Cancer-associated Fibroblasts Induce The Stemness Characteristics Of Cancer Stem Cells In Hepatocellular Carcinoma Via Notch Signaling

Objectives: Human hepatocellular carcinoma(HCC)is the fifth most prevalent cancer and serious threat to the life and health of our people.The main reason is the high rate of recurrence and metastasis after surgery,which is related to the heterogeneity of liver cancer and the existence of a small amount of cancer stem cells(CSC).Previous studies have indicated that cancer cells may acquire a CSC phenotype through dedifferentiation in the tumour microenvironment(TME).Cancer-associated fibroblasts(CAFs)are important components of tumor microenvironment.They can induce the stemness of cancer cell.It was demonstrated that tumor microenvironment associate cells regulated the function of cancer cells via Notch signaling.We have previously demonstrated that Notch signaling promotes CSC characteristics in CD90+ cells in HCC and CAFs can activite signaling in HCC cells.Does CAFs participate in the induction and maintenance of stemness in HCC cells? How does the Notch signaling play a role in the stemness of HCC cells induced by CAFs? Our study intends to clarify the mechanisms of how CAFs activate Notch signaling to induce the stemness of HCC cells.CAFs constitute a supporting niche for cancer stemness,and targeting this paracrine signaling in tumor microenvironment may present a new therapeutic strategy for HCC cancer stem cells.Methods: CAFs were isolated from fresh HCC tissues and characterized with the α-SMA,FAP and vimentin by western blot and immunofluorescence.Then,we utilized transwell co-culture to examine the functional role of CAFs in regulating the stem cell-like properties of HCC cells.We evaluated the effect of CAFs on the stemness of HCC cells by sphere formation assay,colony formation assays,matrigel invasion and transwell migration assay and tumor formation assay.The expression of stem cell related genes(Nanog,Sox2 and OCT4)and Notch signaling components(Notch1,NICD and Hes1)were tested by western blot and q RT-PCR analysis.Additionally,the cytokines and signaling pathways that mediate CAFs-induced stem cell-like properties in HCC cells will be screened by Human cytokine array,Elisa,western blot and q RT-PCR analysis.Moreover,we will clarify the key role of Notch signaling pathway in the process of CAFs inducing the stemness of HCC cells using lentivirus knockout and pharmacologic blockage.Moreover,we will use immunohistochemistry(IHC)to detect the the different expression of α-SMA and the main components of Notch signaling in clinical HCC tissue compared with adjacent non-tumor counterparts in 88 HCC patients,and analyzed the correlation between α-SMA,NICD with the clinic-pathological prognosis.Results:(1)CAFs enhanced the sphere-forming,colony-forming,tumour-formation and migration and invasion abilities of MHCC-97 H cells and HLE cells.Stem cell related genes(Nanog,Sox2 and OCT4)were overexpressed in MHCC-97 H and HLE cells co-cultured with CAFs.Mice injected with MHCC-97 H and PLC/PRF/5 cells in combination with CAFs generated 3-and 5-fold higher mean tumor volume than control mice.(2)Human Cytokine Array assay showed that CAFs secreted high amounts of IL-6.ELISA assay also showed that MHCC-97 H cells co-cultured with CAFs secreted higher amounts of IL-6 than MHCC-97 H cells alone.IL-6 increased sphere and colony formation as well as migration and invasiveness of MHCC-97 H cells,and Western blot analysis demonstrated that IL-6 up-regulated Nanog,Sox2,and Oct4 protein levels in MHCC-97 H cells.(3)IL6 up-regulated Notch signaling pathway components(Notch1 and Hes1)in MHCC-97 H cells.CAFs or IL6-induced sphere-forming,colony-forming,tumour-formation and migration and invasion abilities of MHCC-97 H cells were reduced when Notch signalling was blocked by sh NOTCH1.Knockdown of Notch1 abolished CAFs or IL6-induced overexpression of Notch signalling components(Notch1,NICD,and Hes1)and stemness-associated genes(Nanog,Sox2,and Oct4)in MHCC-97 H cells.(4)Cryptotanshinone decreased phospho-STAT3 levels and expression of Notch signaling-associated components(Notch1,NICD,and Hes1)in MHCC-97 H cells treated with IL-6.Furthermore,inhibition of STAT3 phosphorylation decreased sphere and colony formation as well as migration and invasion of MHCC-97 H cells treated with IL-6.Cryptotanshinone also reduced liver tumor growth in NOD/SCID mice injected with MHCC-97 H cells and IL-6.(5)There was a strong correlation between the high α-SMA expression in CAFs and high expression of n NICD in HCC cells.High expression of n NICD in HCC tissues strongly correlated with venous infiltration(P < 0.0001)and lymph node metastasis(P = 0.003).Hence,high n NICD expression indicated poor prognosis in HCC patients.Conclusions: In this study,we found that CAFs induce expression of stemness-associated transcription factors such as Nanog,Sox2 and Oct4 in HCC cell lines.CAFs modulated stem cell-like properties of HCC cells by secreting IL-6,which activated Notch signaling via STAT3 phosphorylation.Moreover,high nuclear expression of NICD in tumor cells correlated with poor prognosis of HCC patients.Therefore,we postulate that CAFs promote HCC progression by modulating IL-6/STAT3/Notch signaling.The tumour microenvironment involves a complicated mixture of tumour cells within the extracellular matrix(ECM)and various types of stromal cells.This study improves the regulatory mechanisms of cancer stem cells from the perspective of tumor microenvironment and cancer stem cells,and is expected to provide new ideas for the complete eradication of liver cancer stem cells and prevention of liver cancer.Due to limited time,we only explored the role of IL6,the most secreted cytokine of CAFs,in the induction of the stemness in HCC cells.Further studies are needed to clarify the combined effects of other CAF-related cytokines and multiple cells in the tumor microenvironment on the regulation of stem cell-like characteristics of HCC cells.