Role Of Tumor-derived Reg3A In Educating Dendritic Cells To Promote Immune Escape Of Pancreatic Cancer And Its Mechanism

Objective To study the effect and its mechanism of tumor-derived Reg3A(Regenerating islet-derived protein 3A)on dendritic cells(DCs)and pancreatic cancer(PaC)progression from the perspective of tumor immunity,to further reveal the key role of Reg3A in the inflammation-linked pancreatic carcinogenesis.Method siRNAs or plasmids were used to silence or over-express Reg3A in human PaC cell lines BxPC-3,AsPC-1,SW1990 and PANC-1.Then,human peripheral blood-derived DCs were co-cultured with PaC cells in a Transwell co-culture system for 48h.The expression levels of surface molecules CD80,CD83 and CD86 on DCs were detected by flow cytometry by direct fluorescent immunostaining to examine the phenotype of DCs.The apoptosis rates of DCs were detected using Annexin V-FITC/PI double-labeled flow cytometry.Levels of FITC-dextran uptake by DCs were measured using flow cytometry to detect the endo/phagocytotic function of DCs.The mixed lymphocyte reaction(MLR)was used to determine the immunostimulatory function of DCs.The ability of co-cultured DCs to secrete interleukin(IL)-12p70 and IL-23 was tested by ELISA assay.On the other hand,the apoptosis rates and cell cycles of PaC cells co-cultured with DCs were detected by flow cytometry to study the effect of co-culture with DCs on the apoptosis and proliferation of PaC cells in vitro.The in vivo effect of tumor-derived Reg3A-educated DCs on PaC progression was studied by intraperitoneal-transfer-in-PaC-implanted SCID mice Teconstituted with human T cells.As for the mechanisms,the expression of signal transducer and activator of transcription 3(STAT3),toll-like receptor 4(TLR4),etc.on tumor-derived Reg3A-educated DCs or PaC cells was detected using immunofluorescence laser co-localization and Western blot,etc..Results Tumor-derived Reg3A was found to block the differentiation of resting/immature DCs to activated/mature DCs,increase the endo/phagocytotic function of DCs and inhibit DC-induced T-lymphocyte proliferation,protect DCs against apoptosis,reduce the IL-12p70 production but enhance the IL-23 production of DCs,thus educate DCs,contribute to immunosuppressive tumor microenvironment and facilitate PaC escape and progression.The in vivo promotive effect of tumor-derived Reg3A-educated DCs on PaC progression was demonstrated by intraperitoneal transfer in PaC-implanted SCID mice reconstituted with human T cells,when compared with that of control DCs.As for the mechanisms,tumor-derived Reg3A upregulated the expression of STAT3 in educated DCs.The co-localization and potential interaction of Reg3A and TLR4 were found in PaC cells and DCs.In addition,tumor-derived Reg3A could upregulated the p38MAPK/IL-6 and Cyclooxygenase 2(COX2)/Prostaglandin E2(PGE2)pathways in PaC cells.Conclusion Reg3A continuously released by PaC cells could act as a tumor-derived factor(TDF)to educate DCs,the most important antigen presenting cells,contribute to immunosuppressive tumor microenvironment and facilitate PaC escape and progression.The possible mechanisms involved the effects of Reg3A on the expression of STAT3 and TLR4 in tumor-derived Reg3A-educated DCs,as well as on the p38MAPK/IL-6 and COX2/PGE2 pathways in PaC cells themselves.