Cancer Immunology, Immunotherapy

Background Immune checkpoint blockade (ICB) has become the standard of treatment for metastatic melanoma (MM). However, patients with melanoma brain metastases (MBM) are often excluded from clinical trials. Here we analyse patients within the real-world OxCITE cohort receiving ICB treatment for metastatic melanoma, with a focus on MBM, aiming to discern outcomes, prognostic factors and peripheral biomarkers. Methods Patients eligible for ICB treatment were recruited prospectively. Demographic, clinical and radiological data were collected from electronic patient records. Plasma samples were collected prior to the first cycle of treatment for plasma proteomic analysis. Results 307 patients were included, 81 of whom had MBM on commencing ICB. No difference in overall survival between patients with and without brain metastases was noted on multivariable analysis (HR=1.1, 95% CI 0.73-1.6, p=0.68), whereas hepatic metastases were associated with increased risk of death (HR 1.7, 95% CI 1.2-2.4, p=0.0019). Within the MBM group, steroid treatment (HR=2.5, 95% CI 1.3-5.0, p=0.006) was associated with poor OS, whilst treatment with single agent ICB (HR=1.9, 95% CI 0.86-3.9, p=0.11), symptomatic first presentation of MBM (HR=1.5, 95% CI 0.76-2.8, p=0.25) and anti-seizure drug treatment (HR=1.74, 95% CI 0.89-3.4, p=0.10) were not. Amongst all patients carriage of a BRAF mutation was associated with increased risk of MBM (OR 2.49, 95% CI 1.4-4.4, p=0.0007), and increased propensity to new brain metastases on ICB treatment (OR 2.5, 95% CI 1.1-5.9, p=0.036). Analysis of blood and plasma samples demonstrated that patients with MBM have altered haematological profiles, with raised NLR and increased plasma glial fibrillary acidic protein levels, reinforcing the distinct systemic features of MBM. Conclusions In this real-world cohort of patients receiving ICB for metastatic melanoma, the presence of MBM did not associate with worse OS. Conversely, baseline corticosteroid use was adversely prognostic. Intriguingly, plasma GFAP was significantly elevated in patients with MBM and correlated with the burden of intracranial disease, highlighting a potential role for GFAP as a candidate biomarker for MBM in high-risk surveillance. Our observations indicate the adversely prognostic weight historically attached to M1d disease is no longer applicable in the ICB era.

Chimeric antigen receptor (CAR) T cells are patient-derived T cells engineered to recognize specific antigens on malignant cells, representing a major breakthrough in personalized cancer immunotherapy. While CAR-T cell therapy is an established treatment for relapsed or refractory hematologic malignancies, understanding the rationale and methodology behind its development is essential for training the next generation of oncology researchers. Here, we describe a novel, hands-on small-enrollment laboratory course designed to teach upper-level undergraduate and graduate students about immunotherapies and CAR-T cell technology by paralleling a typical bench-scale development process. Students isolated T cells from porcine peripheral blood mononuclear cells (PBMCs) via immunomagnetic selection, followed by flow cytometry to assess purity, viability, and yield. Students then used pre-engineered B7H3 CAR-T cells to perform co-culture assays with U87 glioblastoma cells to evaluate tumor cell killing efficacy. Cytotoxicity was qualitatively evaluated using immunofluorescence microscopy, while IFN-gamma release was quantified using Enzyme-linked Immunosorbent Assay (ELISA). Student performance was evaluated through electronic laboratory notebooks, discussion questions, a capstone project, and student-generated data. Our results demonstrate students high academic performance across assignments and the successful execution of complex laboratory workflows. Collectively, these findings establish the feasibility and implementation of a research-integrated curriculum providing foundational exposure to immuno-oncology research tools and CAR-T cell production techniques.

Background There are currently no available biomarkers that predict the clinical outcome of ulcerative colitis (UC) patients after anti-TNF withdrawal. Here we developed a semi-supervised machine learning framework on RNA-seq data derived from rectal biopsies to predict relapse following anti-TNF discontinuation. Methods We included UC patients (n=136) enrolled in the prospective, randomized controlled BIOSTOP trial. Patients were assigned to a withdrawal group (stopped treatment immediately) or a maintenance group (stopped treatment after 2 years). A machine learning framework was trained on gene expression data from mucosal biopsies collected at baseline from the withdrawal group and validated in the maintenance group using baseline samples (test cohort) and 2-year follow-up samples (interference cohort). Results A semi-supervised machine learning model, combining a shared UMAP manifold representation with LabelSpreading on a targeted 38-gene panel achieved an AUC of 0.794, a sensitivity of 90.3%, and a specificity of 73.1% on the test cohort, and an AUC of 0.711, a sensitivity of 82.9%, and a specificity of 42.3% on the inference cohort. Applying Shapley Additive exPlanations (SHAP) we found that IL1B, TYROBP, REG1B, S100A8, and TNF showed the highest predictive importance for relapse risk, whereas MUC2, HMGCS2, and CDKN1A displayed the highest predictive importance for sustained remission. Conclusion The high sensitivity makes the model a promising screening tool to prevent withdrawal-related relapse, though its moderate specificity underscores the need for multi-center validation in larger, more diverse cohorts before clinical deployment.

