Whether pyroptosis is controllable and reversible remains an enigma. Here we revealed that autophagy could eliminate the pore-formed N terminus of GSDME (GSDME-NT) located on membranes at different locations, suppressing pyroptosis. Crucially, GSDME-NT on the plasma membrane was eliminated through endocytic internalization, where GSDME-NT-laden vesicles were targeted and degraded as intact units. Specifically, GSDME-NT pores on the plasma membrane induced endocytosis, generating endocytosed but leaky vesicles carrying GSDME-NT. Leakage prevented acidification, necessitating further degradation through autophagy. Upon endocytosis, GSDME-NT on the vesicle membrane was labelled with ubiquitin by calcium-activated E3 ligase NEDD4L. These labelled vesicles were recognized by TOLLIP, guiding subsequent autophagosome formation, and enabling further acidification, fusion with lysosomes and eventual GSDME-NT degradation. Furthermore, in several tumour models, either disturbing autophagy or interfering with the recognition of GSDME-NT vesicles by targeting TOLLIP increased tumour cell pyroptosis, activating antitumour immunity and promoting chemotherapeutic efficacy.

A critical determinant of T cell anti-tumor function lies in their spatial interactions with adjacent cells, a dimension that remains largely elusive to conventional analytical approaches. Spatial multi-omics technologies are advancing our understanding of cancer immunity by resolving cellular interactions within intact tumor tissue contexts. Integrative profiling of spatial genomics, transcriptomics, proteomics and metabolomics enables multidimensional characterization of these biological processes, which respectively links clonal architecture to immune selection, delineates functional state transitions, quantifies cellular crosstalk and effector function, and identifies biochemical barriers. We propose an organizational framework where intratumoral immunity is governed by regions, which control immune cell access and retention under microenvironmental constraints, and functional niches, where proximity-dependent multicellular interactions drive immune activation, help, killing, or suppression. Translationally, we outline a paradigm in which discovery-grade spatial multi-omics define minimal architectural metrics measurable in clinical formalin-fixed, paraffin-embedded samples under robust quality control, with large-scale validation establishing reproducible biomarkers for patient stratification and companion diagnostics, potentially supporting mechanism-anchored intervention design and microstructural-guided immunotherapy.

Despite promising data showing that circulating tumour DNA (ctDNA) dynamics during treatment can inform real-time tumour response and recurrence risk1, how best to translate these insights into actionable clinical decision-making remains unclear. Here we report results from the EP-STAR trial-a multi-centre, ctDNA-driven, risk-adapted, non-randomized phase II study ( NCT04072107 ; ClinicalTrials.gov) testing whether a risk-adaptive treatment (RAT) strategy guided by on-treatment ctDNA dynamics can meaningfully improve survival, using nasopharyngeal carcinoma as a model. Eligible patients were enrolled and began treatment with standard-of-care gemcitabine-cisplatin neoadjuvant chemotherapy (GP-NAC; the P in this abbreviation stands for platinum)2, followed by RAT or standard-of-care chemoradiotherapy guided by ctDNA clearance trajectory during GP-NAC. Protocol-eligible patients who did not receive RAT, drawn from a prospectively registered ctDNA biomarker cohort ( NCT03855020 )3, served as a non-randomized, contemporaneous no-RAT external cohort. The primary end-point was failure-free survival (FFS) in the RAT group. After a median follow-up of 47.3 months, the 3-year FFS was 89.1% (83.2-95.0%) in the RAT group (n = 110). Patients who received RAT showed significantly improved FFS (P = 0.003, log-rank test) compared with the no-RAT external cohort (hazard ratio = 0.41 [0.23-0.75]; P = 0.004, Cox regression model). The RAT strategy was well-tolerated with no treatment-related deaths. Collectively, these data show that a ctDNA-driven RAT paradigm could be a promising strategy to improve survival, challenging the conventional fixed-course, static treatment approach.

