OncoImmunology

INTRODUCIONTumor cell infiltration in regional lymph nodes is a strong prognostic marker, guiding treatment decisions in breast cancer. While the immune cell composition in primary tumors has been more widely explored in later years, the immune cell composition of the sentinel node (SN) and axillary lymph nodes (ALN) remains understudied. A better understanding of how primary tumor and metastatic tumor cells alter the nodal immune microenvironment can shed light on metastasis and cancer progression to unveil new treatment strategies. MATERIALS AND METHODSFrom a prospective clinical cohort of 458 treatment-naive patients with primary operable breast cancer, we performed comprehensive immunophenotypic analysis using mass cytometry analysis of non-metastatic (SN-) and metastatic (SN+) and ALN (ALN+) lymph nodes. RESULTSAs expected, patients with ALN+ cases had a shorter time to distant metastases than SN+ and SN- cases. We identified an exhausted T-cell phenotype and an increase in Germinal Center B (GC B) cells and plasma cells in ALN+ samples compared to SN- samples, both in the whole cohort as well as when investigating estrogen-receptor positive (ER+) patients only. There were no differences in immune cell composition across breast cancer (BC) subtypes within SN-samples. SN+ samples from triple negative BC (TNBC) showed a trend towards increased abundance of GC B and plasma cells, similar to more advanced ALN+, suggesting that smaller TN metastases may trigger an immune activation at an early stage of dissemination. Further analysis of SN- samples from ER+ patients revealed a subset of patients where the immune response had a more exhausted T-cell phenotype. This group was enriched for lymph nodes that were deemed negative by ordinary pathology examination (microscopy) but had detectable tumor cells by CyTOF analysis. CONCLUSIONThe immune profiles of SN and ALN samples from breast cancer patients are highly diverse, showing limited associations to BC subtype, clinical parameters or patient outcome. Metastatic tumor cells play a significant role in driving T-cell exhaustion and immunosuppression. Notably, in approximately 50% of the ER+ samples, T-cell exhaustion was detectable. This coincides with the presence of tumor cells identified by CyTOF, which were likely missed by conventional pathological examination. These findings suggest that small tumor deposits alter the immune composition, and the immune profile reveals the presence of tumor cells.

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.

Anaplastic thyroid carcinoma (ATC) is one of the most lethal malignancies. Immune dysregulation is believed to play an important role in ATC. Here, we aimed to characterize the systemic inflammation and the function of circulating immune cells of patients with ATC. First, we retrospectively assessed biochemical parameters of patients with ATC and observed that high systemic inflammation correlated with worse survival. Next, we prospectively investigated the inflammatory proteome, single-cell peripheral blood mononuclear cell transcriptome and epigenetic changes. Circulating concentrations of proinflammatory cytokines were increased in ATC patients. This proinflammatory profile was apparent at the level of gene transcription and chromatin accessibility, especially in monocytes. These findings were substantiated by an increased capacity of peripheral blood mononuclear cells of ATC patients to produce IL-6, IL-8 and lactate. As IL-6 is known to promote tumor cell survival, we assessed its capacity to influence ATC cell proliferation. Blocking IL-6/gp130/Jak/STAT3 pathway inhibited proliferation of ATC cell lines in vitro. In conclusion, these findings show that ATC is characterized by inappropriate systemic inflammation and epigenetic and transcriptional reprogramming of circulating monocytes. Proinflammatory cytokines released by monocytes support survival and proliferation of ATC tumor cells, suggesting a therapeutic potential of targeting this pathway in ATC patients.

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.

