Single-cell profiling reveals lineage-specific fibroblast stromal subtypes drive ECM remodeling and immune modulation in the hepatocellular carcinoma tumor microenvironment.

HCC is characterized by extensive ECM remodeling, primarily mediated by stromal cells. While cancer-associated fibroblasts are known contributors to tumor fibrosis, their transcriptional diversity and role in immune modulation within the HCC tumor microenvironment remain poorly resolved. Integrative analysis of six public HCC scRNA-seq datasets was employed to identify ECM-active stromal cells. High-ECM cells were re-clustered and fibroblast subtypes were defined through differential expression, pathway enrichment, lineage scoring and cell-cell interaction modeling. Spatial transcriptomic mapping and tissue-level profiling were performed to validate and prioritize ECM-related genes for experimental analysis. COL1A1 and COL3A1 were selected for siRNA-mediated knockdown in HepG2 and HepB3 cells followed by RT-qPCR, Western blot, proliferation, colony formation and wound healing assays. ECM activity analysis identified fibroblasts as the most ECM-enriched stromal population. Reclustering of ECM-high stromal cells identified eight distinct subtypes and targeted fibroblast clustering revealed five functionally diverse states. GO and spatial transcriptomic analysis confirmed subtype-specific functions and localization. In silico tissue profiling further prioritized COL1A1 and COL3A1 as pan-mesenchymal ECM genes enriched in fibrogenic fibroblast subsets. CellChat analysis revealed myofibroblasts and inflammatory CAFs as dominant signal senders. Knockdown reduced COL1A1 and COL3A1 expression at both mRNA and protein levels, and enhanced HCC cell proliferation, migration and colony formation. This study identifies transcriptionally and functionally distinct fibroblast subtypes in HCC and highlights COL1A1 and COL3A1 as key matrix-regulatory genes expressed in fibrogenic stromal subsets.