Cancer Immunology, Immunotherapy

The development of cancer immunotherapies is hindered by the lack of human-relevant models that accurately translate to patient outcomes. We combine patient-derived colorectal tumor organoids (PDOs) and cancer-associated fibroblasts (CAFs) into floating extracellular matrix drops to form miniature colorectal tumors. These SHaking Organoid COcultures (SHOCOs) maintain immune cells in numbers, states and functional interactions that are more physiologically accurate than traditional PDO-based co-culture models. Immunocompetent SHOCOs treated with T-cell bispecific antibodies exhibited a robust anti-tumor response, in a concentration- and duration of treatment-dependent manner. By varying the stromal content, we found that fibroblasts present a physical barrier that hinders intratumoral T-cell infiltration. We also demonstrate that tumor-associated stroma can be exploited therapeutically in potentiating anti-tumor immune responses. SHOCOs could aid the battle against cancer both by providing fundamental insights into immune and stromal tumor biology, and by catalyzing the discovery of novel therapeutic approaches. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/729459v1_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@1aed309org.highwire.dtl.DTLVardef@a11696org.highwire.dtl.DTLVardef@1d1d74dorg.highwire.dtl.DTLVardef@18e2c3f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 0C_FLOATNO / Graphical Abstract: Illustration of the SHOCO Model. (A) Overview of the components used in creating SHOCOs and the complex tumor microenvironment they replicate. Epithelial cells (pink), CAFs (red), CD8 T cell (blue), CD4 T cell (green). Characteristics of SHOCOs: (B) Emphasizing the role of CAFs in contracting and creating a denser microenvironment. (C) CAFs act as a physical barrier, preventing CD8 T cells from reaching tumor niches. (D) SHOCOs maintain a substantial myeloid population (violet) capable of performing antigen presentation. (E) T cells within SHOCOs mount an anti-tumor cytotoxic immune response as a result of immunotherapy. Granzymes (orange), cleaved Caspase-3/7 (yellow). C_FIG

Melanoma remains one of the most treatment-refractory malignancies due to immune evasion, high mutational burden, and profound tumor heterogeneity. Although immune checkpoint inhibitors have transformed frontline management, a substantial proportion of patients develop resistance or experience relapse, underscoring the need for alternative and complementary immunotherapeutic strategies. Tumor-infiltrating lymphocyte (TIL) therapy and engineered viral vector-based immunotherapies represent mechanistically distinct yet clinically promising approaches for advanced melanoma. This systematic review and Bayesian meta-analysis evaluated the comparative efficacy of TIL therapy and engineered viral vector immunotherapies in advanced melanoma. A structured search of PubMed, Embase, Scopus, and Web of Science (2015-2025) identified 13 eligible studies, including four randomized controlled trials and nine prospective single-arm studies, reporting objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and treatment-related adverse events. Eight studies met criteria for inclusion in the Bayesian quantitative synthesis of ORR outcomes. Risk of bias and certainty of evidence were assessed using Cochrane and GRADE frameworks. TIL therapy demonstrated substantial standalone efficacy, particularly in PD-1-refractory populations, with reported ORRs reaching 49%, median PFS of 7.2 months, and OS extending to 25.8 months. Viral vector-based therapies, including talimogene laherparepvec (T-VEC) and RP1, showed more modest monotherapy activity but demonstrated improved responses when combined with immune checkpoint inhibitors. Among the studies included in the Bayesian quantitative synthesis, the pooled ORR estimate was 37.8% (95% highest density interval [HDI]: 30.6%-45.3%). Sensitivity analysis excluding the small-sample Cui et al. (2022) study yielded a similar pooled estimate of 38.3% (95% HDI: 30.4%-46.2%). Exploratory meta-regression supported the overall robustness of the findings. Certainty of evidence for ORR was moderate, whereas survival and safety outcomes were downgraded due to heterogeneity, sparse reporting, and inconsistent endpoint definitions. Collectively, these findings support complementary rather than competing roles for TIL and engineered viral vector immunotherapies within evolving melanoma treatment paradigms. The results further highlight the potential importance of biomarker-guided sequencing strategies, including viral immune priming followed by adoptive cellular therapy, as a framework for optimizing personalized immunotherapy in both refractory and earlier-line melanoma settings.

