Spatial Transcriptomics and snRNA‐seq Expose CAF Niches Orchestrating Dual Stromal‐Immune Barriers in Hepatocellular Carcinoma

HmmyCAFs may form a triple immunosuppressive niche: possibly secrete ECM (POSTN, etc.) as physical barriers to block CD8⁺ T cells, induce CD8⁺ T exhaustion via those molecules, and use HIF‐1α‐driven metabolism to create acidic, nutrient‐poor microenvironments that suppress T cells. Abstract Hepatocellular carcinoma (HCC) exhibits profound spatial heterogeneity driving therapeutic resistance, while the role of cancer‐associated fibroblasts (CAFs) in orchestrating immunosuppressive niches remains incompletely defined. This study integrates single‐nucleus RNA sequencing (snRNA‐seq) and spatial transcriptomics (stRNA‐seq) to map the cellular and molecular landscape of HCC. snRNA‐seq identifies key cell populations—including fibroblasts, T_NK cells, and endothelial cells—using canonical marker genes. Spatial transcriptomics maps gene expression across tumor regions (core, invasive front, stroma) via the robust cell type decomposition (RCTD) algorithm. Immunofluorescence validates collagen deposition and POSTN spatial distribution, confirming T‐cell exclusion patterns. The analysis identifies hypoxic metabolic myofibroblasts (hmmyCAFs) as central regulators of the tumor microenvironment. hmmyCAFs enrich at the invasive front, forming collagen‐rich barriers that physically exclude CD8⁺ T cells. Simultaneously, they secrete POSTN to suppress immune checkpoint signaling and drive hypoxia‐mediated glycolytic reprogramming of T‐cell metabolism. Clinically, hmmyCAF activity and POSTN expression correlate with reduced progression‐free survival and immunotherapy resistance. This multimodal study defines hmmyCAFs as triple architects of physical immunosuppression, molecular regulation, and metabolic remodeling. By linking collagen remodeling, POSTN‐mediated checkpoint inhibition, and hypoxia‐driven metabolic reprogramming to clinical outcomes, hmmyCAFs and POSTN may serve as potential indicators for evaluating the efficacy of immunotherapy in HCC. HmmyCAFs may form a triple immunosuppressive niche: possibly secrete ECM (POSTN, etc.) as physical barriers to block CD8⁺ T cells, induce CD8⁺ T exhaustion via those molecules, and use HIF-1α-driven metabolism to create acidic, nutrient-poor microenvironments that suppress T cells. Abstract Hepatocellular carcinoma (HCC) exhibits profound spatial heterogeneity driving therapeutic resistance, while the role of cancer-associated fibroblasts (CAFs) in orchestrating immunosuppressive niches remains incompletely defined. This study integrates single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptomics (stRNA-seq) to map the cellular and molecular landscape of HCC. snRNA-seq identifies key cell populations—including fibroblasts, T_NK cells, and endothelial cells—using canonical marker genes. Spatial transcriptomics maps gene expression across tumor regions (core, invasive front, stroma) via the robust cell type decomposition (RCTD) algorithm. Immunofluorescence validates collagen deposition and POSTN spatial distribution, confirming T-cell exclusion patterns. The analysis identifies hypoxic metabolic myofibroblasts (hmmyCAFs) as central regulators of the tumor microenvironment. hmmyCAFs enrich at the invasive front, forming collagen-rich barriers that physically exclude CD8⁺ T cells. Simultaneously, they secrete POSTN to suppress immune checkpoint signaling and drive hypoxia-mediated glycolytic reprogramming of T-cell metabolism. Clinically, hmmyCAF activity and POSTN expression correlate with reduced progression-free survival and immunotherapy resistance. This multimodal study defines hmmyCAFs as triple architects of physical immunosuppression, molecular regulation, and metabolic remodeling. By linking collagen remodeling, POSTN-mediated checkpoint inhibition, and hypoxia-driven metabolic reprogramming to clinical outcomes, hmmyCAFs and POSTN may serve as potential indicators for evaluating the efficacy of immunotherapy in HCC. Advanced Science, Volume 12, Issue 48, December 29, 2025.