OncoImmunology

BackgroundTumor-infiltrating lymphocyte (TIL) therapy has demonstrated clinical efficacy in malignant melanoma; however, inefficient ex vivo expansion remains a major limitation. We previously showed that stimulation of tumor-infiltrating B cells via CD40-CD40L axis improves TIL expansion, and that direct activation of the 41BB-41BBL pathway on T cells enhances CD8 T cell outgrowth. We hypothesized that adding simultaneous targeting of both pathways would augment the growth and activity of CD8+ cytotoxic T cells. We conducted a study with the objective of determining the feasibility of dual stimulation with human tumors as justification for a Phase I trial. MethodsCD40L variants were generated by yeast display selection and evaluated for B cell binding and activation. The effects of CD40L variants on TIL expansion were evaluated using tumors derived from standard of care resections using fragment method. Based on these findings, a bi-specific molecule was designed and generated fusing a CD40L variant and 41BB to the N- and C-termini of a trimeric leucine zipper. The effects of the bi-specific molecule (termed CD40LEPC6-41BBL) on TIL expansion were evaluated in TIL cultures derived from lung tumor and melanoma fragments. TIL phenotypes were assessed by flow cytometry, including high-dimensional FlowSOM analysis, and tumor reactivity by autologous tumor co-culture assays. ResultsEach of our engineered CD40L variants bound B cells and induced CD80/CD86 expression at levels comparable to wild-type CD40L. Supplementation of TIL cultures with CD40L variants increased the success rate of TIL expansion compared to control. We then developed a bi-specific CD40LEPC6-41BBL molecule capable of binding to both B and T cells. Addition of CD40LEPC6-41BBL significantly increased total TIL yield and improved expansion success rates in both lung tumor and melanoma cultures. In particular, CD40LEPC6-41BBL promoted preferential expansion of CD8 T cells. High-dimensional analysis revealed enrichment of CD8 T cell clusters expressing CD39, CD69, TIM3, and CD56 in cultures supplemented with CD40LEPC6-41BBL. Furthermore, treated cultures displayed increased frequencies of CD27 CD4 T cells. Functional assessment suggested a trend toward enhanced tumor reactivity in melanoma-derived TIL products expanded with CD40LEPC6-41BBL. ConclusionsSimultaneous stimulation of CD40 and 41BB pathways using a novel bi-specific molecule resulted in qualitative and quantitative enhancement of TIL products. These findings support dual targeting of tumor-infiltrating B cells and T cells as a promising strategy to optimize TIL manufacturing for adoptive cell therapy in Phase I trials.

Natural killer (NK) cells at the maternal-fetal interface harbor a specialized decidual phenotype that promotes placentation and immune tolerance. Although it has been postulated that tumors can exploit similar NK cell programs to augment the tumor microenvironment, this mechanism has not been investigated in melanoma. Using flow cytometry and multiplex immunohistochemistry, we identified CD56BrightCD16-CD49a+CD9+ decidual-like NK (dl-NK) cells within melanoma tumors; these cells were rare or absent in peripheral blood from either patients with melanoma or healthy donors. These dl-NK cells were detected within and surrounding melanoma tumors in both male and female patients and across melanoma subtypes and across primary, lymph node, and distant metastatic sites. In an exploratory cohort of patients treated with tumor-infiltrating lymphocyte therapy, higher dl-NK frequency was observed in the patient with progressive disease. Because CD9 is a defining feature of decidual NK cells, we examined the relationship of CD9 with the dl-NK phenotype. Tumor CD9 expression was positively associated with intratumoral dl-NK frequency (Pearson rho = 0.40, p = 0.056). Direct co-culture of healthy donor NK cells with CD9-expressing melanoma cells induced robust CD9 acquisition by CD56Bright NK cells, whereas separation by a transwell abrogated this effect, demonstrating a requirement for direct cell-cell interaction. Collectively, these findings identify a dl-NK cell population within melanomas and suggest that direct interactions with melanoma cells may contribute to acquisition of this phenotype, revealing a potential immunologic parallel between the melanoma microenvironment and the maternal-fetal interface.

