Identification Of New Subpopulations Of MDSCs In The Immune Microenvironment Of Pancreatic Cancer And The Mechanism Of CAF Promoting Differentiation And Migration Of MDSCs

Part ?: Phenotypic identification of MDSCs in pancreatic ductal adenocarcinoma and its immunosuppressive mechanismOBJECTIVE:To verify the phenotypic characteristics of MDSCs in pancreatic ductal adenocarcinoma and their role in tumor microenvironment.Methods: 1.The pathological characteristics of chronic pancreatitis(CP),pancreatic ductal adenocarcinoma(PDAC)and adjacent non-neoplastic pancreatic tissues(ANPT)were analyzed by H&E staining,and the infiltration of MDSCs in three tissues was analyzed.2.Immunohistochemistry and flow cytometry were used to analyze the number,distribution and phenotypic characteristics of myeloid-derived suppressor cells(MDSCs)in peripheral blood and tumor tissues of patients with PDAC.3.Western blot,flow cytometry,cytokine microsphere assay and T cell inhibition assay were used to explore the specific functional mechanism of MDSCs.4.Collect clinical samples to analyze the relationship between MDSCs and the prognosis of PDAC patients.Results:1.Mononuclear lymphocyte envelope was found around CP and ANPT nerves,mainly consisting of CD3 + T cells and CD20 + B lymphocytes.When perineural invasion(PNI)occurred in PDAC,no lymphocyte envelope was found around the nerve,but the number of MDSCs increased significantly,indicating that MDSC may drive out peripheral immune cells and promote the occurrence of PNI.2.Compared with healthy people,the number of n-MDSCs in peripheral blood and tumor tissue of PDAC increased significantly,and two new subgroups of CD13hi-nMDSCs and CD13low-nMDSCs were identified in n-MDSC.Although CD13hi-nMDSCs and CD13low-nMDSCs increased in peripheral blood and tumor tissues of patients with PDAC compared with healthy volunteers,the number of CD13hi-nMDSCs was significantly higher than that of CD13low-nMDSCs,so the increase of CD13hi-nMDSCs was the main factor in PDAC.3.CD13hi-nMDSCs expressed higher levels of Arginase-1(Arg-1)than CD13low-nMDSCs in PDAC tumor microenvironment,which had stronger inhibitory effect on T cell proliferation.Therefore,CD13hi-MDSCs subgroup played a major immunosuppressive role in PDAC tumor microenvironment.4.The number of CD13hi-nMDSCs in peripheral blood of patients with PDAC is negatively correlated with the survival time of patients.Surgical treatment of patients with PDAC can change the composition of n-MDSCs subgroup.The proportion of CD13hi-nMDSCs decreases and the proportion of CD13low-nMDSCs increases.Therefore,the immunosuppressive activity of n-MDSCs decreases after operation,which also shows that the number and proportion of MDSC subgroups are closely related to the progress of PDAC.CONCLUSION:This study identified two new MDSC subpopulations: CD13hi-nMDSCs and CD13low-nMDSCs,in which the CD13hi-nMDSCs subpopulation is the most important cell population of all PDAC subpopulations of PDAC,which is abundantly aggregated in PDAC.And the expression of high levels of Arg-1 plays an immunosuppressive role in the tumor microenvironment,which is closely related to the prognosis of patients and the occurrence of PNI.Part ?: Mechanism of differentiation and migration of CAFs andMDSCs in pancreatic cancer tumor microenvironment.OBJECTIVE:To explore the mechanism of CAFs promoting the large-scale amplification and aggregation of MDSCs in the PDAC tumor microenvironment.Methods:1.Cancer-associated fibroblasts(CAFs)were isolated and purified from pancreatic cancer tissues of patients with PDAC.The phenotype and purity of CAFs were identified by immunofluorescence and flow cytometry.2.Q-PCR and ELISA were used to screen the related cytokines of CAFs which were significantly up-regulated relative to the two control cells(HDF-a,HFF).3.The co-culture system of CAFs and PBMC and the migration model of MDSCs in vitro were constructed to study the specific mechanism of these factors involved in regulating the differentiation and recruitment of MDSCs.Results:1.Primary isolated CAFs are of high purity and express the activation markers a-SMA and FAP.2.Three cytokines with up-regulated CAFs expression were screened by Q-PCR and ELISA,namely IL-6,SDF-1 and MCP-1.3.IL6 secreted by CAFs can promote the differentiation of immature immune cells into CD13hi-MDSCs in PBMC.This function may be mediated by STAT3 pathway,and the production of CD13hi-MDSCs can be reduced by using STAT3 blocker;SDF-1 can participate in the recruitment of CD13hi-MDSC and promote the migration of CD13hi-MDSC.After SDF-1 neutralizing antibody or blocking CXCR4 receptor,the migration of CD13hi-MDSCs is reduced.CONCLUSION:This studydemonstrates that CAFs can promote PBMC differentiation into CD13hi-MDSC via the IL6/STAT3 pathway;promotes migration of CD13hi-MDSC via the SDF-1/CXCR4 pathway.