Fig 2: SCAFs promote cancer stemness via the SOX4-CTHRC1-Notch1 axis. A GSEA revealed enrichment of the Notch pathway from the Enricher database in the group with high-CTHRC1 expression in the TCGA-LIHC cohort. B, C Western blot analysis of Notch1, NICD, Hes1, and Hey1 expression in HCC cells, respectively. D Western blot analysis of Notch1, NICD, Hes1, and Hey1 expression in mice tumor tissues, respectively. E Migration and invasion capabilities assessed by Transwell assay in indicated cells. Scale bar, 20 μm. F, G Self-renewal capacity evaluated through sphere formation assay in indicated cells. Scale bar, 100 μm. H, I Statistical analysis of Transwell migration and invasion assays. J, K Statistical analysis of sphere formation assays. L Venn diagram of predicted transcription factors. M Correlation analysis between selected transcription factors, CTHRC1 expression, and the CSscore. N Representative immunofluorescence images showing p21 and SOX4 expression. Scale bar, 20 μm. O, P qRT-PCR (O) and western blot analysis (P) of SOX4 expression in indicated cells, respectively. Q–T qRT-PCR (Q, S) and western blot analysis (R, T) of SOX4 and CTHRC1 in indicated cells, respectively. U qRT-PCR was used to detect the SOX4 in the ChIP assay. Data presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical significance

Fig 3: CTHRC1 is overexpressed in SCAFs. A qRT-PCR analysis of the expression of 10 crucial genes. B Comparative mRNA expression of SERPINE1, CTHRC1, and MARCKSL1 across diverse cell types in the GSE149614 dataset. C Correlation between SERPINE1, CTHRC1, and MARCKSL1 expression and senescence score determined by ssGSEA in the TCGA-LIHC dataset. D The association between SERPINE1, CTHRC1, and MARCKSL1 expression and senescence score calculated via AddModuleScore in the GSE149614 dataset. E The secreted concentrations and intracellular expression levels of CTHRC1 in CAFs and SCAFs were measured by ELISA. F Representative immunofluorescence images for p21 and CTHRC1 in CAFs and SCAFs. Scale bar, 20 μm. G Western blot analysis of CTHRC1 expression in CAFs, SCAFs, and tumor tissue. H Representative mIHC images showed expression of α-SMA, p21, and CTHRC1 in HCC. Scale bar, 20 μm. Data presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical significance

Fig 4: High expression of CTHRC1 is associated with HCC malignancy and clinical prognosis. A Comparative mRNA expression analysis of CTHRC1 across 16 cohorts from the HCCDB database. B CTHRC1 protein expression levels quantified from the CPTAC database. C CTHRC1 mRNA expression in HCC patient samples (n = 10). D Western blot analysis of CTHRC1 protein expression in HCC patient samples. E Representative IHC images of HCC and adjacent normal tissues. Scale bar, 100 μm. F Correlation analysis between CTHRC1 and Notch1/NICD protein expression levels. G CTHRC1 expression data from the UALCAN TCGA database demonstrating association with advanced tumor grade and clinical stage. H, I Kaplan-Meier survival analyses showing correlation between elevated CTHRC1 expression in HCC tissues and reduced overall survival from our cohort (H) and GEPIA database (I), respectively. Data presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical significance