Tumor-secreted SPP1 activates CD44-Hedgehog signaling in hepatic stellate cells to promote fibrosis

Background: Advanced hepatic fibrosis is a pivotal event leading to cirrhosis and hepatocellular carcinoma (HCC). We identified osteopontin (SPP1) as a fibrosis-associated gene through transcriptome and immunohistochemical analyses and investigated its role in intratumoral fibrosis and HCC development. Methods: Gene expression analysis was conducted using publicly available datasets to screen for fibrosis-related genes. Immunohistochemical staining was performed on HCC samples to assess SPP1 expression and its correlation with fibrosis and prognosis. The biological effects of SPP1 overexpression (SPP1-OE) in HCC cells were examined in xenograft mouse models. Co-culture assay with hepatic stellate cells (HSCs) and single-cell RNA sequencing (scRNA-seq) analysis were used to explore HCC-HSC interactions. Results: Transcriptomic and prognostic analyses revealed that SPP1 was significantly upregulated in fibrotic HCC tissues and associated with unfavorable outcomes. Immunohistochemical analysis confirmed a strong correlation between SPP1 expression and intratumoral fibrosis. In xenograft models, SPP1-OE HCC cells exhibited enhanced tumor growth and extensive fibrosis. Co-culture assay demonstrated that SPP1-OE cells stimulated HSCs, and gene set enrichment analysis and differential gene expression analysis elucidated activation of the Hedgehog signaling pathway and upregulation of GLI1 in HSCs. scRNA-seq analysis discovered SPP1-CD44 signaling transduction as a key mediator of HSC activation. Pharmacological inhibition of GLI1 with the SMO inhibitor vismodegib suppressed HSC activation in vitro and reduced fibrosis and tumor growth in vivo. Conclusions: 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 Hedgehog signaling may provide a promising strategy to mitigate fibrosis and improve HCC patient outcomes.