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.

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.

BackgroundMutation-derived neoantigens, typically identified in primary tumors, are emerging therapeutic targets for personalized cancer vaccines and adoptive T-cell therapies. However, clinical efficacy of neoantigen-directed therapies in patients with metastatic disease remains limited, partly due to inter-site genetic heterogeneity. We investigated whether ubiquitous neoantigens-derived from mutations shared across all tumor sites-could provide more effective, durable targets, particularly in patients undergoing resection of metastatic lesions. MethodsWhole-exome and RNA sequencing were performed on 14 tumor samples (primary and 13 synchronous nodal metastases) from a treatment-naive patient with pancreatic neuroendocrine tumor (PNET). Ubiquitous mutations were identified bioinformatically, and their immunogenicity assessed using in-vitro stimulation of autologous peripheral blood mononuclear cells followed by IFN-{gamma} ELISpot assay. Neoantigen-specific T-cell clonotypes were further identified by HLA-tetramer staining and single-cell RNA/TCR sequencing. Neoantigen-reactive clonotypes identified in peripheral blood were tracked across multiple metastatic sites using bulk TCR{beta} repertoire sequencing. ResultsAmong 1,195 non-synonymous mutations detected, eight were shared across all 14 tumor sites. Of these, one encoded a neoantigen that elicited a reproducible IFN-{gamma} ELISpot response in peripheral blood, confirming its immunogenicity. Further, we identified the corresponding neoantigen-reactive TCR clonotypes in blood. Comparison with bulk TCR{beta} repertoires from eight metastatic sites showed that these clonotypes were present in every site analyzed, with evidence of local clonal expansion. ConclusionThis study provides direct evidence that a single ubiquitous mutation-derived neoantigen can generate systemic T-cell responses and clonotype expansion across multiple metastatic sites in a TMB-low, TIL-low tumor. Our findings support incorporating mutation-sharing status across metastases as a key criterion for neoantigen selection in cancer vaccines and adoptive T-cell therapies. This approach could inform the design of neoantigen-directed immunotherapies in metastatic PNET and potentially other metastatic solid tumors. What is already known on this topicNeoantigen-directed therapies, such as personalized cancer vaccines or adoptive T-cell transfer, can induce anti-tumor responses but have shown limited success in metastatic disease. One major barrier is genetic heterogeneity between tumor sites, suggesting that targeting ubiquitous mutations-those shared across all tumor sites-may improve the efficacy of such therapies. What this study addsIn one patient with metastatic pancreatic neuroendocrine tumor involving 13 lymph nodes, we identified eight ubiquitous mutations, one of which generated a detectable neoantigen-specific T-cell response in blood. The corresponding T-cell clonotypes were found across all metastatic sites analyzed and showed evidence of clonal expansion, providing direct evidence of systemic and local recognition of a shared neoantigen in a TMB-low/TIL-low cancer. How this study might affect research, practice or policyThese findings support incorporating mutation sharing across metastases as a key criterion in neoantigen selection for cancer vaccines and adoptive T-cell therapies. This strategy could enhance the relevance and durability of neoantigen- directed approaches in patients with metastatic disease.

The role of UBE2N in myeloid cell-mediated immune suppression in cancer remains undefined. Here, we examined the function of UBE2N in myeloid cell-mediated tumor progression using a temporally inducible myeloid-specific knockout model (LysMCreERUbe2nfl/fl). Temporally induced deletion of Ube2n in myeloid cells (Ube2nMyeKO) significantly hindered growth of YUMM1.7 melanoma. This was accompanied by reduced myeloid cell burden within the tumor microenvironment. We observed altered abundance of PD-1, PD-L1, and SPP1 in the Ube2nMyeKO tumor microenvironment at the tissue level. In vitro analysis showed that knock-in expression of a catalytically deficient UBE2NC87S mutant in bone marrow-derived macrophages (BMDMs) markedly decreased expression of Spp1. We observed decreased SPP1 secretion in Ube2nMyeKO BMDM-conditioned media (CM). Treatment with Ube2nMyeKO BMDM-CM decreased co-expression of PD-1, TIM-3, and LAG-3 on chronically stimulated T cells. Antibody-mediated neutralization of SPP1 in Ube2nWT BMDM-CM decreased PD-1 expression on CD8+ T cells. Together, these findings suggest a role for myeloid UBE2N in YUMM1.7 progression.

