Cancer-associated Fibroblasts Promote The Stemness Of Hepatocellular Carcinoma Cells Via Secreting STC1

Objective: Liver cancer stem cells(LCSCs)are the initial driving force of metastasis,relapse,and drug resistance of hepatocellular carcinoma(HCC).One of the core signal pathways to promote and maintain HCC stemness is the Notch1 signaling pathway.Cancerassociated fibroblasts(CAFs)are the important components of the tumor microenvironment(TME),which can remodel the TME and regulate tumor stemness.Stanniocalcin-1(STC1)is a secreted glycoprotein in the TME,which is closely related to CAFs and cancer stemness.However,the relationships between STC1,CAFs,Notch1 signaling pathway and HCC stemness are still unclear.This study aims to explore the role of CAFs and STC1 in HCC stemness,with a view to finding new strategies for HCC therapy.Methods: We analyzed the single-cell sequencing data of HCC by bioinformatics analysis and combined ELISA,Western Blot(WB),and immunofluorescence(IF)to explore the relationship between CAFs and STC1 in HCC.The effects of CAFs and STC1 on the stemlike biological behavior and the expression of Nanog,OCT4,and SOX2 of HCC cells were detected by sphere formation assay,sorafenib resistance assay,colony-formation assay,transwell migration and invasion assay,mouse orthotopic liver xenograft tumor model,and WB.The role of STC1 in CAF-induced stemness promotion was also explored by STC1 neutralizing antibody(STC1 Ab)and lentivirus.We explored the signaling pathway through which STC1 regulated the HCC stemness by bioinformatics analysis,pathway inhibitor,lentivirus,immunohistochemistry(IHC)and,co-immunoprecipitation(Co-IP)technology.Then,regulate the expression of Notch1 in HCC cells and detect the change in STC1 expression.The specific site of the Notch1 signaling pathway regulating STC1 expression was clarified through dual-luciferase reporter assay.Finally,tissue microarray IHC and ELISA were used to detect the relationships among STC1,Notch1 signaling pathway,and prognosis of HCC patients.Results: The expression and secretion levels of STC1 in CAFs were significantly higher than that in normal fibroblasts and HCC cells.CAFs and STC1 could promote HCC stemness and the promotion effect of CAFs on HCC stemness could be inhibited by STC1 Ab and lentivirus.CAFs and STC1 could activate the Notch1 signaling pathway and the effect of STC1 on HCC stemness could be inhibited by pathway inhibitor and lentivirus.STC1 and Notch1 were positively correlated in HCC samples.Co-IP showed that STC1 directly bound the Notch1 molecule.Transcription factor CSL bound to the-152/-143 bp region of STC1 promoter directly.The results of IHC showed that STC1 and the Notch1 signaling pathway were highly expressed in HCC,and the highly expressed STC1 and Notch1 signal were related to the clinicopathological characteristics and poor prognosis of HCC patients.The level of serum STC1 in patients with advanced HCC was significantly higher than that in patients with early HCC.Conclusions: This study confirmed that CAFs could promote the stemness of HCC cells through the STC1-Notch1-STC1 axis mediating the continuously amplified STC1 high expression and the Notch1 signaling activation in the TME,which was expected to become a new target for the treatment of HCC.