Circulating tumor cells (CTCs) are key drivers of distant metastasis, while platelets facilitate this process by protecting them from immune surveillance. However, the detection of CTCs exhibiting platelet markers and their communication with monocytes has not been thoroughly investigated. This study seeks to identify the intercellular communication of CTCs that have acquired platelet traits by integrating single-cell RNA-seq data of 377 CTCs and 2,634 white blood cells. Trajectory analysis identified a subpopulation of CTCs with enhanced platelet-related functions, including platelet aggregation and resistance to NK cell-mediated cytotoxicity, termed Platelet-Programmed CTCs. The Dampened Weighted Least Squares method was implemented to estimate the platelet-programmed tumor cell proportion in 1,102 bulk RNA-seq samples from primary breast cancer tumors. A strong inverse association was found between platelet-programmed tumor cell proportion and overall survival (Cox-PH p-value =0.00419, HR=6 per 10% increase in proportion, 95% CI:1.76-20.7). However, this association did not differ significantly between early- and late-stage patients. Cell-cell communication analyses and molecular docking revealed the CD40LG-ITGA5:ITGB1 pair as a potential immune checkpoint candidate for monocytes in cancer (Hex score=-656.83; TNF-TNFR reference complex=-515). However, further experimental and clinical validation is required to translate these findings. In conclusion, these findings deepen our understanding of the immune evasion mechanisms of CTCs by acquiring platelet characteristics. This immune checkpoint axis offers a novel avenue for further experimental validation and future intervention.

Validated biomarkers for identifying patients with early-stage melanomas at high risk of metastasis are limited. We have previously shown that loss of Activating Molecule in Beclin-1-Regulated Autophagy (AMBRA1) and loricrin in the epidermal microenvironment is associated with tumour recurrence. Here we show that endothelial AMBRA1 deficiency contributes to vascular dysfunction and melanoma progression. Melanoma-conditioned media or exposure to TGF{beta}1-3 ligands induced post-transcriptional loss of AMBRA1 in endothelial cells. Transcriptomic profiling in both in vitro and human melanoma single-cell RNA sequencing datasets showed activation of cell-cycle and metabolic programmes alongside suppression of endothelial junction, adhesion and immune-supportive pathways in AMBRA1-deficient endothelial cells. Functionally, AMBRA1 loss increased endothelial cell proliferation, accelerated early tubulogenesis, impaired three-dimensional spheroid adhesion, stabilised endothelial-to-mesenchymal transition-associated transcription factors Slug and Snail, and reduced Claudin-5, VE-cadherin and N-cadherin, indicative of endothelial plasticity and dysfunction. Furthermore, loss of AMBRA1 in a subset of intratumoural and peritumoural blood and lymphatic vessels correlated significantly with metastatic progression in a cohort of 121 non-ulcerated primary AJCC stage I/II melanomas classified as AMBLor at-risk. Collectively, these findings position endothelial AMBRA1 as a regulator of vascular plasticity, dysfunction, and immune-supportive endothelial function in early-stage melanomas, and a marker of a permissive microenvironment associated with metastasis.