BackgroundStrategies to define human monocytes by flow cytometry vary considerably across studies. Recently, toll-like receptor 2 (TLR2) has been proposed as a marker to identify "all monocytes" in human peripheral blood. However, the TLR2-defined monocytes also contained a previously ignored TLR2posCD14dim/negCD16neg population, which we termed the unclassified subset (UCS). MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors and patients with multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS) were analyzed by multiparameter flow cytometry using TLR2pos gating. PBMCs from additional healthy donors were analyzed to characterize the UCS population, including the impact of using either TLR2pos or a negative selection-based gating strategy. ResultsThe TLR2pos CD14dim/neg CD16neg UCS population was present in healthy controls, MGUS, and MM patients. The UCS expressed the monocyte-macrophage scavenger receptor CD163 and was significantly reduced in MM patients compared to healthy donors (P<0.002). Further phenotypic characterization in healthy blood donors revealed that approximately 80% of UCS cells expressed CD163 at levels comparable to classical monocytes, yet phenotypically resembled CD163pos dendritic cells (DCs). Importantly, gating strategies influenced the composition of the UCS: negative selection-based gating captured all DC subsets, whereas TLR2pos gating primarily included CD1cpos DCs that were highly CD163pos. ConclusionsThese findings demonstrate that circulating CD163pos CD1cpos DCs are included in the TLR2pos cell population previously described as exclusively monocytes, highlighting the impact of gating strategy on monocyte subset identification. Further, the lower level of TLR2pos CD14dim/neg CD16neg CD163pos cells in MM patients may represent decreased levels of circulating DCs that may contribute to the immune dysregulation in this disease.

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.

Introduction: Tumor-reactive T cell receptors (TCRs) are promising candidates for the manufacturing of safe and effective adoptive TCR-T cell therapies, but their efficient identification remains a major challenge. We previously established an antigen-agnostic TCR-repertoire analysis workflow that prioritizes candidate TCRs based on intratumoral clonal expansion and high tumor-to-non-tumor (T/N) frequency ratios. Here, we investigated whether T/N-selected clonotypes show independent transcriptional and spatial features consistent with tumor association. Methods: Bulk and single-cell TCR repertoire sequencing of paired tumor and adjacent non-tumor tissues was used to identify clonotypes with high T/N ratios across 15 cancer cases, comprising six non-small cell lung cancers, five pancreatic cancers, two breast cancers, and two colorectal cancers. Candidate clonotypes were further characterized by paired single-cell TCR and gene expression profiling and, in two PDAC cases, by Xenium spatial transcriptomics to assess their cellular phenotype, spatial localization, and proximity to malignant cells. Results: High T/N ratios consistently marked T cell clonotypes with features of tumor reactivity across the studied solid tumor entities. The analysis included Cluster-A, a lead T cell cluster comprising highly similar TCRs shared among 14 HLA-A*02:01-positive patients in the present cohort. Cluster-A clonotypes were consistently enriched in tumor relative to matched non-tumor tissue, and previous functional studies demonstrated tumor recognition by Cluster-A members. In a focused analysis of a pancreatic cancer case, Cluster-A CD8 T cells were selectively detected in tumor tissue, preferentially localized near malignant epithelial cells, and displayed an antigen-experienced effector-memory phenotype with prominent expression of granzymes A and K (GZMA/GZMK). Importantly, in another PDAC case that lacked Cluster-A clonotypes, these features extended to independent clonotypes, revealing a continuous tumor-association axis in which increasing T/N ratios were associated with progressively closer localization to malignant cells and increased GZMA/GZMK expression. Thus, repertoire enrichment, spatial tumor proximity, and effector-associated transcriptional states converged across independent tumor-enriched clonotypes. Outlook: These findings support the T/N ratio as a robust primary criterion for identifying candidate tumor-reactive TCRs. Integration of single-cell and spatial transcriptomics provides supporting biological evidence of tumor association and a framework for prioritizing therapeutic TCR candidates when functional validation is limited.

