrane-destabilizing activity. This property prompted us to hypothesize that such membrane-destabilizing activity could be harnessed and selectively confined to the acidic tumor microenvironment (aTME) to achieve tumor-selective oncolysis. Herein, we report the rational design of tumor acidity-activatable oncolytic lipid nanoparticles (aoLNPs) through programmed structural modulation of ionizable lipids. By designing and screening an ionizable lipid library with systematic variations in alkyl tail length and number, we found a clear correlation between the total tail carbon number and the pH-dependent cytotoxicity of the corresponding lipid nanoparticles. Subsequent library expansion led to the identification of aoLNPE14A6-2 as the optimal candidate, which exhibited selective cytotoxicity toward cancer cells at pH 6.8 and demonstrated potent antitumor efficacy in vivo. Mechanistic investigations revealed that aoLNPE14A6-2 selectively induces lysosomal vacuolation and endoplasmic reticulum stress under tumor acidic conditions, ultimately leading to plasma membrane rupture. Notably, aoLNPE14A6-2 did not cause obvious chronic toxicity or structural damage to normal tissues after intravenous injection. This study expands the application of ionizable lipids for selective oncolytic therapy.

Disruption of calcium ion (Ca2+) homeostasis has emerged as a promising strategy for tumor therapy. However, the intricate regulation of Ca2+ signaling and the limitations of single-dimensional modulation often hinder therapeutic efficacy. Here, we developed a Janus nanomotor platform that orchestrates mechanochemically coupled multidimensional modulation of Ca2+ overload for enhanced tumor therapy. Utilizing a liquid-nano-liquid interface-mediated anisotropic encapsulation strategy, amorphous calcium carbonate (ACC) nanoparticles were asymmetrically coated with mesoporous polydopamine (mPDA) and subsequently functionalized with l-arginine (l-Arg) and hyaluronic acid (HA), forming the Janus ACC@SiO2&mPDA-Arg-HA nanomotors that combine structural asymmetry, NO-driven propulsion, and tumor-targeting capability. Within the tumor microenvironment (TME), degradation of the ACC provided a sustained exogenous Ca2+ reservoir. Simultaneously, the endogenous catalytic conversion of l-Arg into NO triggered self-propulsion, mechanically stimulating the cell membrane to activate Piezo1 channels and promote extracellular Ca2+ influx. In parallel, NO acted as a gaseous chemical messenger to trigger ryanodine receptors (RyRs)-mediated Ca2+ release from the endoplasmic reticulum (ER). The mechanically and chemically coupled regulation induces persistent Ca2+ overload, leading to mitochondrial dysfunction and apoptosis. Our study presents a paradigm of mechanochemical coupling for multidimensional signal modulation, offering a framework for engineering nanomachines that reprogram intracellular signaling in cancer therapy.

Living cell therapy and nonliving drugs are both key approaches for treating solid tumors. Engineering living cells with nonliving drugs harnesses their complementary advantages to enhance therapeutic outcomes. However, inappropriate engineering methods or design strategies can compromise the synergy between cells and drugs. In this review, we provide a decision framework that matches engineering strategies to cell types and drug properties, yielding baseline-qualified engineered cells. To address barriers specific to solid tumors, we then discuss strategies for equipping cells with additional functions that enable deeper penetration, resistance to immunosuppression, and controlled release. We also discuss emerging strategies to increase their safety during clinical translation. This review aims to guide the rational design of next-generation engineered cell therapies to achieve improved clinical outcomes.