Background The clinical significance of clonal hematopoiesis of indeterminate potential (CHIP) in melanoma remains incompletely defined, particularly with respect to CHIP genotype, clone size, and somatic mutations (e.g BRAF mutations). We integrated human cohort data and a syngeneic melanoma mouse model to evaluate whether CHIP is associated with melanoma risk, tumor growth, and differential clinical outcomes. Methods We analyzed CHIP prevalence and survival in a large treatment-unselected melanoma cohort (n=2,480), evaluated tumor growth in a syngeneic BRAF-mutant (BRAFmut) melanoma murine model of TET2-CHIP and DNMT3A-CHIP, and assessed survival outcomes in an immune checkpoint inhibitor (ICI)-treated advanced melanoma cohort (n=361). Associations with progression-free survival (PFS) and overall survival (OS) were evaluated using Kaplan-Meier analyses and multivariable Cox proportional hazards models. Results CHIP was enriched among patients with treatment-unselected melanoma compared with age/sex-matched healthy controls, and larger CHIP clone size showed an age-adjusted association with inferior OS. In a syngeneic BRAFmut melanoma murine model, TET2-CHIP, but not DNMT3A-CHIP, was associated with significantly increased primary melanoma tumor growth. Among patients with ICI-treated advanced melanoma, CHIP was associated with worse OS compared with patients without CHIP. TET2-CHIP had the strongest adverse association with survival, whereas DNMT3A-CHIP was not significantly associated with PFS or OS. Conclusions CHIP is enriched in melanoma and exploratory analyses demonstrate genotype-specific differences in melanoma tumor growth and clinical outcomes. These findings support further investigation of genotype-specific CHIP profiling as a potential biomarker for melanoma risk stratification and immunotherapy outcomes.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is the paradigmatic immunotherapy-refractory cancer, with a 5-year survival of approximately 12% and minimal benefit from immune checkpoint blockade (ICB). The dominant mechanistic explanation classifies PDAC as a T cell-excluded "cold" tumor, implying that no functional anti-tumor T cells are available for checkpoint release. Whether this Block-strategy view is correct has not been re-examined under integrated evasion-framework analysis. MethodsWe applied a previously developed 16-module immune evasion framework to TCGA-PAAD (n=183), integrated with hub-cytokine analysis (IL-10/TGF-{beta}), Kv1.3-immune channelome data, and clinical trial mapping (12,007 trials). Single-cell validation used two independent PDAC cohorts retrieved through TISCH2: PAAD_CRA001160 (Peng 2019, 35 samples [24 PDAC + 11 adjacent normal], 57,443 cells) and PAAD_GSE154778 (Lin 2020, 16 samples, 14,953 cells), examined for CD8A, TOX, PRF1, KCNA3, and FAP expression by cell type. ResultsPDAC scored highest in CAF Wall (z=0.768) and Platelet Cloak (z=0.663) modules; strategy classification yielded Brake -- not Block -- driven by a positive KCNA3-survival relationship (HR=0.649, 95% CI 0.43-0.97, p=0.037). Single-cell qualitative analysis of TISCH2 violin plots showed that CD8 exhausted T cells (CD8Tex) carried (i) high CD8A, (ii) the highest TOX expression among annotated cell types, (iii) preserved PRF1, and (iv) high KCNA3 expression. FAP was strongly localized to fibroblasts (peak [~]3.0 vs. <0.5 elsewhere). The pattern was reproduced in the second cohort. The optimal three-module attack (MHC restoration + CAF disruption + VEGF blockade) suppressed 10 of 16 evasion modules in silico (62.5%); zero of 370 PDAC immunotherapy trials test this combination. ConclusionsPDAC may not be T cell-cold but T cell-trapped: CD8 T cells with intact Kv1.3 channels appear immobilized behind a FAP-positive cancer-associated fibroblast wall. ICB monotherapy is mechanistically insufficient because the brake is engaged on T cells that cannot reach the tumor. The framework predicts that triple-targeted intervention -- checkpoint release + CAF wall disruption + vascular normalization -- is the minimum effective strategy. This is a hypothesis-generating computational analysis; prospective experimental and clinical validation are required.

