Expression Of PD-L-1 In Ovarian Cancer And Its Synergistic Anti-tumor Effect With PARP Inhibitor

Ovarian cancer has a complex pathogenesis,a wide variety of pathologies and a short expected survival period.In 2018,there are 295,414 new cases（1.6%）of ovarian cancer worldwide and 184,799 deaths（1.9%）.According to the American cancer society（ACS）,ovarian cancer is the most lethal of gynecologic tumors.Surgery and first-line chemotherapy may help some ovarian cancer patients achieve clinical remission,but the recurrence rate is as high as 70%.The effect of treatment for advanced tumor is poor.Long-term survival is still less than 20%.Serous ovarian cancer accounts for 75%in ovarian cancer,and high-grade cancer is the most common histological type,accounting about for 70%.Relapse and drug resistance are the main causes of poor prognosis in patients with advanced stage.The improvement of treatment strategy for advanced ovarian cancer is urgent.PD-L1 also known as surface antigen differentiation cluster 274（CD274）or B7homologous protein 1（B7-H1）,is a protein encoded by the CD274 gene.As one of the most important immune checkpoints,PD-L1 can be constitutively low expressed in APCs and a variety of non-hematopoietic cells,including vascular endothelial cells,pancreatic cells,etc.It can also be induced expression by inflammatory cytokines such as interferon（interferon,INF）,tumor necrosis factorα（tumor necrosis factor-a,TNF-α）and vascular endothelial growth factor（vascular endothelial growth factor,VEGF）.PD-L1 is expressed not only in melanoma,but also in lung cancer,bladder cancer,colorectal cancer and other tumor tissues of different tissue sources.PD-L1 and PD-1 combine to transmit inhibitory signals,which can induce apoptosis and dysfunction of T cells,and express more leukin-10（Il-10）,and then inhibit the activation of tumor antigen specific CD8⁺T,proliferation and anti-tumor function to achieve tumor immune escape.Nowadays,the development of these immunotherapy agents has been increasing worldwide.Poly（ADP ribose Polymerase,PARP）inhibitors approved by the FDA in 2014.It is used to treat advanced ovarian cancer patients who have received three or more chemotherapy regimens and have genetic mutations in the germ Line or somatic breast cancer susceptibility genes（breast cancer susceptibility gene,BRCA）.However,it was limited to patients with BRCA gene mutation.More than 80%of inherited ovarian cancers were associated with BRCA1/2 germline mutations.BRCA 1/2 mutations account for 75%of inherited ovarian cancer gene mutations.BRCA1 mutation carries 59%lifetime risk of ovarian cancer,while the lifetime risk of ovarian cancer in BRCA2 mutation is 16.5%^[15].The multi-center large sample study data of BRCA mutation of ovarian cancer in China showed that,the BRCA mutation rate of germ cells was 28.5%,BRCA1 mutation rate was20.82%,BRCA2 mutation rate was 7.63%.The histological types of BRCA mutant ovarian cancer patients were mostly high-grade serous carcinoma（73.0%）,and 85.5%belong to advanced disease（III/IV period）.These limits the efficacy of PARP inhibitors for ovarian cancer.In this study,we detected the expression of PD-L1 and its relationship with the prognosis of ovarian cancer,and explored the effect and its potential mechanism of PARP inhibitor combined with PD-L1 monoclonal antibody in the treatment of ovarian cancer.We hoped to provide experimental basis for the immunotherapy of ovarian cancer.Part Ⅰ,to investigate the expression of synergistic stimulation molecule PD-L1 in ovarian cancer and its relationship with prognosis.PD-L1 expression in paraffin-embedded tissues on 77 cases of ovarian cancer was detected by immunohistochemistry,10 cases of ovarian benign tumor tissues were taken as the control group.The correlation between the PD-L1 expression and the clinical pathological parameters was analyzed by chi-square test.Kaplan-meier method was used to compare the effects of different PD-L1 expression levels on postoperative total survival（OS）and progression-free survival（PFS）.The co-expression of PD-L1,CD4⁺T and CD8⁺T in TILs was detected by confocal laser.Flow cytometry was used to isolate TILs and analyze the expression of PD-L1.It was found that PD-L1 was mainly expressed on the cell membrane and cytoplasm of ovarian cancer.The expression of PD-L1 on ovarian cancer cells was statistically significant with the FIGO stage（P=0.026）.However,there was no statistical significance with other clinicopathological parameters（P>0.05）.Kaplan-Meier Survival Analysis showed that OS was significantly shortened in the high expression group of PD-L1 and the low expression group,the difference was statistically significant（P=0.0005,HR=2.689）.Multi-factor Cox proportional risk model showed that,high expression of PD-L1（HR=2.275,P=0.023）and FIGO stage（HR=11.229,P=0.024）were independent risk prognostic factors for survival of ovarian cancer patients.Hierarchical analysis showed that,OS was significantly shortened in the high expression group of PD-L1 compared with the low expression group of serous ovarian cancer patients,the difference was statistically significant.PD-L1⁺molecules were co-expressed with CD4⁺T cells or CD8⁺T cells on the TILs of ovarian cancer,respectively.Flow cytometry showed that,the average expression of PD-L1⁺in CD4⁺T cells was 36.95%,the average expression of PD-L1⁺in CD8⁺T cells was 23.53%.Pearson correlation analysised that the expression level of PD-L1⁺in ovarian cancer showed a negatively correlation with the number of CD8⁺T cells,but there was no statistically significant（P=0.0078,r=-0.9272）.Part Ⅱ,to explore the potential mechanism of PARP inhibitor combined with PD-L1monoclonal antibody in the treatment of ovarian cancer.Ovarian cancer cell lines ID8（BRCA wild type,mice source）,uwb1.289（BRCA mutant,human source）and SKOV3（BRCA wild type,human source）were selected,the expression level of PD-L1were detected by Western Blot after the intervention of the PARP inhibitor,the expression