Low SPP1 and PLAUR expression correlated with poor prognosis in TCGA-PAAD and two key LR pairs mediated crosstalk between cancer cells and CD8+ Tex cells, confirmed by immunofluorescence, spatial mapping, and protein docking.
Abstract
Summary Pancreatic cancer resists immunotherapy due to a suppressive immune microenvironment where CD8+ T cells play a key role. Using single-cell RNA sequencing and spatial transcriptomics, we characterized CD8+ exhausted T (Tex) cells in pancreatic ductal adenocarcinoma (PDAC). We generated single-cell profiles from PDAC tumors and matched peripheral blood mononuclear cells, and performed T cell sub-analysis. We found CXCL13 upregulated and GZMK downregulated in CD8+ Tex cells. Cell-cell interaction analysis showed that T cells most frequently interacted with myeloid cells and cancer cells via ligand-receptor pairs; INHBA+ macrophages and cancer cells communicated most with CD8+ Tex cells. Two key LR pairs (SPP1-integrin α4β1 and PLAUR-integrin α4β1) mediated crosstalk between cancer cells and CD8+ Tex cells, confirmed by immunofluorescence, spatial mapping, and protein docking. High SPP1 and PLAUR expression correlated with poor prognosis in TCGA-PAAD. These findings provide a resource for understanding CD8+ T cell exhaustion in PDAC.
This study identified UBE2C+ proliferative tumor cells as a functionally relevant malignant subpopulation in SCLC and links this state to immune-stromal communication networks within the tumor microenvironment and provides a systems-level framework for investigating the cancer-immunity regulome in SCLC.
Hong-Ling Jia, Yongxuan An, Bing Chen et al.· Frontiers in Immunology· 0 citations
CXCL13+ T cells and LAMP3+ dendritic cells (DCs) are pivotal players in orchestrating anti-tumor immune responses, particularly within tumor tertiary lymphoid structures (TLS). However, their heterogeneity, differentiation trajectories, and clinical relevance in bladder cancer remain incompletely defined. This study integrated single-cell RNA sequencing (scRNA-seq) data (16 bladder cancer patients, 113,905 post-quality-control cells) and spatial transcriptomics to characterize CXCL13+ T cell/LAMP3+ DC subsets, their differentiation pathways (via Velocyto trajectory analysis), and intercellular crosstalk (via receptor-ligand mapping). A risk model (DTscore) was constructed using marker genes of these cells and validated in the IMvigor210 (atezolizumab-treated bladder cancer) and TCGA-BLCA cohorts. scRNA-seq clustering identified 10 immune and 3 nonimmune cell types, with T cells stratified into 8 subpopulations (including CD4+CXCL13+ T cells and CD8+CXCL13+ T cells) and DCs into 9 subgroups (including LAMP3+ DCs). Receptor-ligand mapping and spatial transcriptomics confirmed functional crosstalk between CXCL13+ T cells and LAMP3+ DCs via key pairs (e.g., CCR7-CCL19, CXCR5-CXCL13, PDCD1-CD274) within TLS. The DTscore was developed using 8 marker genes (TSHZ2, ALOX5AP, GADD45G, TXN, CHN1, CCL19, CXCL13, ICA1) and exhibited robust prognostic and predictive performance: In the IMvigor210 cohort, high DTscore correlated with significantly poorer overall survival (OS) and a 3.27-fold lower immunotherapy response rate (11% vs. 36%, p = 4.23e-07); multivariate Cox regression confirmed DTscore as an independent OS predictor (hazard ratio = 1.97, p < 0.001). DTscore retained prognostic value in TCGA-BLCA (OS: p = 0.003; disease-specific survival: p < 0.001) and effectively predicted atezolizumab response even in the "immune desert" phenotype (p = 0.04). Combining DTscore with tumor mutational burden/tumor neoantigen burden yielded an AUC of 0.8122 for response prediction. Additionally, high DTscore was associated with higher OS hazard ratios in patients with wild-type TTN, RB1, EP300, or FGFR3 (all p < 0.01), while FGFR3 mutations correlated with lower immune checkpoint/CXCL13 expression. This study delineates the heterogeneity and interactions of CXCL13+ T cell/LAMP3+ DC subsets in bladder cancer TLS and validates DTscore as a robust tool for predicting OS and immunotherapy response, offering a potential guide for personalized bladder cancer treatment.