BackgroundAntibody-mediated blockade of innate receptor-MHC-I interactions represents a promising strategy to enhance anti-tumor immunity, particularly against metastatic cancers resistant to conventional checkpoint inhibitors. In this study, we investigated the effects of the pan anti-MHC-I monoclonal antibody M1/42, which targets MHC-I interactions with Ly49, selectively expressed on murine NK cell subsets. MethodsWe administered M1/42 to mice and assayed the proliferation and activation immune cells. Anti-tumor activity of growth and metastasis of checkpoint inhibitor-resistant pancreatic ductal adenocarcoma (PDAC) and B16F10 melanoma were assessed, complemented by extensive cellular phenotypic and RNA expression analysis. Binding and cryo-electron microscopic (cryo-EM) and X-ray crystallographic structural studies of M1/42 complexed with the mouse MHC-I molecule, H2-Dd, examined the Ab interaction site in comparison with those of Ly49 inhibitory receptors. ResultsM1/42 administration in mice robustly unleashed the proliferation and activation of natural killer (NK) cells, memory CD4+ and CD8+ T cells, dendritic cells, and macrophages in both lymphoid and non-lymphoid tissues, independent of Fc{gamma} receptors. M1/42 significantly restricted the growth and metastasis of checkpoint inhibitor-resistant pancreatic ductal adenocarcinoma (PDAC) and B16F10 melanoma in the liver and lungs, accompanied by increased tumor infiltration of effector CD8+ T cells, reduction of T regulatory cells, and a pro-inflammatory cytokine milieu. The anti-tumor effects of M1/42 depend on NK cells and are associated with upregulation of genes involved in antigen processing, interferon gamma responsiveness, and Th1 cytokine production, while downregulating inhibitory PD1/11 signaling. Structural analysis indicated that the effect of M1/42 on Ly49/MHC-I interactions was not due to direct steric competition. ConclusionsCollectively, these findings demonstrate that M1/42 unleashes coordinated innate and adaptive immune responses, overcoming tumor-induced immunosuppression and resistance to checkpoint blockade. This approach represents a paradigm shift in cancer immunotherapy, offering potential for more effective treatment of metastatic cancers that evade immune surveillance through MHC-I modulation. KEY MESSAGESO_ST_ABSWhat is already known on this topicC_ST_ABSA pan anti-mouse MHC-I mAb (M1/42) blocks interaction with several NK inhibitory receptors (Ly49A or Ly49C) resulting in NK cell activation and anti-viral and anti-tumor responses in vitro and in vivo. Other pan anti-human MHC-I mAbs (DX17 and W6/32) function similarly, blocking LILRB inhibitory receptor interaction of myeloid cells and NK cells. These stimulate human immune cells in humanized mouse models. What this study addsThis study analyzes the effects of the pan anti-mouse MHC-I mAb on NK and myeloid cell activation in detail, in the absence of T or B cells, and independent of FcR interaction. Additionally we analyze several mouse models of metastatic tumor progression, indicative of the progressive activation not only of the innate immune response, but also adaptive responses. The molecular mechanism of the mAb blocking of inhibitory receptors is revealed by cryo-EM and X-ray structures of M1/42 Fab/MHC-I (H2-Dd) complexes. How this study might affect research, practice, or policyElucidation of the details of the inhibitory effects of the mouse pan anti-mouse MHC-I mAb provides not only a more advanced understanding of the murine model system, but suggests additional functional avenues to be explored using the parallel an anti-human MHC-I mAbs.

Brain metastases (BrM) are associated with poor prognosis. A better understanding of anti-tumor immune responses in the context of immune specialized microenvironment of the brain is required to develop improved therapeutic strategies for this disease. We demonstrate that the conventional dendritic cells type 1 (cDC1) gene signature positively correlates with a prolonged BrM-dependent survival in melanoma and breast cancer patients. Furthermore, intracranial anti-tumor immune responses in preclinical BrM models consistently rely on cDC1s for tumor growth control, BrM-dependent survival and maintenance of the intra-tumoral CD8+ T cell pool, in contrast to variable, cancer type-dependent cDC1 roles in extracranial tumors. This is underpinned by tumor site-specific cDC1 molecular profiles with distinct Toll like receptor repertoires, upregulation of co-stimulatory molecules and IL-12, and enhanced type-I-IFN signaling in intracranial cDC1s, with the latter driving increased cDC1 activation. cDC1s also promote the conversion of progenitor exhausted CD8+ T cells to transient effectors, which is further enhanced by immune checkpoint blockade therapy. These findings pinpoint cDC1s as a major cell population of interest in the development of future immunotherapies for BrM.

Importance: While gut dysbiosis is known to impair response to immune checkpoint inhibitors (ICIs), the relative clinical impact of antibiotic timing (pre- vs. post-ICI initiation) remains unclear. Objective: To evaluate whether antibiotic timing differentially influences overall survival (OS) in a large, multi-institutional pan-cancer cohort. Design, Setting, and Participants: This retrospective cohort study utilized deidentified electronic health record data from six academic medical centers within the University of California Health system. We included 21,108 adults with any malignancy who received PD-1, PD-L1, or CTLA-4 inhibitors between January 2014 and December 2024. Exposures: Antibiotic exposure windows were categorized as pre-only (-60 to -1 days), post-only (+1 to +60 days), both windows, or none. Main Outcomes and Measures: The primary outcome was overall survival (OS) calculated from the first ICI dose. Multivariable Cox proportional hazards models adjusted for demographics, tumor type, line of therapy, and baseline health indicators (albumin, NLR, and recent hospitalization). Results: Among 21,108 patients, 17.3% had pre-only exposure, 13.3% had post-only exposure, and 60.6% had no exposure. In the multivariable model, post-only exposure (HR, 1.27; 95% CI, 1.20-1.35) and combined pre- and post- exposure (HR, 1.31; 95% CI, 1.23-1.40) were significantly associated with higher mortality. Pre-only exposure was not significantly associated with OS (HR, 1.04; 95% CI, 0.99-1.10). Subgroup analyses by tumor type showed consistent trends across major malignancies, including head and neck (Post HR, 1.46) and renal cell carcinoma (Post HR, 1.26). Conclusions and Relevance: In contrast to some smaller studies, this large-scale analysis indicates that antibiotic exposure after ICI initiation carries a greater risk than exposure prior to treatment. These findings highlight the need for rigorous antibiotic stewardship strategies specifically during the early phases of immunotherapy treatment.

