Investigating the role of the TGF-β–SLC20A1 axis in the spatial heterogeneity of hepatocellular carcinoma through single-cell and spatial transcriptomics

Background: Hepatocellular carcinoma (HCC) is a highly heterogeneous malignancy characterized by marked cellular and spatial diversity within the tumor microenvironment (TME). The transforming growth factor-β (TGF-β) signaling pathway plays a dual role in the initiation and progression of HCC. However, the spatial distribution characteristics and key regulatory mechanisms of TGF-β signaling within HCC tissues remain inadequately elucidated. Methods: This study integrated spatial transcriptomics (ST), single-cell RNA sequencing (scRNA-seq), and bulk RNA-seq data to systematically characterize the spatial heterogeneity of the TGF-β signaling pathway in HCC. By combining non-negative matrix factorization (NMF), CellChat-based cell-cell communication analysis, and multi-algorithm machine learning approaches, we identified key driver genes closely associated with TGF-β activity. Subsequently, CCK-8 assays, colony formation, wound-healing, and Western blot experiments were performed in HCC cell lines to validate the biological functions of the identified gene. Results: The results revealed that the TGF-β signaling pathway exhibited the highest activity at the tumor-stroma interface, which was enriched with cancer-associated fibroblasts (CAFs), immunosuppressive cells, and genes related to extracellular matrix (ECM) remodeling. CellChat analysis showed that TGF-β-TGFBR ligand-receptor interactions between tumor cells, CAFs, and immune cells were markedly enhanced, contributing to the formation of a localized immunosuppressive microenvironment. Machine learning analysis identified SLC20A1 as a key regulatory factor. Functional assays demonstrated that SLC20A1 enhances the proliferation, migration, and epithelial-mesenchymal transition (EMT) of HCC cells, whereas its knockout significantly suppresses these malignant phenotypes. Conclusion: This study represents the first comprehensive integration of spatial and single-cell transcriptomics to uncover the spatial organization of TGF-β signaling in HCC and to identify the TGF-β-SLC20A1 axis as a critical driver of tumor invasion at the tumor-stroma interface. Our findings provide new mechanistic insights into tumor-stroma interactions and suggest a potential therapeutic target for precision treatment of HCC.