Study On The Regulation Of GCMSCs On PD-L1 To Promote Gastric Cancer Progress And Its Mechanism

Objective: The expression of Programmed death-ligand 1(PD-L1)is one of the main causes of gastric cancer(GC)immune escape.However,the exact mechanism for regulating PD-L1 expression in GC cells remains unclear.Mesenchymal stem cells(MSCs)have attracted much attention as a cell component in tumor microenvironment(TME)due to their broad immunosuppressive potential and the abilities to promote angiogenesis,tumor growth,epithelial-mesenchymal transition(EMT)and metastasis.This present study is designed to investigate GCMSCs regulates the PD-L1 expression in GC cells to promote tumor progression and explore the specific molecular mechanism.Methods: GCMSCs were isolated by adherent method.The characterization of GCMSCs was determined by surface markers(CD105,CD90 and CD29)and differentiation assays in vitro.The expression of PD-L1 in GC tissues and adjacent normal tissues was detected by Immunohistochemistry(IHC).The PD-L1 levels in different GC cell lines were tested by Western blot.Conditioned medium of bone marrow MSCs(BMMSC-CM)and conditioned medium of GCMSCs(GCMSC-CM)were collected and then treated GC cells for 24 h.The PD-L1 level was tested by Flow cytometry(FCM)and Western blot and the secretion of soluble PD-L1(s PD-L1)was detected by ELISA.Cytotoxicity assay was used to evaluate the effects of GCMSCCM on the resistance of GC cells to cytotoxicity.Immunofluorescence(IF)was used to compare the differences between GCMSCs and GCMSC-CM in up-regulation of PDL1 expression in GC cells and the key molecule in GCMSC-CM which regulated PDL1 in GC cells was screening by Cytokine array.The mechanism of GCMSC-CM promoting PD-L1 expression in GC cells was further clarified by using cytokine neutralizing antibodies,signal pathway inhibitors and cytokine standards.A combination of IL-8 and PD-L1 neutralizing antibodies was used to observe the effects of GCMSCs on the resistance of GC cells to cytotoxicity.The stem cell-like properties of GC cells were evaluated according to the expression of stemness markers(CD44,Sall4,Nanog,Oct4 and Sox2),ALDH activity,migration and sphere formation abilities.GC cells were transfected with PD-L1 lentivirus(p LV-PD-L1-puro-GFP)or si RNAPD-L1 to observe the effects of PD-L1 on GC cells stemness.The intracellular molecules that binded to PD-L1 and regulated the stemness of GC cells were analyzed by Mass spectrometry and which was further verified by Co-Immunoprecipitation(Co- IP).PD-L1 Negative and PD-L1 Positive cells were sorted by FCM and using for limiting dilution assays to verify the effect of PD-L1 on tumorigenic ability of GC cells in vivo.Results: GCMSCs were isolated from GC tissues successfully.Differentiation assay showed that GCMSCs were able to differentiate into adipocytes and osteocytes in vitro.Immunophenotype analysis displayed that GCMSCs were positive expression of CD105,CD90 and CD29;Nevertheless,negative expression of CD45,CD34 and CD19.The results have shown that GC tissues expressed higher PD-L1 than corresponding adjacent normal tissues in GC patients.The levels of PD-L1 in several kinds of GC cell lines are different and which was higher in SGC-7901 but lower in MGC-803.Compared with BMMSC-CM,GCMSC-CM had stronger ability to up-regulated the expression of PD-L1 in GC cells.GCMSC-CM also promoted the secretion of s PD-L1 by GC cells.The level of PD-L1 was up-regulated in GC cells treated with GCMSCCM for different time and the level of PD-L1 was higher at 24 h.The influence of GCMSCs and GCMSC-CM on PD-L1 up-regulation in GC cells was similar.Cytokine array and cytokine neutralizing antibodies further proved that GCMSC-CM promoted the expression of PD-L1 in GC cells mainly through the secretion of IL-8.GCMSCCM promoted c-Myc and PD-L1 expression in GC cells and the PD-L1 level decreased when c-Myc was inhibited.The inhibition of c-Myc also reversed the promoting effects of GCMSC-CM on GC cells resistance to CD8+T cells cytotoxicity.IL-8 derived from GCMSCs induced PD-L1 expression in GC cells via c-Myc regulated by activating STAT3 and m TOR signal pathways.The results have shown that PD-L1 neutralizing antibody enhanced the sensitivity of SGC-7901 to CD8+T cells,but had little effect on GCMSC-CM promoting the resistance of GC cells to cytotoxicity.However,this resistance was significantly attenuated after the combination blockage of PD-L1 with IL-8.Further studies proved that GCMSC-CM enhanced the expression of stemness markers(CD44,Sall4,Nanog,Oct4 and Sox2),ALDH activity,migration and sphere formation abilities of GC cells.However,these effects were greatly reduced when PDL1 in GC cells were blocked.The down-regulation of PD-L1 in GC cells reduced the expression levels of stemness markers,and the effects of GCMSC-CM on the stemness of GC cells were also significantly weakened.At the same time,the expression of stemness markers,the migration and sphere formation abilities were significantly enhanced in GC cells with PD-L1 overexpressed.Mass spectrometry analyses indicated that PD-L1 in GC cells was binded to the transcription factor CCCTC-binding factor(CTCF),which was stemness-related and involved in tumor cell self-renewal.Co-IP assay was further used to validate the interaction between PD-L1 and CTCF in GC cells.Interfering with the expression of CTCF significantly reduced the levels of stemness markers,diminished the ALDH activity,migration and sphere formation abilities in GC cells.GC cells were treated with GCMSC-CM and PD-L1 Negative and PD-L1 Positive cells were sorted which were using for limiting dilution assays.The results proved that PDL1 Positive GC cells harbored higher stemness and tumorigenicity.Conclusions: GCMSC-CM up-regulated PD-L1 expression in GC cells via IL-8-STAT3/m TOR-c-Myc signal axis and promoted the resistance of GC cells to CD8+T cells cytotoxicity.Combined blocking of IL-8 with PD-L1 effectively inhibited the effects of GCMSCs in promoting GC cells to resist cytotoxic CD8+T cells killing.GCMSCs regulating PD-L1-CTCF to enhance cancer stem cell-like(CSC-like)properties of GC cells.Compared with PD-L1 Negative GC cells,PD-L1 Positive GC cells have stronger ability to promote the occurrence and development of GC.This study provided a new target for the diagnosis and improving the efficiency of immunotherapy in GC,as well as a potential strategy to alleviate therapeutic resistance in GC patients.