Although A2AR is a key immunoregulatory receptor that suppresses CD8+ T cell activation in response to elevated extracellular adenosine in inflamed or hypoxic microenvironments, its role in CD8+ T cell differentiation and cell-fate decisions during chronic viral infection and cancer remains poorly understood. Using A2AR-eGFP reporter mice, we show that A2AR expression is rapidly induced by TCR stimulation and persists under chronic antigen exposure and hypoxia, with sustained expression strongly associated with terminal exhaustion via the canonical Gαs-cAMP-PKA pathway. Paradoxically, A2AR loss does not alleviate exhaustion but instead accelerates differentiation toward the terminally exhausted state. Single-cell multiomics profiling revealed that A2AR deficiency activates CD122 (IL-2Rβ)-dependent signaling, driving T cell exhaustion. Genetic deletion of CD122 in A2AR-deficient CD8+ T cells reduced terminal exhaustion, identifying CD122 signaling as a key mediator of A2AR loss-driven exhaustion. Intriguingly, both sustained A2AR expression and A2AR loss converge to promote T cell exhaustion differentiation through distinct mechanisms. These findings uncover a paradoxical role of A2AR in shaping CD8+ T cell fate choices during chronic infection and cancer.

Chemical labeling of nucleic acids is essential to pinpoint the structure, localization, and function of RNA and DNA. Yet, reversible sequence-independent chemistries that can label native RNA and DNA remain poorly developed. Here, we describe Reversible Uridine Nitrilium-mediated Addition (RUNA), a reversible covalent chemistry that selectively modifies uridine and thymidine residues via N3 deprotonation and reaction with a nitrilium ion intermediate generated from an aldehyde and an isonitrile. The reaction forms a stable N3 adduct that can be quantitatively reversed by hydrolysis. By using reagents that are either membrane permeable or impermeable, we demonstrate the localization and function of DNA on exosomes. Although exosomes harbor nucleic acids, whether the latter are encapsulated in the exosome lumen or are surface-adhered is unknown. RUNA revealed that exosomes display DNA on their outer surface. The abundance of such surface DNA increases upon DNA-damage accumulation in cancer cells that are treated with a PARP inhibitor. This surface DNA drives exosome uptake by M2-polarized macrophages through scavenger receptors and triggers a shift toward an M1-like proinflammatory state. The selective labeling of surface DNA revealed an unexpected mechanism by which exosomes engage innate immune cells. RUNA is a versatile tool to analyze the nucleic acid content and functionality of extracellular vesicles in health and disease.

Extracellular vesicles (EVs) are central mediators of intercellular communication and promising carriers for oncology diagnosis and therapeutic delivery, yet their isolation remains challenged by inadequate specificity, irreversible capture, and vesicle structural damage. Here, we present the Multifunctional Optically Regulated Plasmonic Heating-Enhanced Ultrasensitive Sensing (MORPHEUS) system for programmable and reversible EV manipulation with thermoplasmonics-regulated aptamer switches. Through spatiotemporal control of localized thermoplasmonic heating, MORPHEUS established a temperature-defined "capture window" with enhanced binding kinetics, followed by a mild "release window" enabling 98.94% nondestructive EV recovery. The localized thermoplasmonic heating fine-tuned the conformational dynamics of the CD63 aptamer and free-energy landscape, accelerating association and dissociation kinetics by 3-fold and 415-fold, respectively, for precise binding-to-releasing control over intact EVs. Thermodynamically, the programmed photothermal heating conditions reduced the energy barriers for molecular desolvation and structural rearrangement, enhancing conformational sampling and facilitating interfacial water molecule release, resulting in entropic gains. This dual kinetic-thermodynamic regulation enabled reversible aptasensing regeneration while preserving EV structural integrity, membrane protein activity, and nucleic acid cargo. The platform maintained stable operation over 30 consecutive capture-release cycles with a capture-signal coefficient of variation (CV) of 3.24%. Benefiting from the preserved biological integrity, MORPHEUS-enriched EVs retained efficient drug-loading capability and therapeutic activity in cellular models, highlighting the potential of programmable thermoplasmonic regulation for EV-based diagnostic and therapeutic engineering.