Supplementary Figure S7. IDH1 deficiency promotes GP-induced ferroptosis in NPC cells.

Gemcitabine and cisplatin (GP) serve as first-line induction chemotherapy (IC) for nasopharyngeal carcinoma (NPC), yet predictive markers are lacking. Here we performed multi-omics profiling, including proteomics, phosphoproteomics, genomics and transcriptomics, on 240 patients with NPC who were receiving GP-IC or concurrent chemoradiotherapy (CCRT) alone. Through multi-omic integration, we identified three proteomic subtypes with distinct therapeutic vulnerabilities. The S1 subtype showed a predominant interferon-γ response and had favorable outcomes with CCRT alone. The S2 subtype featured copy-number-driven cell cycle activation, deriving benefits from GP-IC. The immune-exhausted S3 subtype exhibited high IgA+ plasma cell infiltration and was resistant to GP-IC but responded to anti-PD-1 therapy. Single-cell RNA sequencing confirmed interaction between IgA+ plasma cells and CD8+ T cells in nonresponders. Validation from three phase III trials and spatial analyses demonstrated that high IgA+ plasma cell infiltration predicted GP-IC resistance but benefited from anti-PD-1 therapy. This study delineates a subtype-specific landscape of GP-IC response and may inform personalized treatment in NPC.

siRNA sequences used in this study

Supplementary Figure S9. DHODH inhibition enhances GP-induced ferroptosis and antitumor efficacy with systemic safety maintained in NPC cells and xenograft models.

The mechanical properties of the tumor microenvironment serve as crucial physical cues that shape cell fate decisions. However, whether microenvironmental mechanical forces modulate ferroptosis to drive radioresistance remains unclear. Here, using PDMS-based hydrogels with tunable stiffness to establish tumor cell culture systems, we found that tumor cells cultured on stiff substrates were more sensitive to both ferroptosis and radiotherapy. Mechanistically, tumor cells grown on stiff matrices showed increased microtubule acetylation and ER sheet-to-tubule remodeling, which increased the formation of mitochondria-associated membranes (MAMs) and led to mitochondrial succinate accumulation. Succinate in turn promoted CPT1A-mediated succinylation of ACSL3, facilitating its degradation and thereby enhancing tumor cell sensitivity to ferroptosis. Collectively, these findings identify MAMs as intracellular mechanosensitive structures that regulate mitochondrial metabolism and ferroptosis in tumor cells, providing new insights into the mechanical control of ferroptosis and its implications for tumor radioresistance.

Abstract The N-terminal fragments of gasdermin (GSDM-NT) form pores on the plasma membrane that initiate pyroptosis. However, the presence of GSDM-NT pores does not necessarily result in cell death, allowing some cells to survive GSDM-mediated pyroptosis. Understanding the regulators of response to the formation of GSDM-NT pores is crucial for revealing the strategies to harness the potential of pyroptosis for treating cancer. In this study, we found that myosin 1G (MYO1G) enabled cells to resist death driven by membrane rupture. Mechanistically, MYO1G tethered GSDME-NT pores to caveolin 1 (CAV1), thus facilitating CAV1-mediated endocytosis of pyroptotic pores. Pyroptosis triggered the transcriptional upregulation of MYO1G through stabilization of hypoxia-inducible factor 1α (HIF1α), which resulted from the release of intracellular α-ketoglutarate. Moreover, pharmacologic inhibition of MYO1G or cholesterol synthesis promoted pyroptosis, boosted antitumor immunity, and synergized with chemotherapy to eradicate tumors. Clinically, MYO1G was validated as an indicator of poor short-term response to cisplatin-based chemotherapy and unfavorable long-term survival in patients with nasopharyngeal carcinoma. Overall, these findings provide further understanding of a protective mechanism against pyroptosis and identify potential therapeutic targets for cancer treatment. Significance: MYO1G-facilitated endocytosis of gasdermin E pores protects cancer cells against pyroptotic death and contributes to cancer chemotherapy resistance, which provides potential biomarker and therapeutic strategies for cancer.