Immune-related adverse events (irAEs), particularly colitis, are major limitations of immune checkpoint inhibitor (ICI) therapy, but their mechanisms remain poorly understood. Here we show that endogenous autoantibodies (AAbs) can promote ICI-associated colitis through Fc{gamma} receptor-dependent pathways. IgG from melanoma patients treated with pembrolizumab, nivolumab, or ipilimumab, with or without severe colitis, was transferred into wild-type or humanized Fc{gamma}R (hFc{gamma}R) mice receiving comparable ICI therapy. Wild-type mice did not develop changes in the colon. In contrast, hFc{gamma}R mice given IgG from patients with colitis developed colon inflammation marked by a significant increase in submucosal lymphocyte infiltration, goblet cell loss, and circulating cytokines, including IL-1{beta}, IL-17a, and IL-22. Single-cell RNA sequencing identified an IgG-regulated inflammatory network involving IFN{gamma}-producing ILC1, Th1 and cytotoxic T cells, IL-1{beta}+ M1 macrophages, plasma B cells/plasmablasts, and IL-22-producing ILC3-LTi cells. Patient serum autoantibody profiling further identified CCR5 and CXCR4 receptors as candidate immune-related targets associated with ICC susceptibility. Immune-related adverse events (irAEs), particularly colitis, are major limitations of immune checkpoint inhibitor (ICI) therapy, but their mechanisms remain poorly understood. Here we show that endogenous autoantibodies (AAbs) can promote ICI-associated colitis through Fc gamma receptor (FcgR)-dependent pathways. IgG from melanoma patients treated with pembrolizumab, nivolumab, or ipilimumab, with or without severe colitis, was transferred into wild-type or humanized FcgR (hFcgR) mice receiving comparable ICI therapy. Wild-type mice did not develop changes in the colon. In contrast, hFcgR mice given IgG from patients with colitis developed colon inflammation marked by a significant increase in submucosal lymphocyte infiltration, goblet cell loss, and circulating cytokines, including IL-6, IL-17, and IL-22. Single-cell RNA sequencing identified an IgG-regulated inflammatory network involving IFNg-producing ILC1, Th1 and cytotoxic T cells, IL-1betta-M1 macrophages, plasma B cells/plasmablasts, and IL-22-producing ILC3-LTi cells. Patient serum autoantibody profiling further identified CCR5 and CXCR4 receptors as candidate immune-related targets associated with ICC susceptibility.

Background and Aims: Fatigue is a prevalent and disabling symptom in inflammatory bowel disease (IBD), yet its underlying biological mechanisms remain poorly understood. We aimed to characterize fatigue-associated molecular signatures in IBD patients by integrating DNA methylation and mRNA expression analyses. Methods: Peripheral blood was collected from 40 patients with Crohn's disease (CD), 29 with ulcerative colitis (UC), and 10 healthy controls. Fatigue severity was assessed continuously using the Multidimensional Fatigue Inventory (MFI). Epigenome-wide DNA methylation profiling and mRNA sequencing were performed, identifying differentially methylated regions (DMRs) and differentially expressed genes (DEGs) for active and quiescent CD and UC, adjusting for age, sex, and smoking status. Pathway enrichment analysis was performed on genes with differential methylation and expression. Results: In active CD, more severe fatigue was associated with transcriptional suppression of immune and metabolic pathways (246 DMRs; 1,090 DEGs), versus upregulation of mitochondrial and metabolic processes in quiescent CD (200 DMRs; 1,619 DEGs). In active UC, fatigue was associated with anabolic pathway upregulation and epigenetic silencing of neuroactive pathways (6,927 DMRs; 343 DEGs; 56 concordant genes). Quiescent UC showed transcriptional changes without significant epigenetic pathway enrichment (1,710 DMRs; 3,224 DEGs). Healthy controls exhibited a distinct profile spanning metabolic, immune, and neuronal pathways (8,621 DMRs; 395 DEGs). Fatigue-associated signatures were largely non-overlapping across all five groups. Conclusions: Fatigue-associated molecular profiles differed substantially by disease subtype and activity state, highlighting the biological heterogeneity of IBD-related fatigue and laying the foundation for multi-omics approaches to identify biomarkers and potential therapeutic targets.

