The Experimental Study For The Expression Regulation And Combined Intervention Of PD-L1 In Pancreatic Ductal Adenocarcinoma

Background Pancreatic cancer is the worst prognostic malignant tumors in digestive system,whose morbidity and mortality are increasing year by year in China.Due to its low surgical resection rate(less than 20%),insensitivity to traditional radiotherapy and chemotherapy,the effect of clinical treatment to PC is extremely poor.An increasing number of evidence shows that tumor microenvironment(TME)plays a vital role in the development of tumor,at the same time,immunoediting of pancreatic cancer is essential to disease progression as well as resistance to radiotherapy and chemotherapy.Recently,immunotherapy,especially the combination of immune checkpoint antibody treatment has made great progress in a variety of clinical treatment to multiple kinds of tumor,but promoted little in pancreatic cancer.After the examination of a large number of literatures and our preliminary experiments,we found that the expression of PD-L1(programmed cell death ligand-1)was higher in pancreatic ductal adenocarcinoma cells and cell lines than that in normal pancreatic tissues and cells,which suggests relative basic and transformation research might provide new thoughts for pancreatic cancer treatment.Therefore,we designed this project,hopefully,through the exploration of PD-L1 expression level and possible regulatory mechanisms in pancreatic ductal adenocarcinoma cells,to further elucidate its potential clinical significance.We determined to investigate the function and significance of PD-L1 in pancreatic ductal adenocarcinoma in the level of clinical histology,cell biology,molecular biology and model animal.Method1.We used 3 different kinds of antibody(different clones)to detect the distribution and expression of PD-L1 in the collected paraffin specimen,which were after the radical operation and pathological diagnosis as PDAC unequivocally by Immunohistochemical staining.Combined with the clinicopathological parameters,the relationships of PD-L1 expression with clinical index of the patients were analyzed.We further analyzed their survival conditions after the follow up standardized chemotherapy and regular review every three months.2.Investigating the effect of PD-L1 expression on the remodeling of regional immune microenvironment in pancreatic ductal adenocarcinoma cells,and laying emphasis on its relationship with FOXP3+ cells.The expression of FOXP3 in tumor cells is crucial in local Treg infiltration.Observing the relationship between PD-L1 expression and local FOXP3+ Treg cell infiltration in the tumor,and thus to analyze whether these two co-affect the prognosis of pancreatic ductal adenocarcinoma.The relativity of PD-L1 and FOXP3 in protein and m RNA level in fresh pancreatic cancer tissues was further detected and analyzed by Western blot and real-time PCR respectively.The correlation between FOXP3 and PD-L1 in pancreatic cancer in the TCGA database was integrated.3.The correlation of PD-L1 and FOXP3 in protein and m RNA level was examined in the FOXP3 overexpression and knockdown pancreatic cancer cell lines in vitro.The expression of PD-L1 in pancreatic cancer cell membrane was analyzed by flow cytometry,and the relationship with FOXP3 was analyzed.The direct transcriptional regulation of FOXP3 to PD-L1 in pancreatic cancer cell lines was clarified by CHIP and double luciferase assay.4.Observing the antitumor effect of anti-PD-L1 to pancreatic cancer by PD-L1 neutralizing antibody intraperitoneal injection to subcutaneous and orthotopic transplanted tumor models of C57BL/6 mice.Incorporated with the former findings,we used a combination of anti-CCL5 neutralizing antibody to block Treg,to evaluate the anti-tumor effect of combined immunomodulatory therapy,analyze its intrinsic mechanism,and illuminate the core role of tumor derived FOXP3 in pancreatic cancer immune microenvironment remodeling,which is expected to be a molecular marker for local immune remodeling of pancreatic cancer,as well as markers for PD1/PD-L1-based combination immunotherapy screening.Results1.The expression of PD-L1 in normal pancreatic tissue and pancreatic ductal adenocarcinoma cells and its clinical significance.IHC staining of PD-L1 in normal pancreatic ductal epithelial cells was performed in 5 normal pancreatic tissue and 100 pancreatic ductal adenocarcinoma cases,which found that PD-L1 was not or extremely low expressed in the normal pancreatic ductal epithelium,but was significantly increased in some pancreatic ductal adenocarcinoma cells.The results of immunohistochemical staining by three kinds of antibodies showed well consistency.We found that PD-L1 high expression accounted for 26.0%(26 cases)of all cases,while medium expression accounted for 43.0%(43 cases),low expression accounted for 31.0%(31 cases).Through the analysis of the relationship between the expression of PD-L1 and the clinical pathological parameters.We found that the higher T stage of pancreatic ductal adenocarcinoma is correlated with higher expression of PD-L1.The overall survival of patients with low PD-L1 expression in pancreatic ductal adenocarcinoma was significantly longer than that in patients with high expression of PD-L1(median: 37 vs 19,13 months,**p <0.01),meanwhile its recurrence-free survival(median: 29 vs 13,7 months)was significantly longer in patients with moderate and high expression(**p <0.01).2.The expression of PD-L1 in pancreatic ductal adenocarcinoma cells was significantly positively correlated with FOXP3 in tumor cells(R=0.401,**p <0.01).At the same time,there were statistical differences between the number of infiltrated