The aim of this study was to determine whether the frequency of metabolic dysfunction-associated steatotic liver disease (MASLD) and the severity profile of inflammatory bowel disease (IBD) differ across body mass index (BMI) categories. A retrospective analysis was performed using 400 inpatient medical records from 2023-2024 with verified ulcerative colitis or Crohn's disease and sufficient information on anthropometry, hepatic status, and intestinal disease activity. The cohort comprised 118 patients with normal BMI, 176 with overweight, and 106 with obesity. MASLD status was reconstructed from routine clinical documentation and required hepatic steatosis in the appropriate cardiometabolic context. Ultrasonography was the principal source of evidence for steatosis; laboratory findings, non-invasive indices, and elastography were considered supportive when available. Ulcerative colitis activity was assessed with the Mayo Score and Crohn's disease activity with the Crohn's Disease Activity Index; disease-specific results were harmonized into mild, moderate, and severe categories for pooled comparisons. MASLD was identified in 181 of 400 patients (45.3%). Its frequency increased from 20.3% in the normal-BMI group to 49.4% in the overweight group and 66.0% in the obesity group (chi2=49.29; p<0.001). The largest absolute increase, 29.1 percentage points, occurred between normal BMI and overweight. The proportion of severe IBD rose in the same order, from 6.8% to 14.8% and 32.1% across the three BMI strata (chi2=39.45; p<0.001). In unadjusted pairwise comparisons, the odds of MASLD were higher with overweight than with normal BMI (OR=3.83; 95% CI 2.24-6.55) and higher with obesity than with normal BMI (OR=7.62; 95% CI 4.17-13.91); obesity also differed from overweight (OR=1.99; 95% CI 1.21-3.28). These findings demonstrate an ordered BMI-related pattern in which MASLD becomes more frequent and severe IBD represents a larger share of cases. However, MASLD was still documented in approximately one fifth of patients with normal BMI, indicating that BMI is useful for clinical prioritization but not for excluding steatotic liver disease. Because the study was retrospective, liver assessment reflected routine care, and the effect estimates were not adjusted for treatment exposure, disease duration, or other metabolic covariates, the observed relationships should be interpreted as associations rather than causal effects.

Background Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, and emerging T cell engager (TCE) immunotherapies require predictive preclinical models that capture patient-specific tumour biology and tumour-immune interactions. Although patient-derived organoid (PDO)-immune co-culture systems show promise for evaluating immunotherapy responses, many rely on matrix-embedded cultures that limit scalability and reproducibility. We therefore developed and validated a low-matrix organoid-T cell co-culture platform for functional assessment of TCE activity in patient-derived CRC models. Methods Patient-derived CRC organoids representing diverse molecular and genetic backgrounds were co-cultured with activated allogeneic CD3+ T cells in a suspension low-matrix format. Matrix concentration, medium composition, T cell activation status, and assay duration were optimized. Tumour killing, T cells activation, cytokine secretion, apoptosis, and motility were assessed using flow cytometry, live-cell imaging, cytokine profiling, and immunofluorescence. The platform was evaluated using EGFR- and HER2-targeting bispecific T cell engagers across 21 CRC organoid models and multiple healthy donor-derived T cell populations. Results Optimization identified 1% matrix and a 1:1 organoid: T cell medium that preserved organoid integrity while maintaining T cell viability, activation, and motility. The final workflow enabled reproducible co-culture of 5-day mature organoids with 7-day activated T cells for 72 hours. EGFR-targeting TCEs induced tumour killing, T cell activation, and IFN-{gamma} secretion across donor-organoid combinations. Screening of 21 CRC organoid models revealed substantial inter-patient heterogeneity, with 12 models maintaining [&ge;]50% baseline viability and supporting functional TCE evaluation. EGFR- and HER2-targeting TCEs produced potent dose-dependent cytotoxicity, with IC50 values ranging from 0.0188 to 54.77 nM across responsive models and up to 75%-85% tumour killing in the most sensitive organoids. These responses were accompanied by increased effector cytokine secretion. Real-time imaging confirmed dynamic T cell engagement and apoptosis-driven organoid destruction following TCE treatment. Conclusions We establish a robust, scalable, human-relevant low-matrix organoid-T cell co-culture platform for functional screening of T cell engagers in patient-derived CRC models. By enabling integrated assessment of tumour killing, immune activation, cytokine responses, and tumour-immune interactions while preserving inter-patient heterogeneity, this system provides a translational framework for immunotherapy development. The platform may support candidate prioritisation, biomarker discovery, patient stratification, and preclinical evaluation of immune-engaging therapeutics.

Mucosal melanoma is a rare, lethal melanoma subtype with distinct genetics and limited benefit from immunotherapy. To define features associated with mucosal melanoma clinical outcomes, we assembled a large, multimodally profiled cohort spanning primary and metastatic disease. Compared with cutaneous melanoma, primary mucosal melanoma showed greater genomic heterogeneity, ploidy and aneuploidy, and a more dedifferentiated, proliferative transcriptional state. Analysis of genomic sequencing of 339 patients with mucosal melanoma identified four mutually exclusive genomic subtypes: CDK4/MDM2/CCND1, SF3B1-mutated, TP53/ATRX, and mucosal melanoma Triple WT, associated with distinct anatomic sites but similar outcomes. In the tumor microenvironment, we observed reduced T cell infiltration and increased CD163+ tumor-associated macrophages in mucosal melanoma relative to cutaneous melanoma. In matched longitudinal samples, CD163+ tumor-associated macrophages increased with disease progression and immunotherapy resistance. CD163+ tumor-associated macrophages engaged non-regulatory T cells through GAL9-TIM-3 interactions, nominating TIM-3 blockade as a therapeutic strategy for mucosal melanoma. These findings define the distinct aggressive biology of mucosal melanoma and highlight potential therapeutic targets and mechanisms of immune resistance.