BackgroundMesothelioma (Me) is an aggressive cancer with limited response to conventional therapies. The tumors harsh microenvironment contributes to immune escape and therapy resistance and the effects of ICIs on Me are still unclear. Adenosine, an immunosuppressive molecule produced from AMP by the enzyme CD73, accumulates in hypoxic tumor areas. Elevated CD73 and adenosine receptor A2B (A2Br) levels on Me cells are linked to worse patient outcomes, indicating their important role in disease progression and potential as targets for treatment. AimThis study characterizes the Me-ME (micro environment) and evaluates the efficacy of TT-4 (A2B inibitor) and AB680 (CD73 inibitor), alone or with aPD-1, using 3D models in vitro and in vivo. MethodsCD73 and A2B receptor levels were quantified in tumor and normal samples using qRT-PCR and IHC. Cells lines were treated with CoCl2 to mimic hypoxia, then CD73, A2Br and related markers were analyzed. MSTO-211H and REN cells were silenced for CD73, grown as spheroids and adenosine release was measured. Co-culture spheroids of MSTO-211H and Jurkat cells were treated with AMP and CD73 inhibitor, then analyzed for viability and immune markers. An orthotopic Me model was established by injecting AB1-B/c-LUC cells and monitored by in vivo imaging. Proteomic analysis of spheroids was conducted to identify proteins and pathways involved. ResultsHypoxia boosts CD73 and A2Br expression in Me cells, leading to adenosine production via CD73. In 3D co-cultures, AB680 lowered Me cell viability and enhanced activation of Jurkat T cells. In mice, combining aPD-1 therapy with A2Br or CD73 inhibitors strongly reduced tumor growth. Proteomics identified 93 proteins influenced by adenosine signaling through A2B. ConclusionTargeting the adenosine pathway alongside PD-1 blockade offers a promising new immunotherapy strategy for Me.

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.

BackgroundGlioblastoma is an aggressive and incurable brain tumor. Clinical trials of immune checkpoint inhibitors showed no clinical benefit in glioblastoma when given after surgery. However, a clinical trial in which PD1 inhibition was given prior to second surgery did show pharmacodynamic evidence for activity. This suggests the possibility that immune checkpoint inhibitors may be more effective in a setting where large tumors are present. Here we have studied immune responses to large tumors in an autochthonous mouse model of glioblastoma. MethodsGlioblastoma was induced by transfection with oncogenic plasmids injected directly into the lateral ventricle of neonatal mice. Immune responses were assessed using a combination of spectral flow cytometry and immunohistochemistry. ResultsThere was a marked immune response to large tumors, with significant increases in CD4 T cells and dendritic cells. T cell changes occurred primarily at leptomeningeal/perivascular border sites. A large proportion of CD4 T cells expressed PD1 and half of these were regulatory T cells. NK cells were also increased in mice with large tumors, but were predominantly in immature states. The mouse model accurately recapitulates the formation of palisading necroses. These contain apoptotic cells and avidly recruit myeloid cells that are induced to express large amounts of TGF{beta}. ConclusionsLarge glioblastoma tumors generate a border site population of PD1 positive T cells that may explain the pharmacodynamic response in neoadjuvant trials, and a palisading necrosis-driven immunosuppressive mechanism that may explain why responses are insufficient to provide a significant clinical benefit. KEY POINTSThe SB mouse model accurately recapitulates immune features of human glioblastoma Large tumors induce a significant border site immune response Palisading necroses in large tumors counter this with a strong immunosuppressive response IMPORTANCE OF STUDYImmune checkpoint inhibitors have not shown efficacy in glioblastoma when used post-surgery, but do show pharmacodynamic activity when used in patients prior to second surgery (i.e. neoadjuvant). This suggest the possibility that immune checkpoint inhibition is more effective when large tumors are present. Using a clinically-relevant autochthonous mouse model, we show here that large tumors induce an immune response that is evident in leptomeningeal border sites. Large tumors in this mouse model also generate palisading necroses, a well-known diagnostic feature in glioblastoma tumors. These palisading necroses generate large amounts of TGF{beta}, providing a mechanism by which large tumors can suppress border site immune responses. This further supports the concept that palisading necroses are drivers of glioblastoma malignancy and suggests novel strategies to enhance responses to immune checkpoint inhibition in this cancer.

