Targeting SPP 1‐ CD 44‐Hedgehog Axis Elicits Therapeutic Effects in Hepatocellular Carcinoma by Suppressing Intratumoral Fibrosis

Advanced hepatic fibrosis is a major risk factor for cirrhosis and hepatocellular carcinoma (HCC), and it is required to identify a key mediator involved in intratumoral fibrosis and HCC development. Transcriptomic analysis of 372 HCC samples using publicly available datasets revealed that SPP1 was significantly upregulated in fibrotic HCC tissues and associated with unfavorable outcomes. Immunohistochemical analysis of 103 HCC tissues and single-cell RNA sequencing (scRNA-seq) analysis of 228,564 live cells identified SPP1 overexpression in HCC cells, which strongly correlated with intratumoral fibrosis. In xenograft models, HCC cells with SPP1 overexpression (SPP1-OE) exhibited enhanced fibrosis and tumor growth. Coculture assay demonstrated that SPP1-OE cells stimulated hepatic stellate cells (HSCs), and gene set enrichment analysis and differential gene expression analysis elucidated the activation of the Hedgehog signaling pathway and upregulation of GLI1 in HSCs. Cell-cell interaction prediction analysis using scRNA-seq data suggested that SPP1-CD44 signaling transduction might contribute to HSC activation. Pharmacological inhibition of GLI1 with the SMO inhibitor vismodegib suppressed HSC activation in vitro and reduced fibrosis and tumor growth in vivo. These findings indicate that SPP1 promotes intratumoral fibrosis and HCC progression through the SPP1-CD44-GLI1 axis, highlighting its potential as a prognostic biomarker and therapeutic target. Inhibition of SPP1-CD44-Hedgehog signaling may provide a promising strategy to mitigate fibrosis and improve HCC patient outcomes.