MCM enhances ATF4 activation in tumor cells, which facilitates mregDC-mediated formation of central memory T cells, improving long-term protection

BACKGROUND:Adolescents with osteosarcoma face clinical and psychosocial challenges during the transition from adolescence to adulthood. AIMS:This study examined the effects of a nurse-navigated game-based intervention (N2-GBI) on transition readiness in adolescents. METHODS:Participants in the control group received usual care, whereas the intervention group received the N2-GBI programme alongside usual care. Transition readiness was assessed using the Self-Management and Transition to Adulthood with Rx = Treatment Questionnaire as the primary outcome. Secondary outcomes included cancer worry, transition expectation and patient activation, which were measured using the Cancer Worry Scale, Transition Expectation Scale, and Patient Activation Measure, respectively. Outcomes were assessed at baseline (T0), immediately after intervention (T1) and at 12-week follow-up (T2). Generalized estimating equations were used to estimate the intervention effects. RESULTS:A total of 59 adolescents were enrolled. The intervention and control groups comprised 32 and 27 adolescents, respectively, with mean ages of 15 and 13 years, respectively. A total of 19 and 15 participants were male in the intervention and control groups, respectively. Compared with the control group, the N2-GBI group showed significantly greater improvement in transition readiness at T1 (β = 8.83, p < 0.001) and T2 (β = 7.97, p = 0.001). The N2-GBI programme also significantly reduced cancer worry (p = 0.009) and increased patient activation (p = 0.001), but had no significant effect on transition expectation (p = 0.056). CONCLUSIONS:The N2-GBI programme improved transition readiness, reduced cancer worry, and increased patient activation among adolescents with osteosarcoma, but did not significantly improve transition expectation. This intervention may be a useful nurse-led approach to transition preparation in pediatric oncology care. Future studies with larger sample sizes and prolonged follow-up durations are needed.

The human microbiota comprises a diverse and extensive community of microorganisms that participate in intricate interactions with the host, several of which are increasingly acknowledged as key modulators of health and disease. Among these, Fusobacterium nucleatum (Fn), an oral commensal bacterium, has emerged as a significant oncobacterium implicated in tumour progression. The Fn genus comprises distinct subspecies, clades and strains, exhibiting marked phylogenetic and physiological heterogeneity. Consequently, pinpointing the true functional modulators within this complex community and elucidating their mechanisms in various physio-pathological states remains a critical yet challenging endeavour. Moreover, the complete mechanism underlying Fn's function across different spatial locations and physiological states remains to be fully elucidated. This review details the genetic and phenotypic heterogeneity among Fn subspecies, which underlies their differential characteristics and niche adaptation. We further delineate key effectors of Fn, such as adhesins, metabolites and exoproteins, which collectively facilitate host cell invasion, immune evasion and chemoresistance induction. We explore the translational potential of Fn, underscoring its utility as a diagnostic biomarker and a promising target for novel therapeutic strategies.

Supplementary Figure S10. DHODH-associated DNA damage repair and ferroptosis protection are positively correlated across tumors and contribute to poorer survival.