Neoantigens are cancer-specific antigens arising from genomic alterations. Single Amino Acid Variants (SAAVs) represent a primary class of these neoantigens. To evaluate the therapeutic potential of Neurofibromin 1 (NF1)-derived SAAVs - given that NF1 is frequently mutated in malignant brain tumors - we prioritized the 40 NF1 SAAVs determined to be HLA-A*02:01 binders using computational prediction coupled with experimental validation. To validate these predicted neoepitopes, we employed a two-tiered experimental approach in HLA-A*02:01 homozygous U87-MG cells. We first synthesized minigene constructs encoding the predicted neoepitopes, introduced them via lentiviral transfection and confirmed their expression by mass spectrometry (MS). Subsequently, we performed endogenous validation using pan-HLA immunoprecipitation mass spectrometry (IP-MS), confirming 4 (10 neoepitopes) of the 40 candidate SAAVs. We observed a discrepancy between in silico predictions and the observed sequences. Our endogenous peptidomics further revealed conserved peptide motifs and demonstrated that peptide selection for HLA presentation is transient. While our study substantiates the therapeutic feasibility of T-cell immunotherapies targeting NF1 mutations, these results underscore a limitation in current computational prediction. Our study highlights the necessity of experimental validation to refine neoantigen prioritization strategies.

BackgroundChimeric antigen receptor (CAR)-T cell therapies efficacy in solid tumors remains limited, largely due to the profoundly immunosuppressive tumor microenvironment (TME) which drives CAR-T cells to dysfunction and poor persistence. A comprehensive understanding of the dynamic interplay between CAR-T cells and the TME is therefore critical for the rational design of more effective CAR-T strategies for solid cancers. MethodsHere, we performed single-cell RNA sequencing of tumor samples from immunocompetent mice treated with stroma-targeting EDA-CAR-T cells, profiling CAR-T cell states and TME programs at the peak of antitumor response and during subsequent tumor progression. ResultsOur analysis revealed a marked temporal remodeling of EDA-CAR-T cells within the TME, where early antitumor efficacy is associated with concurrent expansion of cytotoxic effector CD8 CAR-T cells and activation of memory CD4 CAR-T subsets. Moreover, EDA-CAR-T cells effectively engaged the myeloid compartment, resulting in strengthened communication networks involving T cell activation. However, by tumor progression, EDA-CAR-T cells suffered a widespread transcriptional reprogramming towards dysfunction, characterized by loss of effector programs alongside induction of exhaustion and immunoregulatory pathways within the TME, including PD-L1/PD-L2 and TGF{beta} signaling, which impairs sustained immune responses. Notably, early CAR-T cell activation led to increased susceptibility to TME-mediated immunosuppression, revealing EDA-CAR-T-specific soluble galectin-mediated cell-to-cell interaction networks. ConclusionsTogether, this works offers a high-resolution view of CAR-T cell dynamics within the solid TME, uncovering cellular and molecular mechanisms of rapid functional decline and identifying regulatory pathways within the TME that can be exploited to improve CAR-T cell therapy efficacy in solid tumors. KEY MESSAGES OF THE ARTICLEO_ST_ABSWhat is already known on this topicC_ST_ABSThe determinants of CAR-T cell therapeutic efficacy in solid tumors remain poorly defined, largely due to the complexity of the immunosuppressive tumor microenvironment. In this effort, it is necessary to perform comprehensive and detailed mechanistic studies that capture CAR-T cell dynamics within the solid tumor microenvironment to understand treatment failure. What this study addsWe performed single-cell profiling of stroma-targeting EDA-CAR-T cells, revealing their dynamic reprogramming toward dysfunction within the solid tumor microenvironment. We dissected CAR-T cell states and their cell-to-cell interactions with the tumor microenvironment across response and tumor progression and identified mechanisms linking CAR-T cell functionality and therapeutic failure. How this study might affect research, practice or policyThis study provides comprehensive mechanistic insights from an immunocompetent model that can be leveraged to identify shared determinants of CAR-T cell functionality in solid tumors and potentially guide the rational development of improved CAR-T cell therapies.

