Aug 2026· Diseases of the esophagus· Vol 39· 0 citations
TL;DR
NRF2-driven immune exclusion is relevant to immunotherapy resistance, while EMT-driven co-evolution with COL10A1+COL11A1+ myCAFs induces desmoplastic reactions induces desmoplastic reactions, which provide new insights into immune evasion mechanisms and metastatic timing in ESCC.
Abstract
Esophageal Cancer: Molecular Biology/Pathology
Spatial transcriptomic profiling was performed using the Xenium Prime 5K platform on treatment-naïve ESCC primary tumors (n=10) spanning mucosal to deep submucosal invasion, matched metastatic lymph nodes (n=11), and normal tissues (n=3). After QC filtering, 8,284,705 cells were classified into nine major cell types. Cancer cells were subclustered per sample using consensus non-negative matrix factorization (cNMF) to delineate spatially distinct subpopulations. To link transcriptomic phenotypes with clonal architecture, LCM-based whole genome sequencing was performed on 20 regions from 3 patients, enabling phylogenetic reconstruction across primary tumors and matched metastatic lymph nodes.
Cancer cell analysis revealed two independent axes of variation. The first axis stratified tumors into NRF2-high and NRF2-low subtypes regardless of invasion depth. NRF2-high tumors showed reduced immune infiltration, consistent with NRF2 pathway enrichment in non-responders from an independent neoadjuvant immunochemotherapy cohort. The second axis reflected invasion depth-dependent progression, where deeper tumors acquired EMT features alongside the emergence of COL10A1+COL11A1+ myCAFs that drove desmoplastic reactions at the tumor-stromal interface. Metastatic lymph nodes showed two distinct stromal patterns: those with desmoplastic reactions and those without. Phylogenetic reconstruction revealed that metastatic lymph nodes lacking desmoplastic reactions branched early from primary tumor clones, consistent with dissemination prior to stromal remodeling.
Integrating spatial transcriptomics with LCM-based WGS, we identified two axes underlying ESCC heterogeneity. NRF2-driven immune exclusion is relevant to immunotherapy resistance, while EMT-driven co-evolution with COL10A1+COL11A1+ myCAFs induces desmoplastic reactions. Phylogenetic analysis confirms that metastatic lymph nodes without desmoplastic reactions arose through early dissemination. These findings provide new insights into immune evasion mechanisms and metastatic timing in ESCC.
Integrating spatial transcriptomics with LCM-based WGS, we identified two axes underlying ESCC heterogeneity. NRF2-driven immune exclusion is relevant to immunotherapy resistance, while EMT-driven co-evolution with COL10A1+COL11A1+ myCAFs induces desmoplastic reactions. Phylogenetic analysis confirms that metastatic lymph nodes without desmoplastic reactions arose through early dissemination. These findings provide new insights into immune evasion mechanisms and metastatic timing in ESCC.
Clear cell renal cell carcinoma (ccRCC) is a highly heterogeneous cancer with complex tumor and immune microenvironment interactions influencing progression and therapy response. Advances in spatial technologies now allow for simultaneous spatial mapping of transcriptomic and proteomic features from patient tissue and phenotyping of blood cells, enabling detailed assessment of disease biology. This study highlights a multi-omic approach to characterizing systemic immune responses, tumor microenvironment niches, immune states and tumor heterogeneity in ccRCC patient samples.
We profiled ccRCC patient samples using peripheral blood mononuclear cells (PBMC) and tumor-derived cells (TDCs) by CyTOF™ technology with a 50-plus-antibody panel. Matched formalin-fixed tumor tissue was examined by a spatial transcriptomic platform, hematoxylin and eosin (H&E) assessment, and Imaging Mass Cytometry™ (IMC™) technology with a 43-marker panel sequentially on the same tissue section. These datasets were analyzed to explore immune and tumor signatures linked to disease states.
We demonstrate compatibility of IMC workflows with H&E and spatial transcriptomic modalities. IMC technology identified spatial distribution and activation states of immune and tumor cells in ccRCC. Combining spatial transcriptomics and proteomics enabled detailed phenotyping of tumor metabolic and signaling states and immune cytokine and transcription factor expression. Comparison of PBMC and TDC composition provided insights into systemic and localized immune responses.
This study highlights the power of spatial multi-omic profiling to unravel the immune and oncologic landscape of ccRCC with high resolution. Identification of spatially defined immune cell states and niches offered insights into tumor immune evasion and resistance mechanisms. These findings pave the way for spatially informed biomarkers and potential precision therapies for ccRCC. For Research Use Only. Not for use in diagnostic procedures.