Intercellular electrical coupling mediated by gap junctions plays a central role in signal transmission in many biological systems. Its disruption contributes to cardiac and neurological disorders, as well as impaired wound healing and tumor progression. Restoring direct electrical communication between cells, however, remains challenging, particularly without genetic manipulation or the delivery of preformed devices across cellular membranes and interfaces. The small conjugated molecule DTTO (2,6-diphenyl-3,5-dimethyl-dithieno[3,2-b:2',3'-d]thiophene-4,4-dioxide) self-assembles inside living cells into supramolecular nanofibers, which can extend between neighboring cells and connect their cytoplasm. Here, we show that these fibers also establish functional electrical coupling between cells: dual patch clamp recordings demonstrate restored signal transmission even when native gap junctions are pharmacologically suppressed, while control experiments show that the recovered signal transmission does not result from nonspecific membrane poration associated with fibers crossing the membrane. Electrical characterization of DTTO fiber networks shows that these structures support charge transport, while humidity-dependent measurements, impedance spectroscopy, and equivalent circuit modeling show that the observed electrical response is shaped by ionic and interfacial contributions from the surrounding environment. Collectively, this work establishes intracellular DTTO self-assembly as a nongenetic strategy to create functional bioelectrical connections in situ and restore electrical communication in diseased and engineered tissues.

The human gastrointestinal tract is colonized by trillions of microbes, collectively termed the gut microbiota, which dynamically shape states of health and disease. This occurs through the modulation of host immunity and through metabolites produced by these microbes, which act both locally and at distal organs. Seminal research has defined that the gut microbiota are essential for successful cancer immunotherapy, with specific microbes and associated metabolites linked to therapeutic outcomes. The mechanisms accounting for this remain preliminary, with additional research implicating diet in shaping the composition and functional potential of the gut microbiota to steer host immunity toward fighting or supporting malignant tumors. The goal of this review is to summarize recent advances in a key communication loop between diet, the gut microbiota, and host immunity as it relates to immune checkpoint blockade in cancer. Further, we discuss gaps in knowledge and future opportunities to harness this knowledge to improve therapeutic strategies.

Solid tumours show substantial mechanical heterogeneity, yet how such cues influence the susceptibility of cancer cells to T cell-based therapies remains unclear. Here we discover that cancer cells grown on soft matrices are less sensitive to chimeric antigen receptor T cell cytotoxicity and exhibit elevated extracellular adenosine triphosphate and sustained calcium activity. To understand the mechanisms underlying this reduced killing, we sought to identify the cells that respond to mechanical softness. We engineered a doxycycline-gated calcium-activated transcriptional mechano-recorder that integrates softness-induced calcium activity over a defined recording window and converts this prior signalling history into a stable fluorescent output that persists for days. Unlike real-time calcium indicators, which report instantaneous calcium activity only at the moment of imaging, this recorder preserves a sortable transcriptional mark, enabling selective labelling and profiling of cells according to their past mechanosensing activity. Transcriptomic analyses showed that recorder-positive cells adopt a stem-like programme, including epithelial-mesenchymal transition, hypoxia responses, oncogenic signalling and elevated stemness markers, across cancer cell lines and patient-derived samples. To render these resistant cells targetable, we rewired the mechano-recorder into a mechano-reprogrammer by replacing the fluorescent output with the clinically validated antigen CD19, enabling softness-responsive cells to be recognized by CD19-directed T cells. This rewired system improved elimination of stem-like cancer cells in culture and animal models, converting mechanobiological resistance into therapeutic vulnerability.

Anthracycline chemotherapeutics are common chemotherapeutics that have substantial toxicities. There is substantial interpatient pharmacokinetic variability, though there is no method to quantify organ or tumor exposure. Here, we exposed an optical nanosensor array to detect each of four anthracyclines. We screened 12 ssDNA sequences paired with seven single-walled carbon nanotube (n,m) species against several concentrations of doxorubicin, daunorubicin, idarubicin, and epirubicin. Complex spectral responses were used to develop machine-learning-based classification models to quantify each anthracycline. The optimized extreme gradient boosting model classified high levels of each anthracycline with 100% accuracy. Principal component analysis distinguished low (≤5 μM) and high concentrations of each anthracycline. Finally, we validated selected ssDNA-(n,m) pair performance in synthetic urine and sweat. Our findings deliver a generalizable optical spectral fingerprinting methodology for hard-to-detect analytes. Their use in clinical biofluids portends the preclinical and potentially clinical pharmacokinetic measurement of anthracyclines to improve efficacy and reduce toxicities.