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.

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by rapid progression and a poor prognosis. CAR-based cellular therapies are promising approaches, and CAR-T cells targeting GD2 have demonstrated transient efficacy. Identifying how tumors evade these treatments is essential for advancing therapy development. In this study, we investigated the mechanisms through which GBM cells evade GD2.chimeric antigen receptor (CAR)-T and CAR-invariant natural killer T (iNKT) in vitro and explored ways to overcome tumor escape. GD2-targeted CAR-T and CAR-iNKT cells were tested in a stepwise in vitro model that repeatedly exposed them to GD2+ cell lines. While CAR effector cells effectively killed GD2+ GBM cells in short-term assays, their anti-tumor efficacy declined after repeated antigen exposures. Tumor escape mechanisms included reduced CAR expression, impaired proliferation, reduced production of cytokine, granzyme, and perforin, tumor downregulation of GD2, trogocytosis, and upregulation of the HLA-E/NKG2A inhibitory compared to MICA-B/NKG2D activation pathways on tumor and immune cells. Increasing effector cell numbers or adding IL-15 +/- IL-7 partially improved CAR persistence but did not fully restore CAR effector functions. By contrast, IL-12 addition optimized tumor-killing capacity by increasing CAR effector cell proliferation, CAR surface expression, IFN-y production, and balancing HLA-E/NKG2A versus MICA-B/NKG2D pathways. In conclusion, GD2.CAR-T and GD2.CAR-iNKT cells effectively target GBM but are susceptible to repeated antigen exposure, which IL-12 could counteract. These findings encourage further development of armored IL-12 CAR-T or CAR-iNKT cells and further investigation of the roles of HLA-E and MICA-B pathways in immunotherapy against GBM.

Chimeric antigen receptor (CAR) T-cells engineered from {gamma}{delta} T-cells show limited clinical benefit in solid tumors such as ovarian cancer (OVCA). Although {gamma}{delta} T-cells can detect malignant transformation and exert cytotoxicity, these properties confer potent preclinical efficacy but limited therapeutic activity and the basis for this discrepancy remains unclear. Here, we established an ex-vivo culture consisting of highly inflammatory patient-derived malignant ascites and hypoxia conditions, to simulate key obstacles encountered by CAR T-cells in the immunosuppressive OVCA tumor microenvironment (TME). Using this condition, we first demonstrated the presence of CAR-expressing {gamma}{delta} T-cells within conventional (conv)CAR T-cells, which displayed greater proliferative capacity than {gamma}{delta}neg T-cells. We therefore produced {gamma}{delta}CAR T-cells for parallel comparison with conventional (conv)CAR T-cells, evaluating differences in gene expression and functionality. Under TME-like conditions, {gamma}{delta}CAR T-cells demonstrated lower viability but consistently outperformed convCAR T-cells, demonstrating favorable gene expression profiles, enhanced cytotoxicity, improved retention of cytokine secretion and degranulation and increased proliferative capacity. Importantly, we characterized an ascites protein profile that impacted the viability of both cell types comparably. Together, these findings reveal specific functional advantages of {gamma}{delta}CAR T-cells over convCAR T-cells in the immunosuppressive OVCA TME-like condition while facing different cellular fitness constraints, providing insights into the use and limitations of {gamma}{delta}CAR T-cells.

