Experimental Study On The Effect Of Neutrophils Recruited By Tumor-associated Fibroblasts On The Proliferation And Migration Of Pancreatic Cancer PANC-1 Cells

Objective More and more evidence shows that tumor microenvironment plays an important role in the evolution of pancreatic cancer.The purpose of this study was to investigate the effect of neutrophils recruited by tumor-associated fibroblasts on the proliferation and migration of pancreatic cancer PANC-1 cells.Methods HL-60 cells were cultured in RPMI1640 medium containing 1.3%DMSO for 5 days to induce them to be neutrophil-like(dHL-60),and an in vitro neutrophil culture model was established.Prepare conditioned medium for pancreatic cancer PANC-1 cells,culture fibroblasts(HFF)with PANC-1 cell conditioned medium for 24 hours to obtain tumor associated fibroblasts(CAF),and then prepare conditioned medium for CAF.Enzyme linked immunosorbent assay(ELISA)detection of granulocyte chemotactic protein-2(GCP-2)in fibroblast(HFF)and tumor-associated fibroblast(CAF)conditioned medium)Content.Western blot was used to detect the expression of CXCR1(chemokine(C-X-C motif)receptor 1)and CXCR2(chemokine(C-X-C motif)receptor 2)in HL-60 cells and dHL-60 cells.In the same way,the above-mentioned PANC-1cell conditioned medium was used to resuspend neutrophils,and placed in a cell incubator for 24 hours to be transformed into tumor-related neutrophils(Tumor associated neutrophil,TAN).Inoculate TAN in the upper chamber of the transwell chamber,prepare the HFF conditioned medium,add it to the lower chamber of the transwell,and divide into TAN control group,TAN + HFF conditioned medium group,TAN + HFF conditioned medium + GCP-2 antibody group And TAN + HFF conditioned medium + artificial recombinant GCP-2protein(rGCP-2)group.The effect of tumor-associated fibroblasts releasing GCP-2 on the migration ability of TAN cells was measured by counting the number of TAN cells entering the lower chamber.ELISA was used to detect the content of A proliferation inducing ligand(APRIL)in neutrophil-like(dHL-60)and tumor-related neutrophil(TAN)conditioned medium.Western blot verification of pancreatic cancer PANC-1 cell APRIL receptor B cell maturation antigen(BCMA)and transmembrane activator and calmodulin interaction factor(transmembrane activator and CAML-interactor,TACI)expression.Pancreatic cancer PANC-1 cells and dHL-60 cells were co-cultured and divided into PANC-1 cell control group,PANC-1 + dHL-60 cell group,PANC-1 + dHL-60 cell + APRIL antibody group and PANC In the-1+ artificial recombinant APRIL protein(r APRIL)group,the CCK8(Cell counting kit-8)method was used to determine the effect of APRIL release by dHL-60 cells on the proliferation activity of pancreatic cancer PANC-1 cells.The transwell cell migration experiment was divided into PANC-1 cell control group,PANC-1 cell+ neutrophil group,and the change of PANC-1 cell migration ability was detected.Results ELISA results showed that both HFF and CAF cells expressed GCP-2,and the level of GCP-2 in the conditioned medium of the CAF group was significantly higher than that of the HFF group(P <0.001).Western blotting confirmed that dHL-60 cells expressed GCP-2 receptors CXCR1 and CXCR2.The results of TAN cells cultured under HFF conditioned medium showed that the number of TAN cells in the TAN + HFF conditioned medium group was significantly higher than that in the control group(P <0.01),suggesting that under the conditions of HFF releasing GCP-2,TAN migration ability Enhanced.The number of TAN cells in the TAN + HFF conditioned medium group was significantly higher than that of the TAN + HFF conditioned medium + GCP-2antibody group(P <0.01),suggesting that GCP-2 was weakened or blocked under the action of the GCP-2 antibody Stimulation,TAN migration ability decreased;there was no statistically significant difference between the TAN +HFF conditioned medium group and the TAN + HFF conditioned medium +rGCP-2 group(P> 0.05).ELISA detected that both dHL-60 and TAN cells expressed APRIL,and the level of APRIL in the conditioned medium of the TAN group was significantly higher than that of the dHL-60 group(P <0.05).Western blotting confirmed the expression of APRIL receptors BCMA and TACI on the surface of pancreatic cancer PANC-1 cells.The CCK8 method showed that the PANC-1 cell activity in the control group was significantly lower than that in the PANC-1 + TAN group(P <0.001)and the PANC-1 +r APRIL group(P <0.05),suggesting that under the conditions of TAN releasing APRIL or adding r APRIL,PANC-1 activity increased;PANC-1 + TAN group PANC-1 activity was significantly higher than PANC-1 + TAN + APRIL antibody group(P <0.05),suggesting that under the action of APRIL antibody,APRIL stimulation was weakened or blocked,PANC-1 activity decreased.Transwell cell migration experiments showed that the number of PANC-1 cells in pancreatic cancer PANC-1 cells + neutrophils group migrated much more than that in pancreatic cancer PANC-1 cells group,the difference was statistically significant(P <0.05).Conclusion This study preliminarily confirmed that tumor-associated fibroblasts can significantly enhance the migration ability of TAN by secreting GCP-2 and promote the recruitment of neutrophils into pancreatic cancer microenvironment to become tumor-related neutrophils;tumor-associated neutrophils can enhance the proliferation and migration of pancreatic cancer cells by releasing APRIL.By studying the interaction between tumor-associated fibroblasts,TAN and pancreatic cancer cells,it may provide new ideas for studying the mechanism of malignant progression of pancreatic cancer and improving the comprehensive therapeutic effect of pancreatic cancer.