Genetic uniqueness of the tumor microenvironment significantly influences cancer growth, survival, and response to therapy, independent of the cancer cells intrinsic properties or the adaptive immune system. Using genetically distinct Rag1-/- mouse models, this study shows that different strains exhibit varied tumor growth kinetics and survival outcomes when xenografted with identical leukemic and solid tumor cell lines. This study further highlights the critical role of the myeloid immune compartment and shows that disrupting both lymphoid and myeloid systems alters cancer progression. These results also reveal that the tumor microenvironment can permanently alter cancer cell phenotypes and significantly affect chemotherapy efficacy, as seen with Cisplatins varying effects across strains. These findings underscore the importance of considering genetic background in preclinical cancer models, suggesting that reliance upon a single mouse strain may lead to incomplete conclusions about cancer biology and treatment efficacy. SUMMARY STATEMENTPre-clinical xenograft mammalian models are used to study human diseases. Here we report that the genetic uniqueness of the tumor microenvironment, independent of the immune system, can determine the fate of cancer progression, survival, and therapy response.

BackgroundTumour-associated macrophages (TAMs) play critical roles within the tumour microenvironment regulating immune evasion and therapeutic response. Previously, we have shown that the combination of Checkpoint kinase 1 inhibitor (CHK1i) with a subclinical dose of hydroxyurea (LDHU) reprograms the tumour immune microenvironment to a pro-inflammatory status. MethodsWe investigated a tumour-restricted Fcgr4 (Cd16.2) expressing macrophage population in multiple murine tumour models and the impact of CHK1i+LDHU on this population, using conventional and imaging flow cytometry as well as single-cell sequencing. ResultsTranscriptional profiling using CITE-seq and single-cell RNA sequencing reveals that Fcgr4 TAMs closely resemble Fcgr4- TAMs but display modest enrichment of interferon-associated and inflammatory gene programs, consistent with a functionally biased state rather than a distinct lineage. Importantly, we show that a highly tumour selective CHK1i+LDHU therapy shifts TAMs toward a more inflammatory phenotype while preserving dominant immunosuppressive features. Depletion of CSF1R macrophages enhanced CD8 T cell activation without influencing tumour growth but significantly augmented therapeutic efficacy of CHK1i+LDHU. ConclusionTogether, these findings define a novel TAM population and establish how targeted therapy reshapes, but does not fully overcome, TAM-mediated immune regulation.

Defining the genetic and cellular programs that allow solid tumours to evade immune control requires preclinical models that preserve the complexity of the human tumour immune microenvironment. Most available systems capture only part of this biology. Organoid cultures and ex vivo tumour fragments can retain patient-derived tumour architecture and associated immune cells, but immune populations are typically maintained only for short periods. These models also cannot capture antitumour immune responses in the physiological setting of a living organism. Patient-derived xenografts propagated in humanized mice offer a potential path to overcome these limitations by combining patient-derived tumour tissue with a reconstituted human immune system. However, few studies have systematically tested whether these models reproduce the diverse immune cell phenotypes present in the parental tumours from which they are derived. This has limited their use for studying tumour-intrinsic mechanisms that shape immune composition and promote immune evasion. To address this gap, we profiled tumour-infiltrating, splenic, and bone marrow immune cells from ovarian, head and neck, and renal PDX models propagated in CD34+ hematopoietic stem cell (HSC)-derived huNOG-EXL mice expressing human IL-3 and GM-CSF. By comparing tumours grown across distinct HSC donor backgrounds with their matched primary tumour samples, we found that tumour-intrinsic factors are a dominant determinant of immune composition in humanized PDX tumours. Across models, these immune infiltrates generally resembled those of the corresponding parental tumours. These findings support humanized PDX models as a platform for functionally interrogating tumour-intrinsic drivers of immune composition and immune evasion in solid tumours.

Background Unplanned hospital admissions in Inflammatory Bowel Diseases (IBD) account for nearly three-quarters of IBD inpatient stays in the United Kingdom. Although costly to services and distressing for patients, research exploring experiences and potential drivers of admissions is limited. We undertook a qualitative study to explore the healthcare experiences and access needs of people with IBD who had unplanned admissions, along with their caregivers and clinicians. Methods Semi-structured interviews with 25 participants from a single tertiary IBD service in England (17 people with IBD, 3 informal caregivers, 5 clinicians) were conducted. We applied thematic framework analysis, guided by the Candidacy Framework, and worked with 2 patient and public contributors to generate final themes. Results We identified four themes: 1) Difficulties in Identifying flares and asserting severity before admission, summarised the prevailing uncertainty in identifying a flare and access to timely IBD care. 2) Navigating a disjointed healthcare system, highlighted how lack of care plans and systemic barriers can delay access. 2) Emergency care access challenges highlighted the gaps in emergency and inpatient care during flares. Whilst 4) fighting for care and individual advocacy needs, described the persistent assertion for care that may disproportionally impact access to vulnerable groups, also highlighting the importance of positive interpersonal relationships. Conclusions Individual, interpersonal and healthcare factors across the patient pathway were perceived to shape access to care in unplanned IBD admissions. Potentially reducing admissions requires proactive strategies, including the integration of patient education, monitoring tools, establishment of specialist rapid-access pathways, and formal psychological support to address barriers to access.

