The Effects Of Exosomes Derived From Gastric Cancer Cells On Chemotaxis And Polarization Of Normal Human Neutrophils And Mechanisms

Objective To investigate whether gastric cancer cells can chemotaxis and polarized normal neutrophils through exosome-mediated information transmission,providing new clues for understanding the mechanism of gastric cancer growth,invasion and metastasis and anti-tumor treatment.Methods Exosomes from human gastric cancer cells(MGC-803,SGC-7901,AGS)and human gastric mucosal cells(GES-1)were isolated and purified by differential hypercentrifugation,and the exosomes were identified by transmission electron microscopy,western blot and nanoparticle tracking analysis.Western blotting was used to compare the contents of the neutrophil chemokines CXCL1,CXCL2,CXCL5,CXCL8 and the polarizing factors TGF-β and IFN-β.The exosomes were used to interfere with normal neutrophils,and the uptake of neutrophils was observed by confocal microscopy.The m RNA and protein levels of neutrophils were detected by q PCR and Western blot.Transwell chamber assay was used to detect the effect of exosomes on the migration of neutrophils.The production of enzymes and factors related to the polarization of neutrophils(TNF-α,ROS,ICAM-1,NE,MMP-9,LOX)were detected by q PCR,Western blot and ELISA.Human multi-pathway phosphorylation microarray was used to detect the changes of related signaling pathways after the intervention of exosomes derived from MGC-803 gastric cancer cells with neutrophils.Results 1.The exosomes isolated and purified from the supernatant of cell culture presented a cup-shaped or double-concave disk shape under transmission electron microscope.Western blot analysis revealed that exosomes had high expression of CD63 and CD9,low expression of tam-actin,and low or no expression of cytochrome C.The particle size of exosomes ranged from 30 nm to 150 nm,and the peak particle size was about 120 nm.2.Western blot detection revealed that the exosomes contained cytokines such as: CXCL-1,2,5,8,TGF-β and IFN-β required for chemotactic and polarized neutrophils.The expression levels of chemokines CXCL1,CXCL2,CXCL5 and CXCL8 in gastric cancer cell-derived exosomes were significantly higher than that in human gastric mucosal cell-derived exosomes(P<0.05).Expression levels of the polarizing factors TGF-β and IFN-β in the gastric cell-derived exosomes were higher than that in the human gastric mucosal cell derived exosomes(P<0.05).The expression of TGF-β in SGC-7901-exosomes and AGS-exosomes was higher than that in MGC-803-exosomes(P<0.05).3.Exosomes were ingested by neutrophils under laser confocal microscopy.With the extension of co-culture time,the uptake of exosomes by neutrophils may increase.4.The Transwell test found that compared with the control group(without exosomes),the experimental group(with exosomes)all promoted the increase of neutrophil migration,P<0.05.Moreover,with the increase of exosome dose,the migration ability of neutrophils was gradually enhanced(P<0.05).Compared with the exosomes derived from human gastric mucosa cells,the cell-derived exosomes of gastric cancer had a stronger effect on the migration of neutrophils(P<0.05).5.The results of q PCR and Western blot on the expression of CXCR1 and CXCR2 of neutrophil surface chemokine receptors showed that the addition of gastric cancer cell derived exosomes significantly promoted the expression of CXCR1 and CXCR2 compared with the control group(without exosomes),P<0.05.With the increase of exosome dose,the expression of CXCR1 and CXCR2 was enhanced(P<0.05).Human gastric mucosal cell-derived exosomes increased CXCR2 expression,P<0.05.6.Different doses of gastric cancer cell-derived exosomes or human gastric mucosa cell-derived exosomes were co-cultured with normal neutrophils.QPCR,Western blot and ELISA detected the production of N1(TNF-α,ICAM-1)and N2(NE,MMP-9,LOX)enzymes and factors related to the polarization of neutrophils.The results showed that with the increase of AGS-exosomes and SGC-7901-exosomes dose(50μg,100μg,200μg),the levels of neutrophils LOX,MMP-9,NE m RNA,protein and secretion in the cell culture supernatum all increased gradually compared with the control group(without Exosomes),and the levels of TNF-α and ICAM-1 all decreased gradually,P<0.05.When the dose of MGC-803-exosomes was 100μg and 200μg,the m RNA,protein and secretion levels of neutrophils LOX,MMP-9,NE all increased compared with the control group,and the contents of TNF-α,ICAM-1 gradually decreased,P<0.05.Conclusion The cell-derived exosomes of gastric cancer contain the cytokines CXCL1,2,5,8,TGF-β and IFN-β required for chemotactic and polarized neutrophils.Exosomes derived from gastric cancer cells can chemotactic neutrophils by enhancing neutrophil migration and expression of chemokine receptors CXCR1 and CXCR2.Exosomes derived from gastric cancer cells can induce neutrophils to play a tumor-promoting role by increasing the expression of LOX,MMP-9 and NE proteins and down-regulating the expression of TNF-α,ICAM-1 and ROS.