Lin Zhou, Jinchao Zhu, Yushan Liu et al.· Molecular Carcinogenesis· 0 citations
Colorectal cancer liver metastasis (CRLM) develops within a liver-specific immune and stromal microenvironment, yet the natural killer (NK)-cell adaptations associated with this metastatic niche remain incompletely defined.
We integrated human single-cell RNA sequencing, an exploratory murine cross-species comparison, spatial transcriptomics, bulk transcriptomic cohorts, donor-level pseudobulk analyses, cell-cell communication, pseudotime and in silico perturbation analyses, together with multiplex immunofluorescence, conditioned-medium assays, ELISA and immunoblotting.
Across 73,486 cells from six anatomical sites, NK cells showed lower relative representation within the recovered T/NK compartment in primary tumors and liver metastases, while the residual compartment showed inflammatory remodeling. NK-cell reclustering identified CD56-associated and CD16-associated CCL4 source clusters that were analyzed jointly as a CCL4-enriched NK-cell transcriptional state characterized by NF-κB, TNF, MAPK and JAK-STAT programs. In paired GSE164522 samples from 10 patients, this state was independently recovered, with nominally higher CCL4-associated signal in liver metastases than in primary tumors that did not remain significant after Benjamini-Hochberg correction. Spatial and communication analyses associated the state with SPP1-positive macrophage and stromal programs without establishing causality. Tissue imaging, conditioned-medium assays, ELISA and immunoblotting provided complementary marker-level and model-system support for CCL4-associated inflammatory activity and NF-κB pathway responsiveness.
CRLM is associated with altered NK-cell representation among recovered cells and a reproducible CCL4-enriched inflammatory NK-cell transcriptional state. Its association with inflammatory and myeloid-stromal programs across independent, spatial and model-system analyses provides a focused framework for future lineage-restricted and
in vivo
studies.
Li-Hong Lin, Michael Xu, Xiao-Chun Gu et al.· Frontiers in Immunology· 0 citations
Abstract Background Clear cell renal cell carcinoma (ccRCC) is characterized by a highly heterogeneous tumor immune microenvironment. However, how tumor-intrinsic programs shape immune cell states remains incompletely understood. We aimed to identify multicellular programs (MCPs) linking tumor and immune compartments and to define tumor-derived signals that modulate immune phenotypes. Methods We applied DIALOGUE to identify MCPs across tumor and non-tumor compartments, including CD8+ T cells, CD4+ T cells, regulatory T cells, myeloid cells, endothelial cells, and fibroblasts. A total of 189,999 cells from 26 patients with sufficient tumor cell representation (>100 cells) were analyzed from our previously reported cohort (Kashima, ASCO 2024). MCP scores were derived from DIALOGUE and summarized at the sample level by averaging single-cell MCP scores. Cell–cell interactions were evaluated using CellChat and NicheNet. Spatial relationships were assessed using G4X spatial transcriptomic data (Yochum, AACR Kidney 2026). Spatial niches were defined using Seurat v5 based on k-nearest neighbor composition, and annotated as stromal, sarcomatoid, or clear cell according to cell-type composition and gene expression features. Results We identified two major MCPs, including tumor–myeloid (MCP1) and tumor–CD4+ T cell (MCP2) programs. The tumor component of MCP1 was enriched for mesenchymal and extracellular matrix (ECM)-related genes, including structural components and remodeling factors (COL5A2, MMP2, MRC2), as well as integrin-associated adhesion and cytoskeletal genes (ITGA3, ITGB1, VCL). Expression of HMGA2 further supported a dedifferentiated, EMT-like state. Clinically, a high MCP1 score was associated with sarcomatoid features (p = 0.04). In the myeloid compartment, MCP1 was characterized by immunoregulatory and tissue-remodeling markers (MRC1, MARCO, VSIG4, MS4A4A), chemokines (CCL13, CCL18, CCL23), and complement-related genes (C2, CFH, CR1), consistent with an M2-like macrophage phenotype. Cell–cell interaction analysis revealed increased communication between MCP1-high tumor cells and MARCO+ and CXCL9+ macrophage populations. Mechanistically, ECM-related signaling pathways, particularly collagen and laminin interactions, were predicted to activate CD44-mediated signaling in macrophages. These findings suggest an ECM–CD44 axis linking tumor mesenchymal programs to M2-like polarization. Spatial transcriptomic analysis further demonstrated preferential accumulation of macrophages in stromal-rich regions. Quantitative comparison across stromal, sarcomatoid, and clear cell regions showed increased macrophage infiltration in stromal and sarcomatoid areas compared to clear cell regions (3900 and 1920 vs 830 cells/mm², respectively). Differential expression analysis of macrophages across these regions showed that macrophages in sarcomatoid areas upregulated immunoregulatory and M2-associated markers, including CD163, MRC1, CSF1R, and TGFB1. CD44 expression was also significantly increased, supporting activation of ECM–CD44-associated signaling. Response to immune checkpoint inhibitors (ICIs) was evaluable in 13 patients. Although MCPs were not significantly associated with response, patients with higher myeloid MCP1 score (> mean), but not tumoral MCP1 score, showed a numerically higher response rate compared to those with low MCP1 score (4/4 vs 5/9 patients). Of note, all 4 responders in the myeloid MCP1-high group received combination therapy with CTLA-4 and PD-1 blockade. This suggests myeloid MCP1 may be associated with response to combination ICI therapy. Conclusions Our study identifies a tumor–immune MCP in which mesenchymal and ECM remodeling programs in tumor cells are associated with M2-like macrophage polarization. These findings highlight an ECM–CD44-associated signaling axis as a potential mechanism by which tumor cells shape an immunosuppressive microenvironment in ccRCC.
