The Preliminary Study On The Effects Of Fractalkine Combined With M2 Macrophages On The Stem Cell Characteristics Of Pancreatic Cancer Cell Line

Objective To investigate the effect of Fractalkine combined with M2 macrophages on the stem cell characteristics of pancreatic cancer cell line.Methods The recombinant lentiviral vector HBLV-h-FKN-GFP-PURO carrying Fractalkine（FKN）gene was constructed and infected into PANC-1 cells,RT-qPCR and Western blot were performed to test the expression levels of mRNA and proteins of FKN,respectively.THP-1 cells,a monocyte of human leukemia,were induced into M2-type macrophages by phorbol 12-myristate 13-acetate（PMA）and interleukin-4（IL-4）in sequence,Western blot was used to detect the expression level of proteins of arginase-1（Arg-1）and inducible nitric oxide synthase（iNOS）.Then the indirect co-culture model of pancreatic cancer cells with different expression level of FKN and M2-type macrophages was established by Transwell chamber system to detect the chemotaxis of FKN to M2-type macrophages,and the co-cultured PANC-1 cells and M2-type macrophages were collected.The proliferation,invasion and migration of PANC-1 cells were detected by CCK-8 method,Transwell chamber test and wound healing assay,respectievely.Western blot was also used to detect the expressions of CD44,CD133,E-cadherin and Vimentin of PANC-1 cells,as well as the expressions of CX3CR1,Arg-1 and CD163 of M2 macrophages.The results were analyzed with one-way analysis of variance（ANOVA）test or analysis of variance by two-way factorial design.Results 1.HBLV-H-FKN-GFP-PURO was successfully transfected into PANC-1 cells,and the mRNA and protein levels of FKN in FKN overexpressed group remarkably up-regulated compared with control groups（P<0.001）.2.THP-1 cells were successfully induced to become M2-type macrophages with high expression of Arg-1 and low expression of iNOS protein(P_Arg-1<0.001,P_iNOS<0.01).3.The recruiting ability of PANC-1 cells to M2-type macrophages was enhanced after FKN over-expression（P<0.001）.4.The expressions of CD44,CD133 and Vimentin protein of PANC-1 cells increased after FKN over-expression or co-cultured with M2-type macrophages,while the expression of E-cadherin protein decreased.There were synergistic effects between M2-type macrophages and FKN on CD44,CD133 and Vimentin expressions in PANC-1cells,but not on Vimentin(P_CD44<0.05,P_CD133<0.05,P_E-cadherin<0.05,P_Vimentin>0.05).5.The proliferation,migration and invasion abilities of PANC-1 cells were increased after FKN over-expression（all P<0.001）,and were more significantly increased after co-culture with M2-type macrophages.There were interactions between M2-type macrophages and FKN for proliferation,migration and invasion abilities of PANC-1 cells（P<0.01,P<0.05,P<0.01）.6.The expression of CX3CR1,Arg-1 and CD163 of M2 macrophages were increased after co-culture with PANC-1 cells,and were more significantly increased after co-culture with PANC-1 cells with FKN over-expression(P_CX3CR1<0.01,P_Arg-1<0.05,P_CD163<0.01).Conclusion 1.The chemokine FKN promotes the recruitment of PANC-1 cells to M2-type macrophages in vitro.2.Both FKN and M2-type macrophages can enhance the proliferation,invasion and migration abilities of PANC-1 cells,promote epithelial mesenchymal transformation（EMT）of PANC-1 cells,and increase the expression of pancreatic cancer stem cell genes.Moreover,there is synergistical interaction between them.3.FKN can increase the expression of CX3CR1 in macrophages and promote macrophage M2 polarization.