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Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Michael J. Cohen, Q. Raza, N. Zabinyakov et al.· Journal of Immunology· 0 citations
Background Bladder cancer (BLCA) is a common malignancy, with muscle-invasive bladder cancer (MIBC) associated with a 5-year survival rate below 50%. Cancer-associated fibroblasts (CAFs) are heterogeneous stromal components of the tumor microenvironment (TME) that contribute to tumor progression, therapy resistance, and immune evasion. However, the molecular basis of CAF diversity and immune regulation in BLCA remains incompletely understood. Methods We analyzed single-cell transcriptomic data from GEO dataset GSE135337 using dimensionality reduction, unsupervised clustering, differential expression analysis, GO/KEGG/GSEA, diffusion pseudotime inference, and LIANA ligand–receptor analysis. TCGA-BLCA bulk transcriptomic data (n = 408 total; n = 401 after stage filtering) were used for score validation. RT–qPCR was performed to assess selected myCAF- and iCAF-associated genes in RT4 and T24 bladder cancer cells. Results Analysis of 9,253 post-QC cells across two non-paired specimens (6,035 adjacent-tissue; 3,218 tumor) yielded 9 DE-marker-annotated cell populations. Among 1,923 retained adjacent-tissue fibroblasts, two transcriptionally distinct states were identified: Homeostatic fibroblasts (n = 1,197; enriched for COL1A1, DCN) and CCL2-high activated-like fibroblasts (n = 726; CCL2 log2FC = 0.806, FDR = 8.08 × 10−11). No fibroblast-like cells were retained in the tumor specimen. Diffusion pseudotime positioned CCL2-high cells at a later within-sample state (median DPT 0.813 vs. 0.672; FBLN1 rho = −0.808). Exploratory LIANA analysis identified 1,347 fibroblast-outgoing ligand–receptor interactions. A pre-specified six-gene myCAF score (ACTA2, COL1A1, MMP11, MYL9, TAGLN, TPM2) tracked pathological stage in TCGA-BLCA (P = 1.60 × 10−12) and showed an unadjusted overall survival association (HR = 1.21, 95% CI: 1.02–1.42, P = 0.025) that attenuated to non-significance after age and stage adjustment (HR = 1.03, P = 0.748); the association was not replicated in GSE31684 (n = 93; log-rank P = 0.443). RT–qPCR confirmed higher expression of ACTA2, POSTN, MMP11, FAP, IL6, and CXCL12 in T24 versus RT4 cells (4.33–16.56-fold; all BH-adjusted q < 0.01). Conclusion Single-cell transcriptomics of publicly available BLCA data identifies two transcriptionally distinct fibroblast states in adjacent tissue and supports an exploratory ligand–receptor interaction framework. A six-gene myCAF-associated score tracks pathological stage but does not independently predict overall survival after covariate adjustment and was not replicated in an independent cohort. These findings constitute an exploratory computational framework warranting prospective validation with primary CAF populations and adequately powered multi-specimen cohorts.
Yan-Dong He, Wen-Long Lu, Guan-Qun Ju et al.· Frontiers in Cell and Develo...· 0 citations
An integrated single-cell atlas defines a critical POSTN+ CAF–APOE+ macrophage unit that is associated with a fibrotic and immunosuppressive TME in advanced prostate cancer, and suggested conserved enrichment and adverse prognostic impact of this stromal-immune axis across diverse tumor types, though tissue-specific context should be considered.
Yangzhou Liu, Ao-Chu Liu, Xing-Lai Dai et al.· Frontiers in Immunology· 0 citations
BACKGROUND AND AIMS
Peritoneal metastases (PMs) represent the most frequent, clinically challenging dissemination pattern in advanced gastric cancer, especially poorly cohesive (PCGC) subtype, and are associated with poor prognosis. We aimed to characterize the transcriptomic landscape of primary gastric tumors (PGTs) and matched PMs to identify differential molecular programs, tumor-microenvironment (TME) features, and potential biomarkers.
METHODS
We analyzed 55 FFPE samples from 23 treatment-naïve patients with synchronous peritoneal-only stage IV PCGC and 10 non-neoplastic gastric controls (NNC). RNA sequencing was analyzed using DESeq2 and GSEA, with cell proportions estimated through immune deconvolution (TIMER/xCell).
RESULTS
A total of 4279 differentially expressed genes were identified across all groups. CLDN18 expression progressively decreased from NNCs to PGTs and PMs. Direct PGT-PM comparison revealed that PGTs were characterized by cytoskeletal and extracellular-matrix remodeling genes upregulation, mitotic/cell-cycle, NOTCH and apical-junction pathways enrichment, and memory T-cell predominance. Compared with PGTs, PMs showed increased IGF1, IGFN1, NTRK2 and adipogenesis-related transcripts, adipogenesis and MAPK7/11-NTRK2 signaling enrichment, and macrophage-dominant and dendritic-cell-dominant microenvironments. epithelial-mesenchymal transition (EMT), inflammatory pathways, and cancer-associated fibroblasts were shared by both tumor lesions. PMs did not match established TCGA or PCGC subtypes, suggesting a distinct molecular identity.