of membrane PD-L1 was detected by FACS.The cell line uwb1.289 and SKOV3 cells were co cultured with human peripheral blood cells,respectively.Transwell assay was performed to observe the effect of PARP inhibitor and PD-L1 monoclonal antibody in invasive ability of ovarian cancer cells.The scratch test was carried out to observe the effect of different treatment schemes on the migration ability of ovarian cancer cells.PARP was knocked down by si RNA transfection,then the expression level of PD-L1 were detected by Western Blot;BRCA1/BRCA2 was knocked down by si RNA transfection,then the expression level of PD-L1 were detected by Western Blot.Western Blot was used to detect the phosphorylation level of the signaling pathway after the intervention of the PARP inhibitor.The uwb1.289 cell lines were co-cultured with CD8⁺T cells,Brd U-labeled,flow cytometry was used to detect the proportion of Brd U positive cells.And the CD8⁺T cell cycle was analyzed by flow cytometry.We found that the expression levels of PD-L1were up-regulated after the intervention of PARP inhibitors at different concentrations in ovarian cancer cell lines ID8（BRCA wild type）,uwb1.289（BRCA mutant）and Skov3（BRCA wild type）,the difference was statistically significant（P<0.05）.FACS detection was performed with fluorescently labeled PD-L1 antibody,which showed that the expression of PD-L1 was up-regulated and increased in a dose-dependent manner.Transwell assay showed that the invasive ability of ovarian cancer cells in PARP inhibitor Olaparib group and Anti-PD-L1 group was poor in comparison to the blank control group,while the invasive ability of ovarian cancer cells in combined group was significantly inhibited compared with that in other groups.Scratch test showed that the migration ability of ovarian cancer cells in PARP inhibitor Olaparib group and Anti-PD-L1 group was weaker than that in control group,while the migration ability of ovarian cancer cells in combined group was significantly inhibited compared with that in individual treatment group.The expression of PD-L1 in PARP knocked down cells was significantly higher than that in parental cells.While,Knocked down BRCA1 or BRCA2 resulted in down-regulated expression of PD-L1 induced by PARP,the difference was statistically significant.after the intervention of the PARP inhibitor,the phosphorylation level of Chk1pathway was significantly increased.After the administration of Chk1 blocker,Olaparib upregulates the PD-L1 effect,which is limited.The difference was statistically significant（P<0.05）.After the intervention of the PARP inhibitor and PD-L1si RNA transfection,the phosphorylation level of Chk2 pathway was significantly increased,the expression level of P53 protein was significantly increased,CDC25a signaling protein was significantly down-regulated,he difference was statistically significant（P<0.05）.In the uwb1.289 cells co-cultured with CD8⁺T cells,the proliferation of CD8⁺T cells was significantly increased after the intervention of the PARP inhibitor and PD-L1si RNA transfection,compared to the individual Olaparib intervention group.In G2-M phase,the proliferation ratio of CD8⁺T cells was significantly increased in cocultured cell after the intervention of the PARP inhibitor and PD-L1si RNA transfection.Part Ⅲ,the synergistic anti-tumor effect of PARP inhibitor combined with PD-L1monoclonal antibody in ovarian cancer was verified in vivo.uwb1.289 cells were used to construct the tumor model of ovarian cancer transplantation in BALB/c NOD mice,ID8cells were used to construct the tumor model in C57BL/6 mice.The blank group,Olaparib group,anti-PD-L1 group,Olaparib+anti-PD-L1 group were processed in groups,plot tumor growth curve.The expression of PD-L1 in tumor tissues was detected by Western Blot;The expression of Ki67 in tumor tissues was detected by IHC.Compared with the control group,the tumor growth volume of the Olaparib monotherapy group,the PD-L1monotherapy group and the combination therapy group all decreased significantly,and the difference was more significant with the extension of the growth cycle.The tumor growth volume of the Olaparib combined with PD-L1 monoclonal antibody group was significantly lower than that of the Monotherapy PD-L1 monoclonal antibody group,and the difference was statistically significant（P<0.05）.With the extension of the growth cycle,the difference became more significant.The tumor mass measurement results showed that,the weight of Olaparib group,anti-PD-L1 group and Olaparib+anti-PD-L1 group was significantly smaller than that of the control group,the difference was statistically significant,and the combined treatment group had more significant efficacy than the anti-PD-L1 group.The level of PD-L1 in transplanted tumor tissue was significantly increased after Olaparib intervention.The results of immunohistochemical staining with Ki67 showed that the proliferation of tumor cells in the control group was active,and the proliferation of tumor cells decreased under the treatment of Olaparib and PD-L1monoclonal antibody,respectively.When the combination of Olaparib and PD-L1monoclonal antibody was given,the proliferation of tumor cells was significantly inhibited.In summary,the co-stimulatory molecule PD-L1 was highly expressed in ovarian cancer tissues.Its expression level is closely related to FIGO stage and prognosis of patients.PD-L1 may be involved in the occurrence and development of ovarian malignant tumors.On ovarian cancer cells,PARP inhibitors may induce up-regulation of PD-L1expression by promoting phosphorylation of Chk1.PD-L1 blocker can reverse the inhibitory effect of PARP on CD8⁺T cells,enhance the anti-tumor effect of PARP inhibitors,which provided a theoretical basis for the synergistic treatment of ovarian cancer by PARP inhibitors combined with PD-L1 monoclonal antibody.PARP inhibitor combined with PD-L1 monoclonal antibody can significantly inhibit tumor volume and mass in the mice model of ovarian cancer.