The nasopharynx constitutes a critical niche in the upper respiratory tract, harboring a diverse microbiota linked to nasopharyngeal carcinoma (NPC), the mechanistic roles of which remain poorly understood. Here, we established the Nasopharyngeal Mucosal and Tumor-resident Bacterial Catalog (NMTBC) that comprises 5311 bacterial isolates representing 127 species, with 1006 of them being fully sequenced and annotated, providing a comprehensive culturable resource facilitating mechanistic dissection of the microbiome-tumor interactions. With NMTBC, we uncovered a Fusobacterium-Prevotella mutualism and revealed heterotypic bacterium-bacterium interactions involving transcriptional reprogramming and metabolic cross-talk. Using single-bacterial transcriptomics, we mapped a high-resolution transcriptomic trajectory, showing the ability of a single strain to differentiate into functionally distinct subpopulations that cooperate to sustain mutualism. By analyzing a multicenter NPC cohort, we showed that Fusobacterium and Prevotella co-colonization in NPC tumors correlated with unfavorable clinical outcomes after conventional radiochemotherapy. Analysis of RNA-seq data from two previous phase 3 clinical trials showed that coenrichment of Fusobacterium-Prevotella predicted better response to anti-PD-1 immunotherapy, highlighting their important role in microbiota-mediated immunomodulation. Overall, this study establishes a comprehensive nasopharyngeal bacterial catalog through culturomics, which offers valuable insights into microbiome-derived biomarker discovery and immunotherapy patient stratification in clinical practice.

Introduction:Accurate dose calculation is essential in breast cancer radiotherapy. This study aimed to evaluate the feasibility and accuracy of cone-beam CT (CBCT) images with HU-relative electron density (HU-RED) correction for dose calculation in left-sided breast cancer radiotherapy. Methods:Twenty postoperative patients receiving adjuvant radiotherapy at Subei People's Hospital of Jiangsu Province (Yangzhou, China) from January 2022 to December 2024 were retrospectively enrolled. A patient-specific HU-RED calibration curve was generated using an improved density override method. Treatment plans created on planning CT (pCT) were transferred to CBCT for dose recalculation with identical optimization parameters. Dosimetric parameters of the planning target volume (PTV) and organs at risk (OARs) were compared. Two-dimensional gamma analysis was performed to assess dose consistency. Equivalence between CBCT- and pCT-based dose calculations was further evaluated using the two one-sided tests (TOST), Lin's concordance correlation coefficient (CCC), intraclass correlation coefficient (ICC), and Bland-Altman analysis. Results:Differences in PTV dosimetric indices (D2, D50, D98, Dmean, HI, CI) between CBCT- and pCT-based plans were small, with mean deviations <1.3% and no statistically significant differences (P > 0.05). OAR parameters, including cardiac Dmean, V20, V30, V50 and lung Dmean, V20, V30, also showed minimal variation (<1.5%), with the largest deviation observed in cardiac V20 (2.1% in a single case). Gamma analysis revealed high agreement between both plans, with passing rates exceeding 90% for both the 3%/3 mm and 2%/2 mm criteria. Equivalence testing demonstrated statistical equivalence between CBCT- and pCT-based dose calculations for all PTV parameters and most OAR metrics. All 90% confidence intervals fell entirely within predefined equivalence margins (Δ), and all TOST P-values were <0.05. Lin's CCC and ICC(A,1) exceeded 0.96 for all parameters, indicating excellent consistency, while Bland-Altman analyses showed minimal bias and narrow limits of agreement. Discussion:CBCT images corrected with HU-RED calibration achieved highly consistent dose calculation results compared with pCT in left-sided breast cancer radiotherapy. This method is feasible for clinical dose verification and may support future adaptive radiotherapy strategies, particularly with the integration of artificial intelligence techniques.