ConspectusNucleic acid nanotechnology has fundamentally transcended the classic paradigm of DNA and RNA as passive carriers of genetic blueprints, which enables the rational design and construction of precise nanostructures with defined shapes, dynamics, and functions. This programmability has revolutionized approaches in biomedicine, facilitating breakthroughs in high-resolution molecular diagnostics, spatially and temporally controlled drug delivery, and the creation of synthetic cellular machinery. However, a central challenge for clinical translation is the inherent immunogenicity of nucleic acid materials. Introducing exogenous DNA or RNA nanostructures risks triggering potent innate immune responses, which can lead to rapid clearance, diminished therapeutic efficacy, inflammation, and toxicity. Rather than pursuing universal immunosuppression, researchers are beginning to rationally exploit defined immunostimulatory pathways, which allows for the strategic incorporation of immune-modulatory cues for vaccine development, immunotherapies, and targeted adjuvant systems.In this Account, we review our efforts to develop framework nucleic acids (FNAs) as a platform with modulable innate immunostimulation for biomedical applications in live cells and in vivo. We briefly summarize structural principles of nucleic acid immune recognition mediated by receptors such as toll-like receptors (TLRs) and cyclic GMP-AMP synthase (cGAS). We highlight that such immune recognition is dictated not merely by the abundance of nucleic acids but by key structural parameters, including size, shape, compactness, and the spatial organization of stimulatory nucleic acid motifs. We illustrate strategies to either enhance or suppress immunostimulation through controlled biodistribution, multivalent ligand display, and dynamic structural reconfiguration. These approaches enable tailored applications such as the development of nanovaccines and cancer immunotherapy, or conversely, anti-inflammatory and antioxidant therapies. Looking forward, we envision FNAs as intelligent tools for precision immunomodulation, bridging nanoscale design with immunological outcomes to advance personalized medicine.

Protein lysine lactylation (Kla) is a newly identified post-translational modification (PTM), in which lactyl groups are transferred to specific lysine residues in proteins. As a crucial intermediary between cellular metabolism and epigenetic regulation, Kla immensely increases the functional diversity of the proteome. This intriguing modification extends beyond histones to non-histone proteins, signaling molecules, enzymes, and substrates. In addition to enzymatic L-lactylation utilizing lactate as a lactyl donor and involving enzymes including writers (lactyltransferases), readers (lactylation-binding enzymes), erasers (delactylases), and lactyl-coenzyme A (lactyl-CoA) synthases, non-enzymatic D-lactylation derived from the glyoxalase II substrate S-D-lactoylglutathione (SLG) has also been identified. Emerging evidence underscores the molecular significance of Kla, including gene transcriptional activation, protein stability, enzyme activity, protein‒protein interactions, protein subcellular translocation, crosstalk with other PTMs, RNA modification, epigenetic instability, and phase separation, in orchestrating diverse biological processes. Functionally, Kla plays a fundamental role in physiology, such as somatic cell reprogramming, as well as embryonic, neural, and cochlear development, by regulating gene expression, cell cycle progression, and signal transduction. Conversely, dysregulated Kla renders extensive impacts on the pathogenesis of various diseases, including cancer, neuropsychiatric disorders, cardiovascular and ophthalmic diseases, and immunoinflammatory and metabolic dysregulation, through modulating immune homeostasis, metabolic adaptation, and epigenetic remodeling. This review systematically elucidates the molecular regulatory mechanisms and biological significance of Kla while comprehensively summarizing its involvement in both physiology and pathology. Furthermore, we emphasize the translational potential of Kla as a diagnostic or prognostic biomarker and therapeutic target, offering novel insights for future research and development of innovative therapeutic strategies.