Dendritic cells (DCs) are central orchestrators of antitumor immunity. Several DC subsets--including conventional type 1 (cDC1), conventional type 2 (cDC2), plasmacytoid DCs (pDCs), and mature DC populations--play distinct roles in immune surveillance, tumor control, immunotherapy response and prognosis. Recent findings suggest that cDC1 are spatially closed to CD8 T-cell and contribute to tertiary lymphoid structure formation in lung cancer. However, how other DC subsets interact with cDC1 to shape the tumor microenvironment (TME) remains largely unknown. Here, we analyzed the spatial distribution of major DC subsets, including cDC1, cDC2, mature DC and pDC, together with CD8 T cells in a cohort of anti-PD1-treated NSCLC patients and we deciphered the corresponding immune microenvironment behavior by paired transcriptomic analysis. We found that, while other DC subsets populated the stroma, cDC2 were localized both in the stroma and in tumor nests. Moreover, unlike other DC subsets, cDC2 abundancy did not affect ICB response both at transcriptomic and in situ analysis. We described spatial organization of DCs in megaclusters characterized by distinct proportions of DC subsets. Patients enriched in megaclusters involving variable proportion of pDC, cDC1 and mature DC, exhibited pro-inflammatory transcriptomic programs while those enriched in cDC2-based megaclusters showed limited immune activation features. Globally, DC in lung cancer were structured around three distinct DC spatial patterns, namely cDC1-driven, cDC2-driven and DC-Scattered, each defined by unique compositions of DC megaclusters, immune features and pathways activation profiles. Among them, the cDC1-driven pattern was associated to prolonged anti-PD1 response in two independent cohorts.

PurposeBladder cancer (BLCA) is a heterogeneous tumor type. Only one third of muscle-invasive (MIBC) patients respond to immune checkpoint inhibitors (ICIs). Reliable resistance markers are needed to guide clinical decisions. We investigated the nerve growth factor receptor (NGFR) in BLCA and analyzed its correlation with disease progression and response to immunotherapy. Experimental DesignWe analyzed NGFR expression in BLCA cell lines, organoids, mouse models and patient samples. The cohorts used were The Cancer Genome Atlas (TCGA), enriched in muscle-invasive bladder cancer (MIBC) (n=407); IMvigor210, representing MIBC patients treated with ICIs (n=348); and UROMOL2, as a non-muscle-invasive bladder cancer (NMIBC)-specific cohort (n=535). IMvigor010 was also included (n=728). Patients were stratified by NGFR expression quartiles. We analyzed survival and tumor subtypes and performed stromal deconvolution and functional profiling. We assessed stemness- and invasion-related features in SCaBER cells. ResultsNGFR marks a basal tumor cell subcluster and is independently associated with poor prognosis in TCGA and IMvigor210. NGFR-high tumors show stromal content enriched in cancer-associated fibroblasts, lower neoantigen burden, higher CD8+ T effector signature together with an immune-excluded phenotype, and a CAF-specific TGF{beta} signature. In the immunotherapy-treated cohort, high NGFR expression was also associated with poorer outcome. Functionally, NGFR appears to promote a stem-like/pro-invasive program in BLCA cells. ConclusionsNGFR identifies a basal-like BLCA subpopulation linked to poor survival, while its association with immunotherapy response requires further validation. In addition, our in vitro analyses support a role of NGFR in stem-like and invasive traits, highlighting its relevance as a biomarker in BLCA.

