| Objective:Chemotherapy resistance in advanced gastric cancer(GC)patients has largely limited the effectiveness of therapy,resulting in disease recurrence and poor prognosis.Stromal cells in tumor microenvironment,especially mesenchymal stem cell(MSC),play significant roles in cancer progress and immune escape.Gastric cancer derived mesenchymal stem cell(GCMSC)is widely believed to promote GC invasion,metastasis and immune escape via up-regulating programmed death ligand 1(PD-L1).However,the mechanism by which PD-L1 mediated by GCMSC might regulate the chemoresistance is unknown in GC.This study aims to explore the role and mechanism of GCMSC-mediated PD-L1 in chemotherapy resistance of GC,providing potential therapeutic schedule to improve therapeutic efficacy in the clinic.Methods:Primary GCMSC was isolated from GC tissues and cultured in vitro.The cell phenotype of GCMSC was detected by flow cytometry and the adipogenic and osteogenic differentiation ability of GCMSC were detected.Half maximal inhibitory concentrations(IC50)of HGC-27 and SGC-7901 cells against chemotherapy drugs cisplatin(DDP),5-fluorouracil(5-FU)and paclitaxel(PTX)were determined by CCK-8 assay.sh RNA,lentivirus transfection,PD-L1 neutralizing antibody,si RNA,17-AAG,and B02 were used to alter the expression of PD-L1,CCCTC-binding factor(CTCF),Decapentaplegic homolog 4(Smad4),Heat shock protein 90(HSP90),and DNA repair related protein Rad51 in gastric cancer cells,respectively.GCMSC conditioned medium(GCMSC-CM)was collected for co-culture with GC cells.The survival rate,apoptosis rate,expression of PD-L1 and other related proteins of GC cells were detected by CCK-8,Annexin V/PI apoptosis analysis,western blot,q RTPCR and immunofluorescence.The intracellular localization of PD-L1 and other proteins in GC cells was observed by western blot analysis and immunofluorescence.The clone formation rate of GC cells was detected by colony-forming assay.Xenograft tumor BALB/c nude mice model were established with regular peritumoral injection of GCMSC-CM and intraperitoneal injection with drugs.Tumor growth was observed and recorded.Immunohistochemistry and western blot were used to detect the expression of related proteins in tumor tissues.GC tissue samples with chemotherapy were collected and made into paraffin sections.The expressions of PDL1 and other related molecules were detected and analyzed by immunohistochemical staining.Results:(1)GCMSC was isolated and identified.After co-culture with GCMSC-CM,GC cells were treated with DDP,5-FU and PTX.We observed higher survival rate,lower apoptosis rate,higher clone formation rate,and higher expression of PD-L1,multidrug resistance related proteins MDR1,LRP and tumor stemness related proteins CD44 and OCT4.The results suggested that the promotion effect of GCMSC-CM on chemotherapy resistance may be related to PD-L1.(2)We observed increased cell survival rate,decreased apoptosis,enhanced clone formation ability,and increased protein expressions of MDR1,CD44 and OCT4 in GC cells over-expressed with PD-L1,which was consistent with that of GCMSCCM group.Knockdown of PD-L1 expression in GC cells increased the sensitivity of GC cells to chemotherapy drugs.Also,knockdown of PD-L1 expression reversed the enhanced chemoresistance of GC cells caused by GCMSC-CM.The effect of PD-L1 neutralizing antibody on GC cells was consistent with that of PD-L1 knockdown.The in vivo experimental results showed that GCMSC-CM weakened the therapeutic effect of 5-FU.The sensitivity to 5-FU was enhanced after the knockdown of PD-L1.Both in vivo and in vitro results showed that GCMSC-CM promoted chemotherapy resistance by regulating PD-L1 expression in GC cells.(3)The CTCF inhibition by si RNA transfection was accompanied by a lower clone formation number and lower expression of PD-L1,MDR1,CD44,and OCT4.Also,drug resistance induced by GCMSC-CM were reversed,suggesting that GCMSC-CM may promote stemness and drug resistance of GC cells by regulating CTCF-PD-L1.(4)Immunofluorescence results showed that GCMSC-CM reduced DDP-induced DNA damage in GC cells.DNA repair related factor Rad51 was found to be related to GCMSC-CM by q RT-PCR.GCMSC-CM up-regulated the expression of Rad51,Mre11 and NBS1,and maintained the stability of Rad51 in GC cells.The use of Rad51 inhibitor B02 demonstrated that GCMSC-CM protected GC cells from DDP through regulating Rad51.(5)Under the influence of GCMSC-CM and DDP,the expression of PD-L1 in the nucleus of GC cells was significantly increased.The interaction between PD-L1 and Rad51 was confirmed by Co-IP experiments.Western blot,q RT-PCR and immunofluorescence showed that PD-L1 knockdown inhibited GCMSC-CMmediated Rad51 over-expression.Smad4 could interact with Rad51.Inhibition of Smad4 could reduce Rad51 expression.HSP90 was up-regulated by GCMSC-CM.Inhibition of HSP90 activity by 17-AAG reversed the up-regulation of PD-L1 and Rad51 by GCMSC-CM,resulting in increased DNA damage induced by DDP,which was verified in in vivo experiment.These results indicated that GCMSC-CM may affect the expression of Rad51 in GC cells by regulating HSP90/PD-L1,thus promoting chemotherapy resistance.(6)Immunohistochemical results of clinical GC samples showed that PD-L1,CTCF,Rad51 and HSP90 were associated with poor prognosis.Also,CTCF,Rad51 and HSP90 were positively correlated with PD-L1.Conclusions:GCMSC-CM enhanced the tumor stemness through regulating CTCF-PD-L1 in GC cells and increased Rad51,a key protein related to DNA homologous recombination repair,protecting GC cells from DNA damage mediated by chemotherapy drugs,which induced increased resistance of GC cells to 5-FU,PTX and DDP.Blocking PD-L1 and HSP90 combined with chemotherapy could improve the therapeutic effect of gastric cancer.This study provides a potential therapeutic strategy for overcoming chemotherapy resistance and improving therapeutic efficacy of GC in the clinic. |
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