Neutrophils Induce EMT By Activating The ERK Signaling Pathway To Promote The Migration And Invasion Of Gastric Cancer Cells

Objective:HL-60 cells were induced by 1.25%dimethyl sulfoxide(DMSO)to establish a neutrophil-like(HL-60N)cell model.The changes of phenotype and functions in neutrophils by gastric cancer cells and the effects to the development and progression of gastric cancer were investigated.Neutrophils from mouse bone marrow were isolated and further tested in vitro.The goal of this study is to provide a theoretical basis for the roles of neutrophils in tumor and provide a new strategy for tumor therapy by targeting tumor microenvironment.Methods:(1)Gastric cancer cells were co-cultured with HL-60N cells.Transwell migration assay and matrigel invasion assay were used to detect the migration and invasion of gastric cancer cells.MTT assay was used to detect the survival of gastric cancer cells.The proliferation of gastric cancer cells was detected by using colony formation assay.(2)HL-60N cells were stimulated with the conditioned medium of gastric cancer cells and the culture supernatant from HL-60N cells were collected and were used to treat gastric cancer cells.Transwell migration assay and matrigel invasion assay were used to detect the migration and invasion of gastric cancer cells.MTT assay was used to detect the survival of gastric cancer cells.The proliferation of gastric cancer cells was detected by using colony formation assay.(3)Quantitative real-time RT-PCR was used to detect the expression of IL-1?,IL-6,IL-8,and TNF-? in HL-60N cells co-cultured with gastric cancer cells or treated with gastric cancer cells derived conditioned medium.Gastric cancer cells were co-cultured with HL-60N cells or treated with the culture supernatant from activated HL-60N cells.The expression of E-cadherin,Vimentin,ZEB-1,Slug,Sox2,Oct4,and Nanog in gastric cancer cells was detected by using quantitative real-time RT-PCR and western blotting.The activation of ERK signaling pathway in gastric cancer cells was detected by using western blot.Human gastric cancer cells were pre-treated with the ERK pathway inhibitor and incubated with the culture supernatant from activated HL-60N cells.Transwell migration assay and matrigel invasion assay were used to detect the migration and invasion of gastric cancer cells.(4)The primary neutrophils were isolated from mouse bone marrow by using cell soring and treated with the conditioned medium of mouse gastric cancer cells.The expression of IL-1?,IL-6,and TNF-a in neutrophils was detected by using quantitative real-time RT-PCR.Mouse gastric cancer cells were pre-treated with the ERK pathway inhibitor and incubated with the culture supernatant from activated mouse bone marrow neutrophils.Transwell migration assay and matrigel invasion assay were used to detect the migration and invasion of mouse gastric cancer cells.Results:(1)Co-culture with HL-60N cells promoted the invasion and invasion of gastric cancer cells while had minimal effect on the proliferation of gastric cancer cells.(2)The culture supernatant from HL-60N cells activated by the conditioned medium of gastric cancer cells promoted the invasion and invasion of gastric cancer cells and had no significant effect on the proliferation of gastric cancer cells.(3)After co-culture with gastric cancer cells or treatment with the conditioned medium of gastric cancer cells,the expression of pro-inflammatory factors including IL-1?,IL-6,IL-8,and TNF-? was elevated in HL-60N cells.After co-culture with HL-60N cells or treatment with the culture supernatant from HL-60N cells activated by the conditioned medium of gastric cancer cells,the expression of E-cadherin was decreased and that of Vimentin,ZEB-1,Slug,Sox2,Oct4,and Nanog was increased in gastric cancer cells.The culture supernatant from HL-60N cells activated ERK signaling pathway in gastric cancer cells.Blocking the ERK signaling pathway activation significantly attenuated the promoting role of the culture supernatant from HL-60N cells on gastric cancer cell migration and invasion.(4)The expression of IL-1?,IL-6,and TNF-? in mouse bone marrow neutrophils was increased after treatment with the conditioned medium of mouse gastric cancer cells.Blocking the ERK signaling pathway activation significantly attenuated the promoting effect of the culture supernatant from mouse bone marrow neutrophils on mouse gastric cancer cell migration and invasion.Conclusions:Neutrophils co-cultured with gastric cancer cells or treated with the conditioned medium of gastric cancer cells express increased levels of pro-inflammatory factors,which in turn activates ERK pathway and induces EMT in gastric cancer cells,thereby promoting their migration and invasion.The interplay between gastric cancer cells and neutrophils is critical for the development and progression of gastric cancer,which may represent a novel target for cancer therapy.