The Mechanism Of All-trans Retinoic Acid Up-regulating Programmed Cell Death Ligand 1 And Its Anti-tumor Effect In Combination With PD-L1 Antibody In Gastric Cancer

Tumor cells evade immune surveillance by binding programmed death ligand(PD-L1)to its receptor,PD-1,and thereby inhibiting T cell function.Reactivation anti-tumor immunity by blocking PD-1/PD-L1 immune checkpoints is a promising cancer treatment strategy.However,the response of PD-L1 antibody is not high in clinical practice,so it is urgent to explore new combination strategy to improve the therapeutic effect of PD-L1 antibody.All trans retinoic acid(ATRA)can be used to treat many types of cancer,but the underlying molecular mechanisms is not fully elucidated.Previous studies of our group have found that ATRA can up-regulate the expression of PD-L1 in gastric cancer cells,but the molecular mechanism remains unclear,and whether the up-regulation of PD-L1 caused by ATRA will affect the anti-tumor effect of PD-L1 antibody is also unknown.Based on this,in gastric cancer the molecular mechanism of the up-regulation of PD-L1 caused by ATRA was further explored in this study,and the anti-tumor effect of the combination of ATRA and PD-L1 antibody was analyzed.(1)ATRA up-regulated the expression of PD-L1 in gastric cancer cellsIn gastric cancer cells(HGC-27 undifferentiated,MGC-803 poorly differentiated,BGC-823 moderately differentiated),Western Blot combined with flow cytometry showed that ATRA could increase the expression of total and membrane protein of PD-L1 in a time-dependent and concentration-dependent manner,and ATRA could also increase the expression of E-cadherin in a concentration-dependent manner.Further detection of PD-L1 and E-cadherin in 12 gastric cancer cells showed a positive correlation between PD-L1 and E-cadherin,with a P value of 0.051.(2)Molecular mechanism of ATRA up-regulating PD-L1 expression.At the cellular level,the regulation of ATRA on PD-L1 was found to be influenced by the protein synthesis pathway by the use of the proteasome inhibitor MG132 and the protein synthesis inhibitor CHX.The QPCR assay showed that ATRA could promoted PD-L1 transcription.In addition,this study further found that ATRA could up-regulate the expression of IFNGR and activate the downstream related proteins of JAK-STAT signaling pathway.When the JAK generics inhibitor(Ruxolitinib,RUX)was used,STAT3 phosphorylation and PD-L1 expression induced by ATRA were partially reversed.When IFNγwas co-treated with ATRA,the expression of p-STAT3and PD-L1 were further increased.These studies indicated that ATRA can up-regulate the expression of PD-L1 through the JAK-STAT3 signaling pathway.(3)Effect of up-regulation of PD-L1 by ATRA on T cell function and anti-tumor effect of PD-L1 antibodyThe vitro T cell killing experiments showed that T cells could play a good killing effect when co-cultured with tumor cells,but when tumor cells were pretreated with ATRA and then co-cultured with T cells,the killing effect of T cells was partially inhibited.In exploring the effect of ATRA on PD-L1 antibody,615 mice and gastric cancer cell MFC were used.In MFC cells,ATRA also up-regulated the expression of PD-L1 through the JAK-STAT3 pathway.In the animal experiment,we used ATRA sustained-release tablets,and found that both ATRA and PD-L1 antibody could inhibit tumor growth.When the two drugs were combined,ATRA could not further improve the anti-tumor effect of PD-L1 antibody,but had a certain inhibitory effect.Western Blot analysis showed that ATRA up-regulated the expression of p-STAT3 and PD-L1.The transcription of IFN-γand GZMB(granulease B)in tumor and spleen were detected by q PCR,and the CD8~+T cell function was significantly increased by PD-L1antibody,while this function was partially inhibited in the combination group.There was no significant difference in the body weight of mice before and after administration,and the mice were in good growth state,indicating that these two drugs had no obvious toxic and side effects.Therefore,we believe that ATRA can up-regulate the expression of PD-L1 by activating p-STAT3 pathway,inhibit the activation of T cells and anti-tumor immune response,and thus antagonize the anti-tumor effect of PD-L1 antibody.In conclusion,ATRA can regulate the expression of PD-L1 by activating the JAK-STAT3 pathway in gastric cancer cells,and the up-regulation of PD-L1 expression by ATRA can inhibit the activation of T cells and the anti-tumor immune response,thus antagonizing the anti-tumor effect of PD-L1 antibodies.