Cuproptosis, a copper-dependent programmed cell death pathway, has emerged as a promising strategy for cancer therapy. While previous reviews have primarily focused on the fundamental mechanisms of cuproptosis and the development of inducing agents, the self-assembly-driven, stimuli-responsive cuproptosis nanoplatforms have remained relatively underexplored. To fill this gap, this review provides a comprehensive analysis of recent advancements in stimuli-responsive, dynamic self-assembly nanosystems for cuproptosis-based cancer therapy and imaging. We begin by elucidating the molecular mechanisms that underpin cuproptosis, followed by a systematic exploration of the design principles behind self-assembly-based cuproptosis inducers. These inducers leverage selective copper-ion coordination chemistry, coupled with diverse intermolecular forces, to enable precise spatiotemporal regulation. Furthermore, we highlight how these nanoassemblies undergo intelligent structural and functional transformations in response to both endogenous and exogenous stimuli, enabling controlled activation of cuproptosis. This review also explores breakthrough applications that integrate cuproptosis with synergistic therapeutic strategies and imaging guidance, highlighting a promising direction for enhancing cancer treatment. Finally, we discuss the critical challenges and barriers that must be addressed to push this emerging field toward clinical translation.

Quenching-resistant near-infrared-II (NIR-II) fluorophores with strong light harvesting capabilities and excellent photophysical properties remain a critical challenge in cancer phototheranostics. Herein, we report a novel acceptor-donor-acceptor (A-D-A) fluorophore, BTP-2TCF, constructed by introducing planar, rotatable tricyanofuran (TCF) acceptors into the core of the high-performance dye Y6. This molecular design imparts obvious anti-quenching behavior to BTP-2TCF nanoparticles (NPs), achieving an improved photoluminescence quantum yield of 1.9% and over a ten-fold enhancement in reactive oxygen species generation compared to Y6 NPs. These improvements are likely attributed to the restricted intramolecular motion of the TCF rotor, which suppresses non-radiative energy dissipation. BTP-2TCF NPs also exhibit a high molar absorption coefficient (>82 000 M-1 cm-1) and brightness (635 M-1 cm-1), outperforming conventional anti-quenching dyes. Their bright NIR-II fluorescence enables high-contrast visualization of mouse vasculature. Guided by NIR-II fluorescence imaging, BTP-2TCF NPs demonstrate superior photodynamic and photothermal anticancer efficacy in vivo. This study highlights the TCF planar rotor as a powerful strategy for developing next-generation quenching-resistant fluorophores for biomedical applications.

Methionine is essential for protein synthesis and serves as a precursor to S-adenosylmethionine, supporting epigenetic modifications and cell growth. Unlike normal cells, cancer cells are dependent on methionine. Therapeutic strategies targeting metabolic vulnerability show promise but face challenges related to tumor heterogeneity and adaptive resistance, limiting clinical translation. This review comprehensively explores methionine metabolism in cancer, detailing its uptake, transport, and metabolic pathways that underpin tumor development. We examine tumor dependency on methionine across various cancer types and subtypes, highlighting oncogene-driven variability, including the pivotal roles of methionine adenosyltransferases (MATs), the methionine salvage enzyme 5'-methylthioadenosine phosphorylase (MTAP), and polyamine synthesis enzymes such as ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase 1 (AMD1). Epigenetic contributions involving DNA, RNA, and histone methylation are discussed, emphasizing their tissue-specific implications, such as the MATI/III:MATII ratio in liver cancer and methionine metabolic features in glioblastoma, including PET imaging applications. This review further addresses methionine metabolic plasticity within the tumor microenvironment, focusing on epigenetic and immune reprogramming, metabolic checkpoint regulation, and the role of methionine in tumor-initiating cells and cancer stem cells. Therapeutic strategies targeting methionine dependence, including dietary restriction, methioninase enzyme therapies, transporter inhibition, and combination regimens aimed at enhancing efficacy, are critically evaluated. Finally, we discuss current challenges and future directions, emphasizing the need for tumor-selective interventions, improved delivery systems, and personalized metabolic diagnostics to translate metabolic vulnerabilities into effective clinical cancer therapies. This integrated analysis underscores the potential of targeting methionine metabolism for advancing cancer treatment strategies.