Katsuhiro Ito, S. Kashima, Rishabh Rout et al.· The Oncologist· 0 citations
Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly immunosuppressive tumour microenvironment (TME), which contributes to its resistance to immunotherapy. Although mast cells (MCs) have been implicated in PDAC progression, their functional heterogeneity and candidate signaling models of immune modulation remain poorly understood. Methods Through an integrated analysis of multiple single-cell RNA sequencing (scRNA-seq) datasets, we identified a significant enrichment of MCs in PDAC tissues and established a characteristic gene signature (TPSAB1, TPSB2, CPA3, HPGDS, KIT, LTC4S) for their precise identification. Results Single-cell RNA-seq analysis categorized MCs in PDAC into resting, activated, and proliferating subpopulations. To functionally validate these transcriptomic predictions, in vitro co-culture experiments showed that pancreatic cancer cells promote the activation and proliferation of MCs. Cell–cell communication analysis suggested that activated MCs preferentially interact with regulatory T cells (Tregs) via the MIF–(CD74/CXCR4) signaling axis. This interaction was associated with an immunosuppressive T-cell landscape, characterized by an expanded population of Tregs exhibiting a highly activated immunosuppressive phenotype. Spatial transcriptomics and immunofluorescence validated the confirmed the spatial proximity of MCs and Tregs in PDAC tissues. Clinically, high expression of MC-Treg signature genes correlated with poor patient survival. Conclusions Our study suggests that MCs are key orchestrators of immunosuppression in PDAC, predicted to interact with Tregs through the MIF–CD74/CXCR4 axis, offering a novel rationale for targeting the MC–Treg axis in future immunotherapeutic strategies.
Mengli Wu, Xuxia Ye, Qi Yu et al.· Annals medicus· 0 citations
Exhausted CD8+ T cells (Tex) within the tumor microenvironment (TME) represents a critical barrier limiting anti-tumor immune responses. Tex cells are characterized by upregulated inhibitory immune checkpoint receptors, reduced cytotoxicity, and functional heterogeneity. Their genomic features and regulatory networks remain poorly defined, and only a minority of patients respond to immune checkpoint blockade (ICB) therapy. Single-cell RNA sequencing (scRNA-seq), through high-resolution transcriptomic profiling, has revealed diverse Tex subpopulations, identified subpopulation-specific marker genes and regulatory pathways. Spatial transcriptomics has further mapped the spatial distribution of Tex and their interaction networks with immune cells, tumor cells, and stromal cells, elucidating the impact of spatial heterogeneity on Tex functionality. Current studies indicate that the exhausted state of Tex is dynamic and modifiable, with functional differences among subpopulations closely associated with tumor progression and therapeutic response. However, the genomic characteristics, epigenetic regulation, and spatial interaction mechanisms of Tex require further exploration. This review summarizes recent advances in high-resolution omics technologies for precisely dissecting Tex heterogeneity, functional features, and interactions with other cells. It emphasizes the central value of optimizing Tex-targeted tumor immunotherapy strategies, providing theoretical foundations and directional guidance for developing more effective anti-tumor immunotherapies.
Zi-Xuan Gou, Xiao-Jun Huang, Xiang-Yu Zhao· Cancer Letters· 0 citations
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