Small bowel gastrointestinal stromal tumors (GISTs) are more aggressive than gastric GISTs, yet the biologic basis for this difference remains poorly understood. We hypothesized that differential expression of immune checkpoints contributes to this site-specific behavior. Bulk RNA sequencing of 42 primary GISTs (36 gastric, 6 small bowel) revealed marked upregulation of VTCN1, which encodes the inhibitory checkpoint B7-H4, in small bowel tumors (log2FC = 7.95, adjusted P < 0.001). In contrast, expression of the therapeutically targeted checkpoints PD-L1, PD-1, and CTLA-4 was comparable between sites. Concordantly, B7-H4 enrichment was accompanied by an immunosuppressive tumor microenvironment, characterized by reduced antigen-presenting cells, fewer effector-memory CD8+ T cells, lower granzyme B expression, and suppression of interferon and inflammatory signaling pathways. Notably, the differences in B7-H4 expression were independent of imatinib-treatment status. These findings were corroborated in an external cohort of 77 untreated GISTs, in which VTCN1 was similarly enriched in small bowel tumors. Independent immunohistochemical analysis of a tissue microarray comprising 68 untreated primary GISTs confirmed the pattern, showing median B7-H4 positivity of 78.6% in duodenal, 20.5% in jejunal/ileal, and 0% in gastric tumors, with staining localized to tumor cells rather than stroma. Collectively, these data identify B7-H4 as a site-specific feature of small bowel GISTs and a potential therapeutic target for tumors that have not responded to conventional checkpoint blockade.

Immunotherapy has transformed treatment for many cancers. In the aggressive and genetically heterogeneous diffuse large B-cell lymphoma (DLBCL), CD19 CAR T-cell therapy is highly effective, whereas immune checkpoint blockade has shown limited benefit. Loss of MHC expression is a common mechanism to escape T-cell cytotoxicity, and loss of MHC class I (MHC-I) and II are frequent in DLBCL. We applied imaging mass cytometry to diagnostic biopsies from younger, high-risk DLBCL patients to map the tumor microenvironment (TME) spatial architecture in relation to tumor cell MHC expression, mutational status, transcriptomic and proteomic profiles. Neighborhood analyses identified four TME subtypes: immune-cell depleted and three immune-infiltrated types (mixed, CD4 T cell-rich, CD8 T-cell/macrophage-rich). Depleted cases had shorter overall survival (p = 0.033) and increased expression of proteins involved in DNA replication and proliferation markers compared to infiltrated cases. Tumor cell MHC-I expression was heterogeneous. Cases with low frequency of MHC-I-pos tumor cells were enriched for the depleted TME type. MHC-I-pos tumor cells were surrounded by CD4 and CD8 T cells and M1 macrophages, whereas MHC-I-neg tumor cells were closer to other MHC-I-neg tumor cells. These findings suggest that TME-based classification incorporating tumor cell MHC-I status may improve individualized immunotherapy selection.

Formation of tertiary lymphoid structures (TLS) within the bladder microenvironment because of chronic mucosal inflammation has been associated with variable clinical outcomes. While the immune cell composition and functional states of TLS have been characterized in both non-invasive and muscle-invasive bladder tumors, the TLS-adjacent tumor epithelial compartments remain poorly characterized. Evaluation of a 16-gene TLS signature in treatment-naive tumor bulk RNA sequencing profiles from 283 non-muscle invasive bladder tumors, from patients treated with Bacillus Calmette-Guerin (BCG) immunotherapy, and 348 muscle-invasive bladder tumors from patients treated with immune checkpoint inhibitor therapy revealed overlapping enrichment of immune exhaustion pathways. High TLS gene expression scores correlated with upregulation of immune exhaustion, hypoxia, and epithelial-to-mesenchymal transition (EMT) pathways in tumors from both cohorts. Spatial whole transcriptomic analysis of tumor sections with high TLS density, revealed enrichment of genes associated with EMT, angiogenesis, extracellular matrix remodeling, and B cell receptor signaling pathways in tumor epithelial regions adjacent to TLS, whereas those distant from TLS exhibited enrichment of IFN-{gamma}, TNF-/NF-{kappa}B, p53, and metabolic pathways. Multiplex immunofluorescence further identified co-localization of exhausted immune cell populations within the core and periphery of peri-tumoral TLS. These findings indicate that a pro-tumorigenic microenvironment associated with disease progression in bladder cancer exists within peri-tumoral TLS and potentially a factor underlying contrasting therapeutic associations potentially driven by live microbial versus targeted immunomodulatory therapy in NMIBC and MIBC.