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm with very heterogeneous clinical and biological behavior. Among molecular variables, TP53 alterations are well-established adverse prognostic markers; however, MYC activation, which has been linked to disease progression, has not been completely defined in terms of clinical and biological impact, particularly in relation to TP53 status. Here, we investigated the effects of MYC overexpression according to TP53 status using clinical and transcriptomic data from CLL patients and novel cellular models. CLL patients with TP53WT and MYC overexpression exhibited significantly shorter time to first treatment and overall survival, indicating an aggressive disease course comparable to that of patients with TP53 alterations. Consistently, MYC overexpression in in vitro TP53WTmodels was associated with increased proliferation, enrichment of AKT/mTOR signaling and upregulation of genes involved in leukemogenesis and tumor progression such as FOXO6. Moreover, MYC overexpression was associated with increased sensitivity to venetoclax in TP53WT cells. By contrast, the concurrence of MYC overexpression and TP53 dysfunction conferred resistance to conventional CLL therapies such as BCL2 or BTK inhibitors. Of note, we identified a glycolysis inhibitor, in monotherapy or combined with BKT inhibitors, as a potential therapeutic strategy for CLL patients harboring MYC overexpression and TP53 alterations.

Background: Non-invasive diagnosis, reliable recurrence surveillance remain critical unmet needs in gliomas. Glioma induces profound systemic immune alterations despite its anatomical confinement to the central nervous system. Circulating immune cells, particularly monocytes, are key mediators of tumor-host crosstalk and may retain tumor-induced transcriptional imprints. However, their potential clinical utility as blood-based biomarkers for detection and monitoring, remain largely unexplored. Methods and findings: In this study, we performed integrated single-cell RNA sequencing of blood immune cells and demonstrated that circulating CD14+ monocytes are significantly expanded in glioma patients, exhibiting features of differentiation arrest and increased transcriptional plasticity. These cells harbor glioma-specific molecular signatures distinct from those observed in healthy controls and patients with other tumors. Leveraging these findings, we developed an ensemble machine learning diagnostic model based on transcriptomic profiles of circulating CD14+ monocytes (training cohort, n=107), which achieved a mean area under the receiver operating characteristic curve (AUC) of 0.971 during cross-validation. In an independent cohort of 567 participants, the model maintained high diagnostic accuracy, yielding an AUC of 0.877 for distinguishing glioma from controls and other tumors. And it achieved a recurrence detection AUC of 0.969 in 51 postoperative samples. Moreover, in a prospective follow-up study involving 30 glioma patients, lower model-derived scores of postoperation were significantly associated with prolonged progression-free survival (log-rank test, P=0.043), supporting its prognostic utility. Conclusion: We demonstrate circulating CD14+ monocytes undergo glioma-specific transcriptional reprogramming, generating systemic tumor-associated signal captured via transcriptomic profiling. This blood-based diagnostic model provides non-invasive, scalable approach for glioma detection, recurrence surveillance, outcome prediction.

BackgroundPrimary and secondary resistance to immune checkpoint blockade (ICB) remains a critical challenge in advanced melanoma. Oncolytic Viruses (OV) selectively lyse tumor cells while generating systemic anti-tumor immune responses with minimal side effects. Yet their clinical use is limited to refractory melanoma patients and are only given in combination with second-line ICB regimens. ICB can both help and hinder OV efficacy depending on the source of checkpoint interactions across the tumor-immune microenvironment (TiME). However, functional checkpoint interactions are typically inferred from gene or protein expression and rarely contextualized within myeloid- and antigen presenting cell-associated immune niches during OV therapy, despite these populations dominating melanoma TiMEs and serving as key regulators of anti-viral immunity. MethodsAn integrated multi-omics framework combining Nanostring GeoMx digital spatial profiling (DSP), COMET sequential immunofluorescence (seqIF) and functional oncology mapping (FuncOmap) was applied to melanoma patient tissues collected pre- and post-neoadjuvant Talimogene Laherparepvec (T-VEC) to characterize immune remodeling and directly quantify checkpoint interaction dynamics associated with pathologic responses to OV therapy. ResultsT-VEC induced broad lymphocyte- and myeloid-associated immune transcriptional activation across melanoma TiMEs; however, pathologic responses could not be defined by bulk transcriptomics or cellular deconvolution alone. COMET seqIF analysis identified that HSV-associated M1/APC-like tumor-associated macrophages (TAMs) were enriched in complete pathologic response (CR) tissues and were a major source of PD-1/PD-L1 interaction niches. While partial (PR) and non-pathologic response (NR) tissues retained melanoma-centered PD-1/PD-L1 interaction niches and were enriched for B cell and M2-like TAM populations. FuncOmap analysis indicated that post-T-VEC PD-1/PD-L1 interaction states were consistently elevated in tumor bed, but not in lymph node tissues, across all pathologic response groups. Suggesting that immune checkpoint interactions may benefit T-VEC therapeutic responses depending on their spatial and immune context relative to OV infection. ConclusionsThese findings highlight the importance of integrated transcriptomic and functional proteomic analyses for resolving the spatial distribution and functional status of immune niches during OV therapy. Resolving PD-1/PD-L1 interaction states to specific M1/APC-like TAM and B cell niches may define mechanisms of responses and resistance to OV therapy.

