The Role Of Tumor Associated Neutrophils In Progression Of Gastric Cancer Bearing Mice Model And Mechanisms

Objective:To explore the role of tumor associated neutrophils(TANs)in progression of gastric cancer bearing mice model and the relationship with lysyl oxidase(LOX),matrix metal oproteinases(MMPs)and vascular endothelial growth factor(VEGF).Methods: Gastric cancer bearing mice model were established by injecting human gastric cancer cell line-AGS into BALB/c-nude mice(model group,n=10).For intervention group(n=10),from 10 days after AGS cells were injected,anti-LY6 G MAb 1A8 were intratumorally injected into mice transplanted tumors to delete the neutrophils.Mice in the control group(n=6)were injected with an equal volume of normal saline at the same locations.CD34 and PAS double staining was used to measure microvessel density(MVD)and vasculogenic mimicry(VM)in tumor tissues.Immunohistochemistry method was used to detecte the number of TANs and the protein levels of LOX,MMP-2,MMP-9 and VEGF in tumors tissues.The correlations between TANs with LOX,MMP-2,MMP-9,VEGF,MVD,VM in tumor tissues were evaluated through Pearson or Spearman’s correlation analysis.Neutrophils were separated from blood or tumor tissues.Flow cytometric method(FCM)was used to analyze the amounts of neutrophils in blood or tumor tissues.And mRNA expression levels of LOX,MMP-2,MMP-9 and VEGF in neutrophils were quantitated by means of real-time PCR assays.The levels of LOX,MMP-2,MMP-9 and VEGF in neutrophils were assessed by western blotting.Results: The rates of tumorigenesis were 100% in both model group and intervention group,there were no statistical difference in mice weight,tumor weight and tumor volume between model group and intervention group(P>0.05).Compared with model group,the number of MVD in intervention group was decreased(P<0.05),and there was no statistical difference in the number of VM between two groups(P>0.05).The number of TANs in intervention group was lower than that in model group(P<0.01);and the percent of TANs in intervention group was lower than that in model group(P<0.01).The protein levels of LOX,MMP-2,MMP-9,VEGF in tumors of intervention group was decreased than that of model group(P<0.05).Compared with model group,mRNA expressions of LOX,MMP-2,MMP-9 and VEGF in TANs were lower in intervention group(P<0.01);and relative expressions of LOX,MMP-9 and VEGF in TANs were also lower in intervention group(P<0.05),but there was no difference of MMP-2(P>0.05).The number of TANs were positively correlated with the levels of LOX(r=0.855,P=0.00),MMP-2(r=0.515,P=0.029),MMP-9(r=0.577,P=0.012)and MVD(r=0.884,P=0.00).There was no correlation between the number of TANs and VEGF(r=0.381,P=0.12),VM(r=-0.45,P=0.061).The percent of neutrophils in blood of model group was higher than that of control group(P<0.01),and number of neutrophils in blood of intervention group was lower than that in control group and intervention group(P<0.01).Compared with control and model group,the mRNA expressions of LOX,MMP-2,MMP-9 and VEGF in blood neutrophils of intervention group were decreased(P<0.05);but compared with control group,mRNA expression of MMP-9 was increased in model group(P<0.05),and there was no statistical difference of LOX,MMP-2 and VEGF in model group(P>0.05);Protein levels of MMP-2,MMP-9 and VEGF in blood neutrophils of intervention group were decreased than that of model group(P<0.05),but there was no difference of LOX among three group(P>0.05).Protein level of VEGF in blood neutrophils of model group was higher than that in control group(P<0.01).Conclusions: The neutrophils in the blood of gastric cancer bearing nude mice were more than those in the normal mice.In gastric cancer bearing mice model,the majority TANs exist in tumor tissues are N2 type and can promote tumor angiogenesis by secreting LOX,MMP-2、MMP-9 and VEGF.