Tumor cells with cholangiocytic phenotype form oncogenic niches with fibroblasts in hepatocellular carcinoma

Background: A subset of hepatocellular carcinoma (HCC) exhibits cholangiocytic features associated with aggressive behavior, poor prognosis, and treatment resistance, but the underlying mechanisms remain unclear. We investigated the characteristics of duct-like (DL) tumor cells and their interactions with fibroblasts in HCC. Methods: Single-cell RNA sequencing data from 40 primary HCC samples were integrated to identify tumor subpopulations and signaling networks. Spatial transcriptomic datasets were analyzed to assess colocalization of DL cells and fibroblasts. Functional studies were performed using liver cancer cell lines cultured on collagen I or treated with cancer-associated fibroblast (CAF)-conditioned medium, followed by molecular analyses. Results: A distinct DL tumor cell population expressing cholangiocytic and stem-like markers was identified. DL cells showed activation of Wnt, MAPK, Notch, and Hippo pathways, whereas non-duct-like cells retained hepatocytic metabolic programs. Fibroblasts preferentially interacted with DL cells through extracellular matrix ligands, especially collagens and fibronectin, binding integrins and syndecans. Spatial transcriptomics demonstrated close colocalization of DL cells and fibroblasts in regions with high oncogenic pathway activity. SPP1 emerged as a major mediator of DL-to-fibroblast signaling. In vitro, collagen I and CAF-conditioned medium promoted cholangiocytic features and suppressed hepatocytic differentiation. Conclusion: DL tumor cells form a reciprocal oncogenic niche with fibroblasts that promotes HCC progression and therapeutic resistance