Research On The Effects And Potential Molecular Mechanisms Of PARP Inhibitor Combined With PD-L1 Blockade In Pancreatic Cancer

Objective:Pancreatic cancer is a highly lethal disease with a low 5-year survival rate and cancer mortality caused by it is increasing every year.PARP inhibitors have been applied in pancreatic cancer as a major breakthrough in targeted therapy.Our research work focused on whether PARP inhibitors can affect PD-L1 expression in Tumors and whether PD-L1/PD-1 immune checkpoint inhibitors can enhance the tumor suppressive effect of PARP inhibitors.Methods:(1)By using flow cytometry,immunohistochemical techniques,the pancreatic cancer cell line SW1990 was observed for functional alterations(i.e.,apoptosis,cycle,proliferation,etc.)in the control and PARP inhibitor pamiparib groups;(2)To examine whether there is a difference in PD-L1 expression between the group given the PARP inhibitor pamiparib and the control group in pancreatic cancer cell lines SW1990,Bx PC-3,and nude mice transplanted Tumors by Western blotting,immunohistochemistry,and flow cytometry analysis;(3)Validation of whether the PARP inhibitor pamiparib leads to upregulation of PD-L1 expression through the PARP1 locus by si PRAP1.Then,using bioconductivity analysis techniques,the signaling pathways leading to PD-L1 upregulation in pancreatic cancer were searched for,and the key pathways leading to PD-L1upregulation were screened by drug screening assays using different inhibitors of signaling pathways,and IHC staining was performed in nude mice transplanted Tumors to further validate the reliability of the signaling pathway findings;(4)The activated T cells and pancreatic cancer SW1990 were co-cultured for 48h and then stained using crystal violet to qualitatively observe whether pamiparib affects the killing function of T cells.CFSE-labeled T cells were then co-cultured with pancreatic cancer SW1990,followed by flow cytometry to analyze whether the PARP inhibitor pamiparib inhibits T-cell proliferation in an in vitro setting;(5)The treatment effects of the control group,the PARP inhibitor group alone,the PD-L1 immune blocker group alone,and the two-drug combination group were tested in a mouse tumor model.After the treatment time point was reached,measurements were taken and photographed.Certain cellular components of the tumor microenvironment of a portion of the isolated mouse Tumors were analyzed by flow cytometry to assess whether drug treatment had altered the tumor microenvironment.Fixation of another portion of the isolated mouse Tumors and confirmation of changes in PD-L1 and the corresponding pathways in the four groups by HE staining as well as immunohistochemistry to further verify the accuracy of the mechanistic study of the cells;(6)RNA was extracted from tumor tissues of mice in four different groups using TRIzol reagent,sequenced and libraries were prepared.The RNA-seq results were used to observe the changes in expression of genes associated with the immune microenvironment.Results:(1)The PARP inhibitor pamiparib affects apoptosis,cycle,and proliferation of pancreatic cancer cells;(2)In pancreatic cancer cell lines and mouse models,PD-L1expression was significantly higher in the group that had used the PARP inhibitor pamiparib compared to the control group;(3)The PARP inhibitor pamiparib causes upregulation of PD-L1 expression not through the PARP site.PD-L1 upregulation caused by PARP inhibitors is due to activation of the JAK2/STAT3 pathway;(4)PARP inhibitors do not affect the killing and proliferative functions of T cells;(5)Combination therapy with PARP inhibitors and anti-PD-L1 resulted in significantly improved treatment efficacy in vivo compared with the two drugs alone;(6)RNA-seq results also showed that both monotherapy and combination therapy had significant changes in immune response compared to the control group,and the dual drug combination significantly increased the infiltration of CD8~+T cells in the immune microenvironment.Conclusion:In pancreatic cancer,JAK2/STAT3 pathway plays an important role in the upregulation of PD-L1 expression by PARP inhibitors.Meanwhile,the study confirmed that the combination of PARP inhibitors with PD-L1/PD-1 immune checkpoint blockers is a promising approach for the treatment of pancreatic cancer.