Background Immune checkpoint inhibitor (ICI) therapies have revolutionized oncology, but identifying patients who benefit remains a challenge, particularly in populations where ICIs are not the standard of care, including microsatellite stable colorectal cancer (MSS CRC) and rare solid cancers. We evaluated real-world performance of the Immune Profile Score (IPS), a validated DNA- and RNA-based molecular signature, in these cohorts. Methods In an exploratory analysis, we analyzed two real-world cohorts: 1) MSS CRC; and 2) MSS, tumor mutational burden-low rare solid cancers treated with off-label ICI. IPS-High and IPS-Low were calculated using a previously validated threshold. Cox proportional hazards models were fit to demonstrate prognostic utility for real-world overall survival (rwOS). In MSS CRC, time-to-next-treatment (TTNT) on prior non-ICI therapy was compared to rwOS on subsequent ICI therapy. Results In MSS CRC (n=46), IPS-High patients (13%) demonstrated clinically meaningful improvement in rwOS versus IPS-Low (Median OS: not reached vs 7.3 months; HR = 0.22, 90% CI 0.04-1.16). While IPS was not associated with TTNT on the preceding non-ICI line (HR = 1.07, 90% CI 0.60-1.91), it was associated with OS in patients receiving later line ICI (HR 0.21, 90% CI 0.04-1.22). In the rare cancers cohort (n=90; 26 unique malignancies), IPS-High (17.8%) was associated with longer OS to ICI therapy (HR = 0.26, 90% CI 0.11-0.61), even when restricted to histologies represented in both IPS groups (HR = 0.18, 90%, CI 0.05-0.62). Conclusions Preliminary evidence suggests that IPS may serve as a generalizable, pan-cancer biomarker capable of stratifying outcomes for ICI treatment in populations traditionally excluded from immunotherapy benefit.

Immune checkpoint blockade (ICB) with ipilimumab and nivolumab is a front-line standard of care in patients with mesothelioma. However, only a minority of patients respond. The tyrosine receptor kinase AXL is a putative ICB resistance factor. We evaluated AXL and programmed death 1 (PD1) inhibition (AXL-PD1) with bemcentinib and pembrolizumab respectively, in a phase IIA clinical trial in patients with ICB naive-relapsed mesothelioma (MIST3, NCT03654833). The study met its primary endpoint with a 12-week disease control rate of 46.2%, 90% confidence interval 29.2-63.8%. Grade 3 or greater adverse events occurred in 53.9% of patients. Multiomic analyses identified tumour-intrinsic and tumour extrinsic features linked to response to AXL-PD1 inhibition. Responders exhibited greater NF2 loss, heightened immune response signalling, and epithelial mesenchymal transition, which was lower in biopsies obtained at the time of acquired drug resistance. Neutrophil- related transcriptional programmes were associated with response to AXL-PD1 inhibition. Neutrophil regulating erythroblast transformation specific (ETS) transcription factors SPI1, ETS2, FLI1 were differentially activated in responding tumours. Gut microbiota rheostat differed between responders and non-responders and positively correlated with neutrophil- associated transcription program, IRF3 and TLR2. In summary, our findings support a hypothesis linking gut microbiota composition, neutrophil- associated tumour biology and response to AXL-PD1 inhibition in mesothelioma.