Immune checkpoint inhibition (ICI) is clinically active against multiple cancers, including urothelial cancer at the non-muscle invasive, muscle-invasive, and metastatic stages. Despite this, large numbers of patients experience disease progression and relapse after treatment with ICI-containing regimens. Tumor antigen-specific T cells are critical to ICI response, however few studies have evaluated the breadth and magnitude of tumor antigen-specific T cell responses with ICI therapy. In this study, we mapped the tumor antigen immunodominance hierarchy in the BBN963 model of murine basal-like bladder cancer for endogenous tumor neoantigens expressed physiologically. We used a high-throughput matrixed ELISpot assay to detect CD8+ T cell responses to predicted BBN963 tumor antigens derived from multiple mutational genomic sources. We found CD8+ T cell responses were directed against a subset of tumor antigens forming a stable and reproducible immunodominance hierarchy across individual mice. Treatment with anti-PD-1 or anti-CTLA-4 did not substantially reshape this hierarchy or broadly shift dominant responses to previously defined subdominant epitopes. Predicted peptide MHC binding stability and affinity was associated with antigen immunogenicity. Cancer-testis antigens, endogenous retroviral antigens, and SNV-derived tumor antigens that were immunogenic were found across tumor subclones. By diversifying the immunogenic antigen repertoire beyond SNVs, we achieved nearly 100% tumor subclone coverage, suggesting that broader antigen selection could help immunotherapy target more tumor subclones. In conclusion, this study supports the stability of the immunodominance hierarchy under ICI therapy and a role for broadening antigen discovery to multiple expressional sources in immunotherapy design.

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: CD8+ tumor-infiltrating lymphocytes (TILs) are established prognostic markers in colorectal cancer, yet the clinical significance of CD103+CD8+ tissue-resident memory-like (TRM-like) T cells in locally advanced rectal cancer (LARC) after neoadjuvant chemoradiotherapy (NACRT) remains unknown. Methods: We quantified CD8+ and CD103+CD8+ T-cell densities in stromal and intratumoral compartments of post-NACRT resection specimens from 40 LARC patients using Cu-Cyto, a deep learning-based imaging cytometry platform. Associations with survival, pathological response, and adjuvant chemotherapy (AC) were examined. Treatment-induced T-cell dynamics were assessed in paired pretreatment biopsies and post-NACRT resections (n = 9). Results: High stromal CD103+CD8+ density independently predicted better 5-year RFS (67.4% vs. 12.1%, p < 0.001) and OS (80.0% vs. 26.6%, p = 0.016); intratumoral density showed no prognostic significance. Pathological response correlated with stromal CD8+ but not CD103+CD8+ density. Paired analysis revealed a selective non-expansion of the CD103+ subset: stromal CD8+ T cells increased significantly after NACRT while CD103+CD8+ density remained unchanged. AC may preferentially benefit patients with low stromal CD103+CD8+ density. Conclusions: Stromal CD103+CD8+ T-cell density is a robust independent prognostic biomarker in rectal cancer after NACRT that appears to reflect pre-existing rather than treatment-induced immunity. Given its stability across NACRT, pretreatment biopsy assessment may provide equivalent prognostic information, with potential implications for patient stratification before treatment initiation.

