Study On The Mechanism Of M2 TAMs Involved In The Malignant Phenotype Of Esophageal Squamous Cell Carcinoma

Objective: In the first place,to explore the correlation between M2 type tumor associated macrophages(M2 type TAMs)infiltration and the prognosis of patients with esophageal squamous cell carcinoma(ESCC),analysis of the clinicopathological significance of M2 type TAMs infiltration in ESCC.In thesecond place,M2 type TAMs were co-cultured with ESCC cell lines in vitro to mimic the tumor microenvironment in the body,and to discuss the the effects of M2 type TAMs on EMT,proliferation,migration and invasion of the ESCC cells.In the third place,collects co-culture supernatanthe and screened differentially expressed proteins factors in the co-culture systems by Antibody microarray,to investigate the influence of M2 type TAMssecreted protein factors on EMT,migration and invasion of the ESCC cells.Meanwhile,the molecular mechanism of M2 type TAMs involved in the regulation of malignant phenotype of esophageal cancer cells was preliminarily investigated.Methods: 1)Meta analysis of the clinicopathological significance of M2 type TAMs infiltration in ESCC.Detection of the infiltration of M2 type TAMs in ESCC tissue array by immunohistochemical staining,Chi-square test was used to analyze the correlation between infiltration of M2 type TAMs and overall prognosis of patients with ESCC.Cross-table statistical analysis was employed to analyze the clinical correlation between infiltration of M2 type TAMs and clinicopathological parameters;2)To construct a co-culture system of M2 type TAMs and ESCC cells,to observe the morphological changes of ESCC cells after co-cultured with M2 type TAMs.QRT-PCR and Western-blot were used to detect the relative expression of EMT related markers in ESCC cells after co-cultured with M2 type TAMs.The proliferation,migration and invasion activities of Eca109 and KYSE-150 were detected by MTT,Wound-healing and Transwell Assay;3)Screening of differentially expressed protein factors after co-culture by antibody chip,to observe the effect of the protein factorsecreted by M2 type TAMs on the morphological changes of ESCC cells.QRT-PCR and Western-blot were used to detect the relative expression of EMT related markers in ESCC cells after induction by protein factors.The proliferation,migration and invasion activities of ESCC cells were detected by MTT assay,Wound-healing assay and Transwell assay after induction by protein factors.Preliminary to discuss the molecular mechanism of M2 type TAMs involved in the regulation of malignant phenotype of ESCC cells.Results: 1)Meta analysis demonstrated that M2 type TAMs infiltration was associated with lymph node metastasis,clinical staging and T staging in patients with ESCC.Immunological staining revealed that M2 TAMs infiltration was significantly higher in ESCC tissues in compared with paired normal control tissues(P<0.05).Clinicopathological analysis showed that there was significant association between M2 TAMs infiltration and lymph node metastasis(P=0.018)and T staging(P=0.021);however,there was no significant difference between M2 TAMs infiltration and clinicopathological parameters,including age(P=0.862),gender(P=0.249),clinical stage(P=0.563),tumor diameter(P=0.898).There was a negative correlation between M2 type TAMs infiltration and prognosis of patients with ESCC(P<0.05)after analysis using Kaplan-Meier survival curve;2)ESCC cells co-cultured with M2 type TAMs result in EMT of ESCC cells,q RT-PCR and Western-blot showed that the relative expression of the epithelial marker was down-regulated,while the relative expressions of the mesenchymal markerswas up-regulated as compared with the non-cocultured cells.The migration and invasion abilities of ESCC cells were increased while the proliferation activity was not significantly changed after co-cultured with M2 type TAMs;3)Antibody microarray screening demonstrated that after co-cultured of M2 TAMs and ESCC cells,the expression of IL-1β(up to 829.43 times)and MCP2(up to 808.41 times)increased significantly,and the difference was statistically significant compared with that of non-coculture group(P<0.05).Cell experiments show that IL-1βand MCP2 were able to facilitate ESCC cells EMT,migration and invasion as compared with the untreated cells.However,there was no significant change in the proliferation ability of ESCC cells after induced by IL-1β.In addition,Western-blot revealed that IL-1βand MCP2 activation of NF-κB signaling pathway,make use of NF-κB pathway inhibitor(BAY11-7082)can weaken the phosphorylation level of the NF-κB signaling pathway.Conclusion: 1)The invasion density of M2 TAMs in ESCC tissues was significantly higher than that in adjacent tissues.M2 TAMs infiltration was significantly correlated with prognosis,lymph node metastasis and T staging of patients with ESCC;2)ESCC cells co-cultured with M2 type TAMs was able to facilitates ESCC cells EMT,migration and invasion as compared with the non-cocultured group;.3)The IL-1βand MCP2 secreted by type M2 TAMs can promote EMT in ESCC cells and enhance the migration and invasion abilities of ESCC cells.The IL-1βand MCP2 secreted by type M2 TAMs may be involved in the regulation of the malignant phenotype of ESCC cells by activating the NF-κB signaling pathway.