CONCLUSIONS
PMs are characterized by TME remodeling and transcriptional reprogramming. Shared EMT and inflammatory pathways act as bridge linking PGT to PM, consistent with adaptive exploitation of physiological peritoneal programs. Collectively, our findings suggest a transcriptomic framework in which PM can be characterized as a distinct niche-conditioned biological entity and support microenvironment-informed diagnostic and therapeutic strategies.
M. Bencivenga, M. Simbolo, Stefano Gobbo et al.· Annals of Surgery· 0 citations
BACKGROUND
Hypopharyngeal squamous cell carcinoma (HPSCC) is a rare, highly aggressive malignancy, with recurrence playing a pivotal role in treatment failure and cancer-related mortality. The tumor microenvironment (TME) characteristics and molecular mechanisms driving recurrence in HPSCC remain poorly defined.
MATERIALS AND METHODS
Primary HPSCC (P-HPSCC) samples (n = 6) and recurrent HPSCC (R-HPSCC) samples (n = 3) were analyzed. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics were employed to dissect the cellular composition and molecular characteristics of the TME. Key regulatory networks were identified through gene expression analysis, pathway enrichment, and intercellular communication profiling. Functional validation experiments were performed to confirm the biological relevance of the pathways identified.
RESULTS
ScRNA-seq analysis revealed that malignant epithelial cells (maEpCs) in recurrent tumors exhibited increased stemness, epithelial-mesenchymal transition (EMT), and immune evasion programs. Mechanistically, IGF2BP2 was selectively upregulated in R-HPSCC and functioned as an m6A reader to stabilize SMAD3 mRNA, thereby activating transforming growth factor beta (TGF-β) signaling and facilitating malignant progression. Integrated spatial transcriptomics and functional assays demonstrated that cancer-associated fibroblasts in recurrent tumors promote progression via FN1-mediated signaling. Concurrently, the recurrent TME underwent myeloid remodeling toward an immunosuppressive state, characterized by increased infiltration of SPP1+ tumor-associated macrophages and further enrichment of LAMP3+ dendritic cells with impaired antigen-presenting capacity, alongside aggravated CD8+ T-cell exhaustion, marked by upregulation of CTLA4 as a potential immune escape driver.
CONCLUSIONS
This study identifies recurrence-associated cellular and molecular features in HPSCC. Recurrent tumors exhibited maEpC reprogramming via IGF2BP2-mediated activation of TGF-β signaling, coupled with FN1-dependent stromal activation and immune evasion marked by myeloid immunosuppressive remodeling and CD8+ T-cell exhaustion, characterized by CTLA4 upregulation. Collectively, these findings reveal a pro-recurrent TME and suggest potential therapeutic targets in R-HPSCC.
Z. Cai, Jinhong Zhang, Yaodong Ding et al.· Cellular & Molecular Biology...· 0 citations
Understanding primary tumor features predicting post-recurrence immunotherapy response in resectable stage III non-small cell lung cancer (NSCLC) could guide adjuvant strategies. We hypothesized that spatial profiling of surgical specimens would reveal microenvironmental determinants of programmed death ligand-1 (PD-L1)-discordant responses to subsequent immunotherapy.
Surgical specimens from eight stage III NSCLC patients with post-recurrence progression were analyzed by Xenium 5K spatial transcriptomics. Patients were stratified into four PD-L1-high (≥50%) non-responders and four PD-L1-negative (< 50%) responders based on primary tumor PD-L1 expression and post-recurrence immune checkpoint inhibitor (ICI) response. Analyses included pathway enrichment, T cell exhaustion mapping, fibroblast subtyping, spatial neighborhoods, and NicheNet inference.
Non-responders displayed elevated glycolysis and hypoxia pathways indicating metabolic reprogramming, while responders did not. Spatial analysis revealed T cell exhaustion proximal to tumors in non-responders with marked attenuation in responders. Fibroblast subtyping identified metabolically active CAFs (meCAFs) enriched adjacent to glycolytic tumors in non-responders, with tighter tumor-meCAF spatial coupling. NicheNet identified meCAF-derived VEGFA as a key signaling mediator targeting tumor cells, with predicted targets including ACKR3, PTGS2, and PLAU, implicating angiogenic-metabolic crosstalk driving immune exclusion.
Spatial profiling reveals that PD-L1-discordant ICI responses are determined by coordinated interactions of metabolically reprogrammed tumors, localized T cell exhaustion, and meCAF-driven signaling. These findings establish meCAF-VEGFA-tumor crosstalk as a therapeutic vulnerability and support spatially-resolved tumor-stroma profiling as a prognostic tool beyond PD-L1 for predicting ICI responses.
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Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Yongjae Kim, Seong-Eun Kim, Bokyung Ann et al.· Journal of Immunology· 0 citations
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