Approximately 30% of patients with acute graft-versus-host disease (GVHD) develop steroid-refractory disease and have very poor outcomes. Ruxolitinib has become the standard of care for steroid-refractory acute GVHD, but it is unclear which patients derive benefit. The MAGIC Composite Score (MCS), an algorithm that combines clinical symptoms and biomarkers, has been validated to predict outcomes at the start of primary GVHD treatment. Here, we evaluated its performance at the initiation of second-line treatment in 278 patients. MCS stratified patients into three risk groups (MCS1-3), with the majority (88%) classified as intermediate or high risk. Increasing MCS score was associated with progressively higher 1-year non-relapse mortality (NRM) rates (16%, 41%, and 73%; p<0.001), lower 1-year survival (77%, 56%, and 24%; p<0.001), and lower complete response (CR) rates at day 28 (47%, 38%, and 20%, respectively; p<0.01). The area under the receiver operating characteristic curve (AUROC) for 1-year NRM was significantly higher with MCS compared to clinical symptoms alone (0.70 vs. 0.63; p=0.023). Among patients treated with ruxolitinib, higher MCS similarly predicted higher NRM and lower survival and CR rates. Patients classified as MCS2/3 had poor outcomes despite ruxolitinib, underscoring the need for novel therapies in this patient population. In conclusion the MCS is an accurate predictor of outcomes for patients who require second-line treatment and may be of use as an eligibility criterion for future clinical trials in this high-risk population.

Tumor-draining lymph nodes (tdLNs) are critical hubs of anti-tumor immunity but are also vulnerable to tumor-mediated immunosuppression. We analyzed T cell and B cell receptor (TCR/BCR) repertoires and transcriptomes from sentinel and non-sentinel lymph nodes of patients with melanoma from a historical pre-immune checkpoint inhibitor cohort (1994-2002) and an independent contemporary validation cohort (2022-2024). Melanoma-positive lymph nodes exhibited increased immune receptor clonality compared with tumor-free nodes. While average clonality showed no consistent association with outcome, the presence of extreme high-clonality outliers in individual lymph nodes was strongly associated with poor melanoma-specific survival. These outliers were characterized by a loss of lymphocyte-related genes and activation markers, an enrichment of melanocytic transcripts, and the suppression of immune signaling pathways, consistent with local immune dysfunction. Increased clonality was confined to lymph nodes and not observed in the peripheral blood. T cell responses to melanocyte differentiation antigens were infre-quently shared between lymph nodes and peripheral blood, highlighting immune compartmentalization.

High-grade serous ovarian cancer (HGSOC) represents 75% of ovarian cancer cases and 80% of deaths, with most patients relapsing despite initial treatment response. The limited effectiveness of immunotherapies in HGSOC indicates urgent need for novel therapeutic approaches. HGSOC patients produce tumor-binding autoantibodies (TBAs) with high tumor selectivity. Since effective antibody-mediated tumor cell killing requires Fc domain interactions with immune cells, we hypothesized that, although TBAs recognize tumor cells, they might still poorly elicit cell killing responses. Using a systems serology approach, we profiled TBA subclass and biophysical interactions with Fc receptors in HGSOC, comparing them to antiviral antibody responses. TBAs were consistently identified within ascites and serum and were heterogeneous in subclass composition. However, TBAs consistently lacked the capacity to bind Fc{gamma}RIIIa despite abundant interaction with Fc{gamma}RIIa and poorly elicited antibody-dependent cellular cytotoxicity, suggesting their Fc features prevent cell killing responses. Restoring Fc{gamma}RIIIa interaction may be a promising therapeutic approach in HGSOC. HighlightsO_LITBAs in ovarian carcinoma patients consistently lack interaction with Fc{gamma}RIIIa C_LIO_LIAscites- and serum-derived TBAs have heterogeneous subclass composition C_LIO_LISystems analysis shows complex serologic differences between TBAs and antiviral responses C_LIO_LIPatient-expressed TBAs demonstrate little antibody-dependent cellular cytotoxicity C_LI