Background: Lung cancer remains the leading cause of cancer-related deaths in the United States with over 124,000 estimated deaths for 2026. Previous studies have found that tumor-adjacent lung tissues may provide additional insight into the immune microenvironment of early-stage NSCLC. Methods: Multiplex immunofluorescence (mIF) imaging was performed on 192 tissues from 101 early-stage non-small cell lung cancer patients including 91 matched tumor-adjacent pairs, using three mIF panels. Spatial analyses were performed to identify distinctions between tissues and identify associations with clinical and genomic features. Results: Tumor tissue showed significantly higher densities of T cells, macrophages, B cells, and memory/regulatory populations than adjacent tissue (p<0.001). Tumors exhibited greater spatial heterogeneity, with higher prevalence of spatial patterning (47.7% vs. 31.2%) and more consistently localized organization, whereas adjacent tissue showed stronger individual-cell clustering. Pairwise colocalization (Ripley's L-cross) revealed selective spatial segregation in tumors (including B-cells from memory/regulatory cells and among cytotoxic T-cell subsets) and reduced immune proximity to malignant cells relative to the strong immune-epithelial association in adjacent tissue. Spatial features were linked to genomic features or patient outcomes: tumor neoantigen burden exhibited a positive association with CD3+ T cells in the tumor, while colocalization between CD45RO+CD57+GZMB+ cells and CD57+GZMB+ cells was negatively associated with overall survival outside the tumor. Conclusion: Tumor and adjacent tissues harbor distinct spatial immune architectures, with spatial features displaying associations with genomic features or patient outcomes. These findings highlight immune microenvironment reorganization and the importance of incorporating spatial context from both compartments into risk assessment in NSCLC.

The sialyl-Tn (STn) glycan antigen is aberrantly expressed in a subset of triple-negative breast cancer (TNBC) and is associated with poor prognosis and immunosuppressive microenvironment. Tumour-derived extracellular vesicles (TDEVs) are emerging regulators of immune escape however the role of glycan-mediated mechanisms remains elusive. Aberrant glycosylation is a hallmark of cancer that extends to TDEVs, yet how tumour-associated glycans within EV cargo modulate cell function remains poorly understood. Here we used engineered MDA-MB-231 TNBC cells to overexpress the glycosyltransferase ST6GalNAc-I, generating STn-positive cells whose EVs were enriched in STn (STn+ EV). The STn+ EVs impaired the maturation of monocyte-derived dendritic cells (DCs), reduced antigen presentation, and diminished CD4{square} and CD8{square} T cell priming, alongside the expansion of regulatory T cells. DCs co-cultured with STn{square} EVs display STn at their cell surface. Notably, STn+ EVs transferred both STn antigen and the ST6GalNAc-I to recipient DCs. Enzymatic removal of terminal sialic acids from STn{square} EVs reversed the immunosuppressive effects, confirming the STn{square}dependent nature of DC dysfunction. These findings add STn to the extensive list of components of EVs molecular cargo that play a role in immune suppression and may contribute for developing precision medicine approaches in oncology.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressive tumor microenvironment (TME) with pancreatic myofibroblasts (PMF) and macrophages being two prominent cell populations essentially impairing tumor responses to (immuno)therapies. L1 cell adhesion molecule (L1CAM) is upregulated in PDAC cells in primary and metastatic tissues and associated with tumor progression and therapy resistance. Using L1CAM as tumor-associated antigen, two bispecific antibodies (bsAB) targeting L1CAM and CD3 were developed in the IgG-(L)-ScFv format and their anti-tumorigenic activity was investigated in different preclinical PDAC models. In 2D models, both L1-bsAB exerted L1CAM-specific anti-PDAC cell activity when co-cultured with activated CD8+ T cells. Strong anti-PDAC cell effects along with elevated release of T cell effector molecules were also observed upon co-culture with peripheral blood mononuclear cells (PMBC) from healthy donors and PDAC patients. Of note, both L1-bsAB were also effective in 3D PDAC cell spheroids and neither impaired by PMF nor macrophages. Finally, application of L1-bsAB on organotypic tissue slice cultures from PDAC tissues comprising the entire complex TME also induced PDAC cell apoptosis and release of T cell effector molecules. Overall, our results highlight relevant anti-PDAC cell activity of L1-bsAB in immunosuppressive contexts supporting their potential as immunotherapeutic strategy for PDAC.