FOXP3+ Treg cells in the tumor with PD-L1 expression levels(F = 8.925,**p <0.01).Additionally,the correlation analysis showed that the expression of PD-L1 in tumor cells was significantly correlated with the local infiltration of tumor cells(**P <0.01).The overall survival(median: 11 vs 26,38 months)(*p <0.05)and no recurrence and metastasis survival(median : 6 vs 20,20 months)(*p <0.05)was significantly lower in patients with high levels of PD-L1 accompanied with high Treg infiltration.These results suggest that the elevated expression of PD-L1 in tumor cells is one of the characteristics of localized immune microenvironment remodeling of pancreatic ductal adenocarcinoma,which is involved in the tumor malignant progression.To further clarify the expression of PD-L1 in pancreatic ductal adenocarcinoma cells and its relationship with FOXP3 in tumor cells,we detected their expression in the frozen matching carcinoma and adjacent non-cancerous tissues with explicit clinicopathological diagnosis by Western blot.The results showed that the expression of PD-L1 and FOXP3 in the tissue was significantly higher in tumor tissue than that in the adjacent non-cancerous tissues,which was positively correlated.The expression of PD-L1 and FOXP3 in m RNA level detected by Real-time PCR were consistent with the protein level.Furthermore,the m RNA expression levels of FOXP3 and PD-L1 obtained by TCGA database was statistical analyzed as a third-party data to support the results of this study.3.FOXP3 acts as a transcription factor involving in the expression of PD-L1 in pancreatic cancer cells.According to the basic expression level of FOXP3 in different pancreatic ductal adenocarcinoma cell lines,we constructed the c-FOXP3 overexpressing cell lines(As PC-1 and Panc-1)and knockdown cell lines(Panc-1 and MIA-Pa Ca2)by lentivirus infection.The expression of FOXP3 and PD-L1 was detected by Western blot.Real-time q PCR was performed to examine the expression of PD-L1 m RNA varied with FOXP3 expression.The expression of PD-L1 on the membrane of tumor cells was detected by flow cytometry,confirming the positive regulation by FOXP3 simultaneously.Bioinformatics analysis suggested that there was a potential target sequence for FOXP3 transcriptional binding in PD-L1 promoter.We further selected human pancreatic ductal adenocarcinoma cell line Panc-1 and C57BL/6 murine pancreatic cancer cell line Pan02 for chromatin immunization Co-precipitation to verify the binding of transcription factor FOXP3 to the target sequence in PD-L1 promoter region.We next constructed the PD-L1 promoter reporter vector to confirm that FOXP3 could directly bind and transcriptional activate PD-L1,while the transcriptional activation was significantly inhibited after the mutation of the binding site in the promoter of PD-L1.4.Constructing C57BL/6 mice subcutaneously xenograft model,demonstrating the antitumor effect of anti-PD-L1 for pancreatic cancer by intraperitoneal injection neutralizing antibodies.The results suggest that anti-PD-L1 can inhibit the growth of transplanted mice;Combined with the results of previous studies,we used anti-CCL5 and anti-PD-L1 neutralizing antibody treatment for combining treatment,the results suggest that combined immunomodulatory treatment has a more significant anti-tumor effect,while the effects was attenuated after removal of CD8+ lymphocytes in mice by anti-CD8 neutralizing antibodies.The detection of the tumor shows that the tumor proliferation index of the anti-CCL5 or anti-PD-L1 neutralizing antibody treatment group and were both lower than those in the control group,while the proliferation index of the combined treatment group was lower than single antibody treatment group.The results of flow cytometry showed that the combination treatment of anti-CCL5 and anti-PD-L1 neutralizing antibody significantly improved the local immune microenvironment of the mouse transplanted tumor.Compared with the control group,the number of CD8+ lymphocytes increased significantly,especially IFN?+ CD8+ lymphocytes;Treg cells and CD8+ lymphocyte apoptosis rate decreased significantly.The mouse survival curves were consistent with the former results.Conclusion1.Compared with normal pancreatic ductal epithelial cells,PD-L1 expression was elevated in some pancreatic ductal adenocarcinoma cells,tumor cells overexpressing PD-L1 prompted poor prognosis of patients with pancreatic ductal adenocarcinoma.2.The expression of PD-L1 was positively correlated with the expression of FOXP3 in cancer cells,and the overexpression of PD-L1 was correlated with localized Treg cell infiltration and poor prognosis.3.The promoter region of PD-L1 has a FOXP3 transcriptional regulatory target sequence.In pancreatic cancer cells FOXP3 can directly bind to the specific sequence of PD-L1 promoter region,activate its transcription,and then increase PD-L1 expression in pancreatic cancer cells to promote its function.4.Anti-PD-L1 neutralizing antibody can inhibit the growth of pancreatic cancer xenografts.The combination of anti-CCL5 and anti-PD-L1 neutralizing antibodies can improve the local tumor immune environment of pancreatic cancer,significantly inhibit tumor growth,validate tumor-bearing mice survival,suggesting that the immunoregulatory therapy based on immune-checkpoint PD-L1-targeted intergation therapy is expected to improve the treatment of pancreatic ductal adenocarcinoma,as well as the regulation role of FOXP3 in the in pancreatic cancer immune microenvironment remodeling,which is expected to serve as a molecular marker for local immune remodeling of pancreatic cancer and a screening marker for the combined immunization treatment of dominant populations.