Background: Inflammatory bowel disease (IBD) has been linked to a two- to eightfold increase in colorectal cancer (CRC) risk. However, particular genes that bridge intestinal dysbiosis and oncogenic transcriptional reprogramming remain unclear. The Hippo/YAP pathway is a major regulator of intestinal epithelial homeostasis whose effector YAP is hyperactivated in CRC, but its transcriptional engagement in IBD mucosa and its link to specific cancer-risk genes has not been thoroughly mapped. Methods: Bulk RNA-seq data were integrated from two independent cohorts: GSE235236 (IBD colon mucosa; UC/CD vs. healthy controls; n = 56) and TCGA-COAD/READ (CRC tumors stratified by YAP activity; n = 592). Differential expression analysis was conducted separately within each cohort using Welch's t-test with Benjamini-Hochberg FDR correction. YAP transcriptional activity was estimated per sample as the mean row-wise Z-score of seven canonical YAP target genes, and TCGA tumors were stratified into Hippo-High (Q4) and Hippo-Low (Q1) groups. Genes showing consistent dysregulation across both cohorts were identified and ranked to define a Dysbiosis-Hippo Response (DHR) module, which was further organized into functional gene archetypes. Results: IBD mucosa showed 2,848 significantly dysregulated genes (FDR < 0.05). Comparison with the CRC cohort identified 120 genes showing concordant dysregulation, forming the DHR module. These genes were significantly enriched in TNF-alpha signaling via NF-kB (adjusted p = 0.006), inflammatory response (adjusted p = 0.013), and Myogenesis (adjusted p = 0.013). DHR gene classification revealed three functional archetypes: (i) innate immune activation (SPI1, OSM, IL11, LILRA2, SLC11A1) reflecting convergent myeloid reprogramming; (ii) epithelial-mesenchymal transition (TWIST1, SNAI1, PRRX2, MMP9) priming invasive potential before malignancy; and (iii) tumor microenvironment immune regulation (TN-FRSF4, TNFRSF18, PDCD1, VASN, SPON2) shaping immune tolerance. Interestingly, the canonical YAP target genes (CTGF, CYR61, ANKRD1, AMOTL2, and FSTL1), sharply upregulated in Hippo-High CRC, were essentially unchanged in IBD mucosa. This signifies that the shared DHR programme operates through non-canonical YAP effectors. And yet, YAP target scores are positively correlated with DHR module expression across IBD samples (Spearman rho = 0.726, p = 2.5 x 10-10). Conclusions: Our findings define a three-archetype DHR module that links gut dysbiosis to YAP-driven oncogenesis at single-gene resolution. The programme shared between IBD and CRC is driven by innate immune activation, EMT priming, and remodeling of the immune microenvironment, not by the canonical CTGF/CYR61 YAP target axis, which appears only at the CRC stage. This gene-level framework provides tractable mechanistic targets for chemoprevention and biomarker development at the inflammation-to-cancer transition.

Pseudoprogression in response to immune checkpoint inhibitor (ICI) therapy can be a complex challenge during treatment response evaluation. Erroneous classification of progression may result in stopping effective treatment for patients, while delayed confirmation of progression can prolong an ineffective treatment course. We develop a mechanistic mathematical model of tumor-immune dynamics under ICI treatment in which observed tumor volume includes contributions from both tumor burden and immune infiltration. We show analytically how pseudoprogression can arise from increased immune infiltration and proliferation as well as continued tumor growth during the ``ramp-up" phase. The model also incorporates circulating tumor DNA (ctDNA) dynamics, which we demonstrate are able to closely fit existing longitudinal tumor and ctDNA data and extract realistic parameter ranges to generate representative in silico data. Using model-generated cohorts, we train a random forest (RF) classifier using longitudinal tumor and ctDNA measurements to distinguish pseudoprogression and true progression, and demonstrate that it improves response classification relative to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1) and provides classification earlier than the immunotherapy-specific iRECIST, even in the presence of tumor measurement noise. This work predicts that paired longitudinal tumor-ctDNA measurements may enable earlier discrimination of true progression from pseudoprogression, and motivates collection of imaging and ctDNA data synchronously during ICI therapy, especially in the setting of suspected disease progression.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is often diagnosed months to years after symptoms begin, and diagnostic delay is associated with complications. We tested whether longitudinal machine learning using electronic health record (EHR) data can identify incident IBD before diagnosis, and quantified lead time and deployment burden. METHODS: We studied adults with an IBD-compatible symptom, sign, or finding at UCSF (2012-2024). Incident cases were confirmed by structured filtering and validated large language model note review. We trained a state space model (Mamba) and comparators on pre-diagnostic EHR data, excluding diagnostic-workup features, and assessed discrimination at 6- and 12-month landmarks. We externally tested Mamba without recalibration at ZSFG, a safety-net hospital serving a demographically and socioeconomically distinct population, and simulated diagnosis-code-only deployment at UCSF. RESULTS: Six months after presentation, among patients still undiagnosed (67% of UCSF test cases), Mamba achieved an AUROC of 0.969 (95% CI, 0.928-0.999) at UCSF and 0.965 (0.952-0.976) at ZSFG. Median lead time was 20 months at UCSF and 15 months at ZSFG. Among future cases with an initially normal colonoscopy (N=32) or a histological finding of acute inflammation without chronicity (N=63), the model flagged 84% and 89% of these patients, respectively, before those procedures, indicating the potential for pre-diagnostic detection. Among 358,886 symptomatic patients not used for training (60 incident cases), the diagnosis-code-only model achieved an AUROC of 0.926; reviewing 20 alerts/month identified 34 cases (PPV, 1.09%; 64-fold above baseline). CONCLUSIONS: Longitudinal machine learning can identify increased IBD risk months to years before diagnosis and may define a population for prospective studies of early IBD biology. Prospective evaluation should determine whether earlier identification reduces diagnostic delay.