PURPOSEPersonalized immunotherapy strategies are urgently needed for patients with locoregionally advanced nasopharyngeal carcinoma (NPC). We aim to identify biomarkers predictive of immunotherapy benefits, using data from the phase III CONTINUUM (ClinicalTrials.gov identifier: NCT03700476) and DIPPER (ClinicalTrials.gov identifier: NCT03427827) randomized clinical trials.PATIENTS AND METHODSTumor samples from 407 patients in the CONTINUUM (discovery cohort) and DIPPER (validation cohort) trials were subjected to RNA sequencing. In the discovery cohort, metabolic gene-based consensus clustering was performed to determine subtypes. A machine learning-based classifier was subsequently developed in the discovery cohort and then applied to the validation cohort to assign metabolic subtypes. Gene set enrichment analyses were used to characterize the biological features of each metabolic subtype. The clinical end point was event-free survival (EFS).RESULTSIn the discovery cohort, three metabolic subtypes were identified with distinct tumor-intrinsic and immune features as well as differential EFS benefits from adding anti-PD-1 to chemoradiotherapy (CRT). Specifically, the MS1 subtype exhibited a significant improvement in 3-year EFS in the anti-PD-1 plus CRT arm compared with the CRT-alone arm (3-year EFS, 90.2% v 69.6%; hazard ratio, 0.27 [95% CI, 0.11 to 0.67]), whereas MS2 (3-year EFS, 94.1% v 93.8%) and MS3 subtypes (3-year EFS, 75.0% v 75.0%) derived no significant survival benefit. The subtype features were preserved in the validation cohort, with consistent prognostic and predictive value. A pooled analysis of both cohorts demonstrated the significant interaction between metabolic subtypes and the treatment effect (Pinteraction = 0.0074).CONCLUSIONIn this biomarker study, we defined metabolic subtypes of NPC that predicted the EFS benefit from immunotherapy. This novel molecular classification provides a promising predictive biomarker for personalized treatment decision for patients with locoregionally advanced NPC.

Chemoresistance is a primary factor limiting nasopharyngeal carcinoma (NPC) treatment. Growing evidence indicates that E3 ubiquitin ligases play a pivotal role in chemoresistance. Here, we identified that the E3 ubiquitin ligase RNF138 is significantly upregulated in NPC patients who do not respond to chemotherapy. Our study reveals that RNF138 promotes the K48-linked ubiquitination of hnRNPA0 at K133, thereby destabilizing WWOX mRNA. The subsequent loss of WWOX protein relieves the inhibition of JAK2 self-phosphorylation, leading to constitutive pathway activation. Consequently, RNF138-JAK2/STAT3 activation suppresses chemotherapy-induced apoptosis via reduced ROS production and promotes immune evasion by upregulating PD-L1. Clinically, high RNF138 expression correlated with poor prognosis and resistance to chemotherapy. In conclusion, this study unveils the RNF138-hnRNPA0-WWOX axis as a driver of JAK2/STAT3 activation, leading to both chemoresistance and immune evasion in NPC. This work positions RNF138 as a valuable biomarker to guide individualized chemotherapy, and highlights JAK inhibitors as a potential targeted therapy for NPC patients.

Supplementary Figure S1. IDH1 overexpression is observed across tumors and facilitates GP resistance in NPC cells.

The efficacy of chemotherapy depends partly on the ability to induce antitumor immunity. A better understanding of how different chemotherapy modes mediate antitumor immune responses could provide insights for developing optimized treatment modalities. In this study, we demonstrated that metronomic chemotherapy (MC), a mode of frequent and regular administration of chemotherapeutic drugs at lower doses, induced robust CD8+ T cell-dependent antitumor immune memory by modulating the activity of mature regulatory dendritic cells (mregDCs). Mechanistically, by imposing more frequent stress on tumor cells, MC induced sustained activation of activating transcription factor 4 (ATF4), leading to metabolic reprogramming of tumor cells and enhanced asparagine (Asn) release into the tumor microenvironment. Functioning as a ligand, Asn directly bound to AXL receptor tyrosine kinase (AXL) on mregDCs, inhibiting AXL kinase activity and downregulating programmed death-ligand 1 (PD-L1) expression. Consequently, mregDCs with reduced PD-L1 expression fostered the generation of more memory-like CD8+ T cells during their interactions. Overall, this study unveils critical biological events driving antitumor immune memory formation under therapeutic stress and provides a rationale for optimizing chemotherapy modalities. SIGNIFICANCE:Metronomic chemotherapy enhances antitumor immunity by rewiring tumor cell metabolism to reprogram mature regulatory dendritic cells and foster memory CD8+ T-cell formation, providing insights for enhancing treatment efficacy.