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.

The standard-of-care for patients with higher-risk non-muscle invasive bladder cancer (NMIBC) after tumour resection is intravesical administration of Bacillus Calmette-Guerin (BCG). While this form of adjuvant immunotherapy has improved recurrence-free and progression-free survival, a large proportion of patients experience recurrences within a year of diagnosis. The reasons for this high rate of early recurrence following BCG therapy remain unclear; however, inadequate activation of systemic immunity may be a contributing factor. To address this, we analysed the transcriptomic and chromatin accessibility profiles of peripheral blood mononuclear cells obtained from patients with NMIBC at single-cell resolution before BCG immunotherapy and after five induction doses of BCG. Monocytes from patients who experienced disease recurrence within a year of initiation of BCG therapy (BCG non-responders) exhibited a pro-inflammatory phenotype consistent with age-related immunosenescence prior to BCG immunotherapy. Moreover, inflammation-associated pathways that were active before initiation of BCG therapy in the BCG non-responders were down-regulated after five instillations of BCG. In contrast, these pathways were quiescent before BCG therapy in patients who remained disease-free for at least a year but were markedly up-regulated after five doses of BCG. Genomic regions with accessible chromatin were enriched in activator protein 1 (AP-1) binding sequences in monocytes from BCG-non-responders prior to BCG therapy. AP-1 is a central regulator of the inflammatory phenotype associated with immunosenescence. Our findings indicate that a pre-existing state of innate immunosenescence underlies early disease recurrence following BCG. Patients unlikely to benefit from BCG may be offered alternative therapies early in their disease journey. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/723215v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1f7c844org.highwire.dtl.DTLVardef@7cea65org.highwire.dtl.DTLVardef@1008d23org.highwire.dtl.DTLVardef@131f973_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: The use of immune checkpoint inhibitors (ICIs) in the treatment of cancer has rapidly expanded over the last decade. However, there are several knowledge gaps in understanding how tumor cells evade the immune system. There is paucity of data in HPV negative oral cancer, particularly of the gingivobuccal region. Understanding the mechanism of immune system evasion in this cancer is vital for improving patient outcomes. Methods: We characterized the baseline immune milieu of oral cancer using immunohistochemistry (IHC) on whole tumor sections from 124 cases. Tumors were classified as hot or cold and further stratified into high-risk and low-risk groups. High-risk patients included those with lymph node metastasis at diagnosis/recurrence or distant metastasis within 2 years of treatment completion. Patients without these features were categorized as low risk. Validation by RNA-Seq and Joint Enrichment Analysis of Oncogenic and Immunologic Pathways was carried out in a subset of 46 cases. Results: Hot high-risk tumors (by IHC) were distinguished by elevated PD-L1 expression and reduced NK-cell, PD1, and CTLA-4 expression. There was no difference in the expression levels of CD3+, CD8+, granzyme, or perforin compared to hot low-risk tumors, findings that align with the definition of hot tumors. RNA-Seq revealed a gene signature associated with exhausted T-cells in hot high-risk tumors. Gene and pathway analyses identified differential upregulation of isoform-specific TOX, TCF, CXCR, RUNX, IRF, BRD and BCL6 genes, implicating immune cell exhaustion and tumor aggressiveness. Significantly downregulated genes included PDCD1, HAVCR2, ZAP70, and STAT, indicative of a disabled immune microenvironment. These findings support that a state of immune exhaustion in HHR tumors is driven by progenitor exhausted T-cells and terminally exhausted T-cells; independent of PD1-TIM3. Conclusion: These findings suggest that combining TOX/TCF/BCL6 inhibitors with immune checkpoint inhibitors in the adjuvant setting might benefit patients with hot high-risk tumors. Given the results, testing for a targeted exhaustion-related gene panel at diagnosis is recommended for oral cancers to stratify tumors as high-risk or low-risk. Larger validation studies and clinical trials are now warranted.