Background: Objective assessment of tumor burden in patients with solid tumors remains inexact. Furthermore, while mutation-based liquid biopsies are specific, they are limited by tumor heterogeneity and the requirement for detectable clonal mutations. Cell-free DNA (cfDNA) concentration, a cost-effective substrate for mutation-focused liquid biopsy, has shown promise as a "molecular tumor burden" biomarker. Methods: We analyzed cfDNA concentration from 1000 unique cancer patients using standardized protocols. Demographics, AJCC stage, clinical anatomic tumor burden and oncologic outcome variables were collected. Associations were assessed with non-parametric tests. Multivariable linear regression and Cox proportional hazards models were used to identify independent predictors and survival associations. Results: CfDNA concentration varied broadly (median 6.25 ng/mL; range 0.5-1132.9). Anatomic tumor burden, including tumor number (rho=0.25, p<0.0001) and largest tumor diameter (rho=0.25, p<0.0001), correlated significantly with cfDNA, especially in stage IV disease. Primary tumor site influenced cfDNA levels, with liver/bile duct cancers having higher cfDNA than other sites (median 13.5 vs 6.1 ng/mL, p<0.0001). Multivariable analysis confirmed overall tumor burden and hepatic tumor location (primary or metastatic) as principal drivers of cfDNA. Critically, cfDNA concentration was an independent predictor of shorter PFS (p=0.001) and DSS (p<0.0001), demonstrating increased value in advanced disease settings, irrespective of treatment intent. Conclusions: CfDNA concentration is a robust, biologically integrated biomarker that provides an objective measure of total systemic tumor burden and prognosis in a large, pan-cancer cohort. By capturing disease activity irrespective of mutational status, it offers a valuable adjunct to targeted profiling, particularly in heterogeneous or advanced malignancies. Although these findings require prospective clinical validation, cfDNA concentration may eventually augment patient selection for aggressive versus palliative interventions, especially in advanced disease.

The mutational landscape of neural innervation axes in cancer has garnered increasing attention due to their significant influence on tumorigenesis, metastasis, and treatment resistance. This study provides an overview of the alterations of neural signaling pathways based on multi-omics data analysis. In particular, we conducted a comprehensive analysis of mutations in key genes associated with neurotrophin signaling, norepinephrine and cholinergic pathways, alongside paracrine and synaptogenic factors. Our findings reveal that 65% of patients in a pancancer cohort exhibit at least one somatic alteration in these pathways, with neurotrophin genes being the most frequently altered. Notably, alterations in these genes (e.g., NGF) correlate with poor patient survival across various cancer types, suggesting their oncogenic potential. We also identified significant co-occurrence of mutations and differential expression patterns from both bulk and single-cell RNAseq data, indicating complex interactions among these pathways that contribute to tumor innervation and neuroplasticity. This study underscores the necessity of understanding the crosstalk between neural signaling and cancer biology to improve patient stratification based on mutational profiles and identify potential therapeutic targets. Further research utilizing advanced genomic techniques is essential to elucidate the mechanistic roles of these pathways in tumor innervation.