Background: Severe hepatotoxicity has been reported when sotorasib follows immune checkpoint inhibitor (ICI) therapy, and fixed washout intervals have been proposed. We assessed prior ICI exposure and the ICI-to-sotorasib interval as separate risk factors. Materials and methods: We searched PubMed, Europe PMC, Embase, Web of Science, Scopus, CENTRAL, and meeting proceedings for comparative studies of grade 3 or higher hepatotoxicity after KRAS G12C inhibitors. Matched risk ratios (RRs) and unadjusted cohort data were synthesized separately, the latter with an exact conditional odds ratio (OR). Certainty was rated with GRADE. Results: Of 530 unique records screened, seven sources were included, all involving sotorasib. In the only matched analysis (meeting abstract, 386 patients per arm), prior ICI exposure was associated with higher risk (13.2% vs 4.4%, RR 3.00, 95% CI 1.76-5.10), whereas matched comparisons at 6 weeks (RR 1.11, 0.61-2.03) and 12 weeks (RR 1.08, 0.52-2.27) detected no difference but could not exclude important effects. All five unadjusted cohorts showed higher risk with shorter intervals, including 21/74 versus 8/75 patients in the earlier versus later interval quartiles of the CodeBreaK 200 trial, although effect sizes were heterogeneous (exact homogeneity P=0.006). Certainty was very low throughout. Conclusions: Prior ICI exposure was associated with higher risk of severe sotorasib-associated hepatotoxicity, with very low certainty. A shorter interval was associated with higher risk in unadjusted data only, and current evidence does not establish a mandatory washout period. Liver-test monitoring per the prescribing information applies to all patients, whereas additional surveillance after ICI exposure remains an untested precaution.

Glioma is a fatal tumor of the central nervous system (CNS) with limited therapeutic options. While immune checkpoint inhibitors (ICI) are gaining traction as a novel alternative treatment for human gliomas (HG), clinical trials have yielded mixed results. A proposed major barrier to ICI therapy in cancer is epithelial-mesenchymal plasticity (EMP)-driven intratumoral immunosuppression. However, studying this mechanism is difficult in ex vivo and murine models, which fail to adequately replicate cancer cell-immune cell interactions. Formalin-fixed, paraffin-embedded (FFPE) patient-derived canine glioma (CG) samples represent an attractive alternative approach. Although mechanistic perturbation is unachievable in archival FFPE tissues, CG is notoriously 'cold' immunologically and is actively being investigated as a comparative model of HG. Moreover, the immune microenvironment in CG advantageously develops under exposure to environmental conditions directly shared with humans. Yet, it remains unknown whether CGs harbor heterogeneous niches combining varied plastic phenotypes and immunosuppressive populations. Defining the immune landscape of CG in relation to EMP is therefore critical to determining its potential as a comparative model for immunotherapy response in HG. By combining standard histology, immunohistochemistry, and tissue microarray-based spatial transcriptomics, we analyzed 30 low-grade (LGr) and high-grade (HGr) FFPE CG cases (n=10 cases/subtype): astrocytomas (4xLGr, 6xHGr), oligodendrogliomas (1xLGr, 9xHGr), and undefined gliomas (3LxGr, 7xHGr). All CG subtypes harbored heterogeneous immunosuppressive niches associated with varied EMP phenotypes. Astrocytomas exhibited a more-mesenchymal identity correlated with recruitment of tumor-associated macrophages (TAMs). Oligodendrogliomas displayed a strong stem-like signature and uniquely upregulated a FOXP3/LAG3/SNAI1 triad, positioned directly at the interface of immunosuppression and EMP. Across all CG subtypes, a more-mesenchymal phenotype co-occurred with TAMs, and intratumoral T-cell dysfunction and exhaustion. We conclude that CG replicates the EMP-immunosuppression heterogeneity seen in humans. Importantly, our results emphasize the existence of subtype-specific mechanisms of EMP and immune evasion, underscoring the major relevance of subtyping for translational applications.