Background: Immune checkpoint inhibitors have transformed cancer treatment, yet a large number of patients fail to respond. Identifying molecular characteristics that predict response before treatment initiation remains an unmet need. Towards that end, this study presents a large-scale integrative analysis of existing single-cell and bulk tissue datasets, aimed at identifying predictive features while providing insights into their cellular origin and potential function within the tumor microenvironment. Methods: A stepwise analysis was performed using single-cell RNA-sequencing data from 60 melanoma patients at baseline, separated into discovery (n=41) and validation (n=19) sets. An integrated bulk transcriptomics dataset (n=128) from melanoma patients and a bladder cancer dataset (n=298) were used for further validation. Results: Integrative analysis of melanoma single-cell datasets revealed that responders exhibit distinct molecular profiles across multiple cell types compared to non-responders. Notably, these included downregulation of the TNFR superfamily and other immunosuppressive genes (TNFRSF18, TNFRSF9, TNFRSF4, LGALS1, BATF, IL12RB2, LINGO1, DUSP4, SDC4, VCAM1) in T-cells. By investigating the findings from the immune cell populations in the bulk tumor context, 13 transcripts were found to be consistently associated with response across all cohorts. These were differentially expressed in T-cells (SELL, EPB41, CD96, UHFR2, LINGO1, LGALS1), B-cells (ALDH5A1), NK cells (PLEC, PDGFRB) and Monocytes (TLR10, ST6GAL1, IKZF1, MPRIP). A predictive model based on these features effectively discriminated responders from non-responders in melanoma (AUC=0.73). The model maintained significant predictive power in an independent bladder cancer dataset (IMvigor210; AUC=0.64). Of high clinical relevance, it demonstrated enhanced performance in identifying responders among patients with low tumor mutational burden (AUC=0.75). Conclusion: Our study reveals pre-treatment molecular features related to immune-cancer crosstalk that are associated with response to immunotherapy. A 13-gene model demonstrates potential added clinical value in stratifying responders, particularly in patients with low tumor mutational burden, meriting further validation.

Macrophage-melanoma interactions critically shape the tumor microenvironment, yet the cytokine networks driving this process remain incompletely understood. Among these, colony-stimulating factors--particularly granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF or CSF3)--regulate myeloid cell behavior during cancer progression, although their specific roles in melanoma remain unclear. Using co-culture systems of melanoma and monocyte-derived macrophages, we found that GM-CSF-primed macrophages induced robust G-CSF secretion and concurrent CSF3 upregulation in both cell compartments. Moreover, transcriptomic profiling of patient-derived tumor-associated macrophages (TAMs) confirmed elevated CSF3 and CSF3R expression in metastatic melanoma. Multiplex immunofluorescence analysis of a stage II-IV primary melanoma cohort (n=84) revealed increased G-CSF and CD114 expression in TAMs and tumor cells from patients who subsequently developed metastasis. High G-CSF levels in either TAMs or melanoma cells emerged as an independent prognostic factor for shorter disease-free and overall survival (p < 0.001). Mechanistically, G-CSF activated STAT1/STAT3 signaling in melanoma cells and promoted proliferation and invasion in a CD114-dependent manner. Of note, G-CSF drove monocyte differentiation toward a distinct inflammatory macrophage state characterized by STAT1/STAT3 activation and a pro-invasive secretory profile. Furthermore, melanoma cells conditioned by G-CSF-differentiated macrophages displayed increased in vivo lung colonization and enriched transcriptional programs linked to invasion and proliferation. Collectively, these findings establish the G-CSF/CD114 axis as a potential clinically relevant driver of melanoma metastasis and identify G-CSF as a novel independent prognostic biomarker. HIGHLIGHTSO_LIDespite its extended clinical use, G-CSF role in human melanoma remains undefined. C_LIO_LIHigh G-CSF expression in primary melanomas correlates with poor patient survival. C_LIO_LIG-CSF promotes melanoma cell invasion and proliferation through CD114 signaling. C_LIO_LIG-CSF drives macrophage differentiation toward a pro-tumoral phenotype. C_LIO_LIMelanoma cells conditioned by G-CSF-primed macrophages are more metastatic. C_LI

