The Effect And Mechanism Of GC-MSCs Stimulated By CD4+T Cells On Gastric Cancer Growth

Objective : Mesenchymal stem cells(MSCs)and immune cells as important components in tumor microenvironment,which are closely related to the growth of tumor.It is not well known that tumor associated MSCs stimulated by immune cells could promotes tumor development.This study focuses on the roles and underlying molecular mechanisms of gastric cancer tissues-derived mesenchymal stem cells-like cells(GC-MSCs)stimulated by CD4+Tcells on the growth of gastric cancer.Methods : CD4+T cells were activated by CD3 and CD28 monoclonal antibody,which were isolated from peripheral blood mononuclear cells(PBMC)of healthy donor by magnetic bead sorting.GC-MSCs were cocultured with CD4+T cells for 24 h and then remove CD4+T cells to collect conditional medium.Gastric cancer cells SGC-7901 were treated with culture supernatant from GC-MSCs stimulated by CD4+T cells.The migration of treated SGC-7901 were detected by Transwell,the apoptosis of treated SGC-7901 were tested by flow cytometry analysis using Annexin V/PI double staining.After GC-MSCs stimulated by CD4+T cells the expression of PD-L1 in GC-MSCs was examined by immunofluorescence,flow cytometry,western blot,q RT-PCR.At the same time,the p-stat3 signaling pathways related to PD-L1 expression was detected by western blot.GC-MSCs pretreated with p-stat3 inhibitor to further observe the expression of PD-L1 in GC-MSCs stimulated by CD4+Tcells.The change of the inflammatory cytokines related to tumor growth in GC-MSCs,including IL-6,IL-8,CCL2,TGF-β,IDO were examined by q RT-PCR.The surface antigens of GC-MSCs stimulated by CD4+Tcells was tested by flow cytometry analysis.Results: Culture supernatant from GC-MSCs stimulated by CD4+T cells promote the migration of gastric cancer cells in vitro,but have no effect on the apoptosis of gastric cancer cells.The growth curve analysis of subcutaneous xenografts showed that GC-MSCs stimulated by activated CD4+T cells have more potent in promoting the growth of gastric cancer cells in vivo than GC-MSCs.The main consequence of GC-MSCs stimulated by CD4+T cells is the upregulation of PD-L1,which is mediated by p-stat3 signaling pathways.Inhibiting the activity of p-stat3 in GC-MSCs can reduce the the expression of PD-L1 in GC-MSCs stimulated by CD4+T cells.In additon,several inflammatory molecules related to tumor growth in GC-MSCs,including IL-6,IL-8,CCL2,TGF-β,IDO also increased.The surface antigens of GC-MSCs stimulated by CD4+Tcells were not changed by flow cytometry analysis.Conclusion: GC-MSCs stimulated by CD4+T cells can significantly promote the development of gastric cancer,which may related to PD-L1 and inflammatory cytokine.This research will provide new experimental evidence for tumor associated MSCs stimualted by immune cells in promoting the tumor development.