Background and aimsCrohns disease (CD) is a highly heterogeneous disease. Patients with similar characteristics at baseline may end up on very different therapeutic and surgical paths. We aimed to identify disease-course trajectory phenotypes from a mix of clinical, treatment, surgical and comorbidity variables, and evaluate whether information already available at diagnosis could anticipate which trajectory a patient would follow. MethodsWe analysed a retrospective cohort of 98 unique CD patients enrolled at the IRCCS Ca Granda Policlinico Hospital, Milan. Factor analysis of mixed data (FAMD) was applied to 30 disease-course variables spanning demographic and Montreal descriptors, extra-intestinal manifestation burden, individual advanced-therapy drug exposures, surgical burden and comorbidity/symptom burden; clusters were then ranked by treatment burden using a line-weighted therapy score built separately from drug line positions and therapeutic-class weights. Clustering itself was carried out in the retained FAMD coordinate space, and cluster robustness was checked with silhouette metrics and bootstrap Jaccard stability. Baseline-associated variables were assessed separately, while advanced-therapy episodes were reconstructed from the therapy timeline. ResultsThree trajectory phenotypes emerged: C1 (n=56), C2 (n=31) and C3 (n=11). C3 was characterised by younger age at diagnosis, a heavier extra-intestinal manifestation burden, more intensive and prolonged therapy, greater biologic exposure and a higher cumulative number of surgeries, with comorbidity burden rising across the gradient. Montreal behaviour and comorbidity burden emerged as the strongest baseline correlate of trajectory membership, ahead of age at diagnosis and extra-intestinal manifestation burden; penetrating (B3) behaviour, notably, was actually more common in C1 than in C3. A model built on the diagnosis-available variables independently linked to trajectory membership (age at diagnosis, disease location and Montreal behaviour) showed moderate discrimination for the C3 trajectory (area under the curve, AUC=0.80, 95% CI 0.67-0.93). A sensitivity analysis that also added extra-intestinal manifestation count and comorbidity burden, whose timing relative to diagnosis we could not confirm, pushed discrimination to AUC=0.90. ConclusionsFAMD-space clustering identified stable, clinically interpretable CD-course trajectory phenotypes, including a small, high-burden C3 subgroup. This C3 trajectory, small, early-onset and treatment-refractory, shares clinical features that overlap with the difficult-to-treat CD phenotype, though C3 was derived by unsupervised clustering rather than validated against that frameworks own criteria; variables available at or near diagnosis showed exploratory discrimination for the high-burden C3 trajectory, though external validation and careful timing verification are needed before any clinical use.

Background: While immunotherapies have transformed outcomes in several adult cancers, more than half of pediatric patients with high-risk neuroblastoma fail to achieve sustained responses, resulting in poor outcomes. Therefore, understanding the phenotypic and spatial organization of immune cells within the neuroblastoma tumor microenvironment is essential to identify mechanisms driving immunotherapy failure. Methods: To identify spatial determinants of the neuroblastoma immune landscape that may contribute to treatment failure, we dissected immunoregulatory mechanisms at single-cell and protein levels in neuroblastoma samples from 28 patients, using imaging mass cytometry (IMC, n=14) and high-dimensional spectral flow cytometry (n=18). Results: We characterized four distinct cellular neighborhoods, ranging from tumor-dominant to stromal and immune niches. Macrophages were broadly distributed across these niches. In contrast, T and B cells were largely confined to immune niches, forming immature tertiary lymphoid structure-like aggregates within the tumor and stromal cores. T cells displayed a CD69+ tissue-resident memory phenotype, with an overall reduced cytotoxic profile (in CD8+ T cells) and increased expression of inhibitory receptors (predominantly in CD4+ T cells), indicative of dysfunctional features. Intriguingly, T cell functional states differed between spatial niches, with a TCF7+/Ki-67+ self-renewing profile within immune niches, and increased granzyme B expression in CD8+ T cells in tumor cores. Interaction analysis pinpointed CD163+ macrophages as the dominant immunosuppressive interaction partner of T cells, particularly within the tumor edge and immune cores. Analysis of inhibitory ligand-receptor interactions identified the TIGIT/PVR/Nectin-2 axis as the main engaged axis between CD163+ macrophages and T cells. Nectin-2 expression on macrophages was particularly high within immune cores, and elevated in macrophages with direct T cell contact, suggesting a high probability of in situ engagement. Consistent with this immunosuppressive role, both CD163 and Nectin-2 expression on macrophages correlated with poor patient survival, substantiating the clinical relevance of inhibitory macrophage-T cell interactions. Conclusions: This study reveals T cell-excluded tumor niches and targetable inhibitory interactions between CD163+ macrophages and T cells. These insights provide a rationale for combinatorial immunotherapeutic strategies that both enhance T cell infiltration into tumor-rich areas and disrupt inhibitory signaling to improve immunotherapy efficacy in neuroblastoma.