ContextImmunotherapy based on immune checkpoint inhibitors (ICI) revolutionized the treatment of triple-negative (TN) breast carcinomas (BC), but remains more challenging in HR+/HER2- BCs. Because invasive lobular carcinomas (ILC) generally exhibit low immune infiltration, ICIs were largely overlooked in this pathological type. The only clinical trial of ICIs dedicated to ILCs showed disappointing results, notably in HR+/HER2- cases. The immune landscape of HR+/HER2- ILCs has been poorly described. High level of tumor-infiltrating lymphocytes (TIL) was associated with worse prognosis in HR+/HER2- ILCs. A better characterization of the immune landscape of HR+/HER2- ILCs could clarify the poor efficiency of ICIs and the negative prognostic value of TILs, and reveal complementary targets able to increase immunotherapy efficiency. MethodWe comprehensively characterized the immune landscape of HR+/HER2- ILCs, comparatively to HR+/HER2- invasive ductal carcinomas (IDC), by applying multi-omics and multi-scale analysis (gene expression at the bulk and single-cell levels, and protein-based spatial analysis) to clinical samples. ResultsWhile the overall level of immune infiltration was comparable between both pathological types, the quality of immune infiltrate differed markedly. Comparatively to HR+/HER2- IDCs, HR+/HER2- ILCs were enriched in immune cells and tertiary lymphoid structures with anti-tumor potential, presented more spatial proximity between cancer cells and CD8+ cytotoxic T cells, and stronger theorical vulnerability to ICIs. However, in HR+/HER2- ILCs, anti-tumor response was defective; CD8+ cytotoxic T cells failed to fully unleash their cytotoxic function and CD4+ helper T cells evidenced a pro-tumoral and naive phenotype. Furthermore, antigen-presenting compartment was defective, altogether embedded in a stronger immunosuppressive environment, enriched in immunoregulatory cancer-associated fibroblasts (iCAF). ConclusionThis study contributes to explain the lesser efficiency of PD-1/PD-L1-based ICIs in HR+/HER2-ILCs by comparison with HR+/HER2- IDCs, by shedding light on a complex ecosystem where tumor cells shape a distinctive stroma that contribute to prevent anti-tumor immune response activation. Altogether, our findings further support the rationale for combining iCAF-targeting strategy with an ad hoc immunotherapy (such as an anti-VTCN1/B7-H4 antibody-drug conjugates for example). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/728418v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@c62294org.highwire.dtl.DTLVardef@86392org.highwire.dtl.DTLVardef@c10748org.highwire.dtl.DTLVardef@c543da_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_ST_ABSWHAT IS ALREADY KNOWN ON THIS TOPICC_ST_ABSO_LIImmune cells infiltrate both HR+/HER2- IDC and HR+/HER2- ILC tumors, but current ICIs are less effective in HR+/HER2- ILCs than HR+/HER2- IDCs. C_LI WHAT THIS STUDY ADDSO_LIThe anti-tumor immune response is mobilized but not effective in HR+/HER2- ILCs. C_LIO_LIA complex ecosystem - composed of immunoregulatory cancer-associated fibroblasts, high levels of TGFa, prostaglandin, acidosis, and a lack of antigen-presenting cells - prevents anti-tumor CD8+ cytotoxic T cell activation in HR+/HER2- ILCs. C_LI HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICYO_LITargeting the PD-1/PD-L1 axis is not the appropriate therapeutic strategy for HR+/HER2- ILCs. A more complex approach should be considered, notably those combining other immune-based strategies and iCAF targeting, which may offer a better chance to eradicate HR+/HER2- ILC tumor cells. C_LI

Background Recent mouse model data demonstrate that chronic colonization with toxigenic Clostridioides difficile promotes colonic tumorigenesis via intraluminal toxin B (TcdB), its main virulence factor. In a prior multisite hospital cohort, we found that history of positive tcdB stool testing was associated with increased CRC risk in a dose-dependent manner, though limited by small sample size. We aimed to validate this association in a larger cohort with extended follow-up and greater geographic distribution using the Veterans Health Administration (VHA) Corporate Data Warehouse (CDW). Methods We conducted a retrospective cohort study among adults receiving care through the VA from 2000-2025 who underwent C. difficile testing. Data collected from the VHA CDW and National Death Index (NDI) included demographics, comorbidities, medications, CRC risk factors, and cancer incidence and death. The first C. difficile test date defined cohort entry; individuals with prior CRC were excluded. Ever C. difficile positivity was defined by a positive PCR or EIA results. The number of positive tests (episodes) was also determined to define recurrent positivity. Follow-up time ended at the first occurrence of CRC incidence or mortality, death from other causes, or censor date. Follow-up time was split for individuals who converted from negative to positive, with follow-up time updated accordingly. Multivariable Cox proportional hazards models were used to estimate hazard ratios (HRs) for C. difficile exposure and CRC incidence and mortality after adjustment for confounders. Tests for linear trend and tests for interaction were conducted to assess effect modification by sex and IBD status, while time-lag intervals were evaluated for 1, 3, 5, and 10 years before the outcome. Results Among 806,844 veterans with C. difficile testing, those with positive tests were more likely to be older, male, to have diabetes, to use aspirin, and to have a lower BMI than those with negative tests. Race and IBD prevalence were similar between the groups. There was no overall association between ever C. difficile positivity and CRC incidence (HR = 0.99, 95% CI 0.93-1.05). However, recurrent C. difficile positivity was associated with increased risk in a dose-response manner [2-3 episodes HR = 1.30 (95% CI 1.16-1.47), and >3 episodes HR = 1.58 (95% CI 1.17-2.14) compared to negative tests; ptrend< 0.001]. Further, ever C. difficile positivity was associated with increased CRC mortality risk (HR = 1.21, 95% CI 1.13-1.30; p < 0.001). Recurrent C. difficile positivity was associated with increased mortality risk but was particularly strong for those with >3 episodes among individuals with IBD (HR=3.84, 95% CI 1.98-7.45). In sensitivity analyses, the increased risk of CRC incidence and mortality attenuated beyond 10 years. Conclusion Prior positive C. difficile testing was associated with increased CRC incidence and mortality in a dose-dependent manner, particularly among patients with IBD. These findings extend animal model evidence, epidemiologically establishing C. difficile presence as an independent risk factor for subsequent colorectal tumorigenesis and supporting investigation into recurrent CDI, especially among patients with IBD, as a potential modifiable CRC risk factor.