Aberrant proximal biomolecular complexes are critical disease biomarkers. The precise in situ imaging of these complexes is essential for deciphering disease pathogenesis and precision diagnostics. However, current in situ analysis technologies are often constrained by limited resolution, diffusion-mediated false positives, or the requirements for rigid conjugation between recognition and amplification moieties, which hampers versatility and multiplexing. Here, we introduce Proximity Anchored Modules Assembly (PAMA), a versatile and multiplexed imaging strategy with a "plug-and-play" architecture. By decoupling target recognition from signal amplification via programmable DNA tracks synthesized by Primer Exchange Reaction (PER), PAMA triggers a polymerase-driven extension exclusively upon dual-recognition events. This mechanism ensures precise proximity-dependent activation, showing a specific signal response to homologous and heterologous targets. We further validated PAMA as a versatile platform for detection of proximal biomarkers by visualizing HER2 receptor dimerization patterns in breast cancer cells and precise discrimination of BCR-ABLP210 fusion gene isoforms in clinical chronic myeloid leukemia (CML) samples. PAMA bridges molecular precision with spatial context through a rapid (approximately 2.5 h) and highly sensitive in situ profiling workflow. Ultimately, PAMA establishes a versatile, modular framework for the precise imaging of diagnostic biomolecular complexes, promising to accelerate both biological discovery and precision clinical diagnostics.

Tumor metastasis is the primary cause of cancer-related mortality. This complex process is orchestrated by the tumor microenvironment (TME) and metabolic reprogramming. Protein lactylation, a newly recognized post-translational modification derived from lactate metabolism, is emerging as a critical regulator of tumor progression and metastasis. This review aims to provide an overview of the current understanding of lactylation within the metastatic process and to offer an updated perspective on its regulatory role in tumor metastasis and its promise as a new therapeutic avenue. The review focuses on both the enzymatic and non-enzymatic mechanisms of lactylation and delineates the enzymatic machinery, including writers, erasers, and readers, that dynamically regulate this modification. It emphasizes how lactylation influences critical stages of metastasis, such as the epithelial-mesenchymal transition (EMT) and invasion, cancer stemness maintenance, immune evasion and TME remodeling, angiogenesis and vascular dissemination, and survival during colonization. Beyond these stage-specific roles, this review discusses how lactylation operates within a broader post-translational modification (PTM) network through crosstalk with acetylation, ubiquitination, and RNA methylation to amplify oncogenic signaling. Finally, the review evaluates emerging therapeutic strategies targeting lactate metabolism, the lactylation machinery, and site‑specific modifications, and addresses persistent challenges including context‑dependent functions, limited causal validation, and technical hurdles in isomer‑specific detection.

Reactive oxygen species (ROS) play a complex dual role in cancer biology. At physiological levels, ROS act as signaling molecules that drive tumorigenesis, metastasis, and therapy resistance by activating oncogenic pathways, such as NF-κB and PI3K/AKT, and fostering an immunosuppressive microenvironment. Conversely, excessive ROS accumulation overwhelms antioxidant defenses, triggering oxidative stress that can selectively eliminate tumor cells. Consequently, manipulating the delicate redox equilibrium has emerged as a pivotal strategy for cancer treatment. This review systematically examines the multifaceted functions of ROS, bridging the gap between fundamental redox biology and clinical application within the Predictive, Preventive, and Personalized Medicine (3PM) framework. Beyond molecular mechanisms, we evaluated the rationale for utilizing mitochondrial redox signatures as intrinsic biological sensors to identify suboptimal health conditions (SHC) and prevent the health-to-disease transition. We elucidate the regulatory networks governing ROS production and elimination, highlighting their dual function in promoting genomic instability versus inducing distinct cell death modalities, including apoptosis, autophagy, necroptosis, and ferroptosis. Special attention is given to ROS-mediated remodeling of the tumor microenvironment (TME), where oxidative stress facilitates immunosuppression. Importantly, we provide expert recommendations on integrating digital health monitoring and patient stratification into clinical oncology. By emphasizing mitochondrial rejuvenation and individualised protection, this review discusses how proactive interventions can restore homeostasis and improve long-term outcomes, offering a cost-effective alternative to reactive treatments.