Abstract Background: Experimental models that retain endogenous tumor immune complexity are needed to investigate heterogeneous responses to immune checkpoint inhibitors (ICIs) in melanoma. We established patient-derived melanoma organoids (PDMOs) to examine retention of parental tumor cellular components and investigate patient-specific and context-dependent responses to checkpoint blockade. Methods: Fresh melanoma specimens (n=50), including primary tumors, neoadjuvant-treated tumors and tumor-infiltrating lymphocyte (TIL) associated samples were processed for Matrigel-embedded culture of patient-derived melanoma organoids (PDMOs). Characterization and functional studies were performed in subsets of established cultures. Immunofluorescence assessed tumor, stromal, immune, and checkpoint-marker expression in PDMOs and matched parental tissues. NGFR expression was evaluated in relation to organoid establishment and growth. Responses to anti PD1 alone or combined with anti-LAG3 or anti-CTLA4 therapies were evaluated using viability and morphological analyses, with selected models undergoing immune phenotyping and multiplex cytokine profiling. Available clinical outcomes were used for exploratory comparison. PDMOs and matched two-dimensional cultures were evaluated under 21% and 5% oxygen conditions. Results: PDMOs were established from 30 of 50 specimens (60%) and retained melanoma, stromal, lymphoid, and myeloid components identified in matched parental tissues during early culture, together with immune checkpoint expression. Higher NGFR (CD271) expression was associated with greater organoid-forming capacity in an exploratory subset. Responses to checkpoint blockade varied across patient-derived models and regimens, with combination blockade not uniformly producing lower viability than PD1 monotherapy. Ex vivo responses showed parallels with available clinical outcomes, with discordant cases also observed. In three selected PDMOs, divergent PD-1 responses were accompanied by differences in T-cell and myeloid representation, MHCII and AXL expression, and inflammatory versus immunoregulatory cytokine profiles. Under normoxic conditions, PDMOs and matched two-dimensional cultures exhibited divergent treatment sensitivities, including clinically responding cases in which sensitivity was retained in PDMOs but attenuated in monolayers. Changing oxygen tension further altered checkpoint sensitivity within individual PDMOs, with the direction and magnitude of the observed shifts varying by model and regimen. Conclusions: Early passage PDMOs retain key endogenous tumor, immune, and stromal components and support functional investigation of patient-derived melanoma biology. Their heterogenous responses across checkpoint regimens and oxygen conditions provide a platform for investigating how tumor-immune composition and environmental context influence treatment sensitivity.

The current treatment regimen for neuroblastoma involves immunotherapy, including a monoclonal antibody that recognizes disialoganglioside (GD2), expressed at high levels on neuroblastoma. GD2 is not present on most normal tissues except for nerves. Thus, anti-GD2 antibody treatment causes substantial, dose-limiting neuropathic pain. B7-H3 is overexpressed on multiple tumor types, including neuroblastoma, and is largely absent on nerves and other normal tissues. We designed a bispecific antibody (bsAb) that requires simultaneous binding of these two tumor antigens to achieve tight binding of tumor cells. Our preclinical research shows that, compared with a monospecific anti-GD2 antibody, the GD2xB7-H3 bsAb has improved tumor specificity, comparable antitumor efficacy, and reduced nerve binding and pain-associated toxicity. Since this bsAb does not bind to nerves, it may permit more tolerable and sustained treatment schedules than are currently feasible with monospecific anti-GD2 antibodies. In addition, its enhanced tumor specificity may support future development as a targeted delivery platform for antibody-drug conjugates or other payload-based therapies, potentially improving both efficacy and quality of life for patients with neuroblastoma.

Although MSI-H/dMMR colorectal cancers and the rarer subset with pathogenic POLE exonuclease domain mutations can benefit from immune checkpoint blockade, MSS colorectal cancer remains largely refractory, raising the question of whether other DNA damage response defects may also confer sensitivity to immunotherapy. Here, we performed a systematic in vivo screen of recurrently altered DNA damage response (DDR) genes to determine whether distinct repair defects modulate response to PD-1 blockade in MSS colorectal cancer. Using a genome-edited murine model in immunocompetent mice, we found that DDR perturbations produced heterogeneous outcomes, indicating that the immunologic consequences of DNA repair deficiency may be pathway-specific. Loss of Brca1, Brca2, Atm, and Fanca enhanced response to anti-PD-1 therapy, whereas Atr and Msh6 deficiency were associated with poor benefit. Brca1/2-deficient tumors showed the strongest regressions, supporting a link between core homologous recombination deficiency and increased immune sensitivity. In an independent cohort of colorectal cancer patients treated with ICB from the MSK-IMPACT Clinical Sequencing Cohort, BRCA1 alterations were associated with improved survival status, while FANCA alterations showed a similar trend. Because ATM is therapeutically targetable, we evaluated pharmacologic ATM inhibition in MSS colorectal cancer. Sustained ATM inhibition induced immune-related transcriptional programs in vitro and, when administered as a pre-treatment in vivo, enhanced the antitumor activity of PD-1 blockade. Together, these findings suggest that selected DDR alterations may represent candidate biomarkers of immunotherapy sensitivity, while ATM inhibition may provide a rational combination strategy to enhance PD-1 blockade in MSS colorectal cancer.