Mechanism Of TAMs Mediated HIF-1α Promoting Invasion And Metastasis Of Esophageal Squamous Cell Carcinoma

Objective: To explore the mechanism of M2 tumor associated macrophages(TAMs)promoting the invasion and migration of esophageal squamous cell carcinoma(ESCC)cells by secreting CXCL1 and mediating the stable expression of HIF-1α.Methods: Human peripheral blood mononuclear cells(THP-1)were stimulated with phorbol ester(TPA)to form M0 TAMs,and then induced into M2 TAMs by IL-13 and IL-4;Bioinformatics technology combined with multi factor detection and transcriptome analysis were used to screen and detect the important cytokines involved in the regulation of malignant biological behavior of esophageal squamous cell carcinoma cells.The expression of key factor CXCL1 was further verified by qRT-PCR,and the secretion of M2 TAMs CXCL1 in co-culture system was detected and verified by ELISA;The co localization and binding of CXCL1-CXCR2 in esophageal squamous cell carcinoma cells were analyzed by immunofluorescence technique;Bioinformatics data were analyzed and Western blot was used to detect and verify the role of CXCL1-CXCR2 ligand receptor binding in promoting the activation of STAT3 and PI3K-AKT signaling pathway and regulating the expression of key proteins HIF-1α,VEGF-A and MMP9 in esophageal squamous cell carcinoma cells;Through the intervention of CXCL1 neutralizing antibody,CXCR2 receptor inhibitor,STAT3 and PI3K-AKT signal pathway inhibitor,combined with CCK8 proliferation test,scratch test,Transwell migration and invasion test,M2 TAMs promoted HIF-1αexpression by secreting CXCL1 mediated STAT3 and PI3K-AKT signal pathway,and the effects of VEGF-A and MMP9 secretion on the proliferation,invasion and migration of esophageal squamous cell carcinoma;The distribution of CXCL1,CXCR2,p-Akt and p-STAT3 in esophageal squamous cell carcinoma and adjacent normal tissues were detected by immunohistochemistry;Combined with the detection of clinical parameters and the distribution of M2 TAMs in the early stage,analyze and verify the role of CXCL1 cooperating with the expression of CXCR2,p-Akt and p-STAT3 in the clinical progression and prognosis of esophageal squamous cell carcinoma.Results: 1.THP-1 has the phenotype of M2 TAMs(high expression of Arg-1 and IL-10,low expression of IL-12).2.Bioinformatics database analysis showed that CXCL1 was highly expressed in esophageal cancer and other tumors,and there was a significant correlation with the expression of HIF-1α,VEGF-A and MMP9(P < 0.05).3.In the co-culture system,compared with esophageal squamous cell carcinoma cells,the expression and secretion level of CXCL1 in M2 TAMs were higher,and the secretion of CXCL1 in M2 TAMs increased significantly after co-culture(P < 0.05).4.CXCL1 secreted by M2 TAMs in the co-culture system significantly increased the number of membrane receptor CXCR2 binding CXCL1 of esophageal squamous cell carcinoma cells,and enhanced the expression level of key phosphorylated proteins of STAT3 and AKT signal pathway in esophageal squamous cell carcinoma cells.CXCL1 neutralizing antibody and CXCR2 receptor inhibitor could significantly inhibit the expression of key phosphorylated proteins of STAT3 and AKT signal pathway in esophageal squamous cell carcinoma cells in the co-culture system,and the HIF-1α The expression of VEGF-A and MMP9 decreased significantly,and the invasion and migration of esophageal squamous cell carcinoma cells were also significantly inhibited(P < 0.05).5.In M2 TAMs co-culture system,the expression of HIF-1α,VEGF-A and MMP9 decreased significantly under the action of STAT3 and AKT signal pathway inhibitors,and the invasion and migration ability of esophageal squamous cell carcinoma cells were also significantly inhibited(P < 0.05).6.The expressions of CXCL1,CXCR2,p-Akt and p-STAT3 in esophageal squamous cell carcinoma were significantly higher than those in adjacent normal tissues(P < 0.05).The distribution of M2 TAMs in esophageal squamous tissue was significantly positively correlated with the expression of HIF-1α,VEGF-A and MMP9(all P < 0.05)Conclusion: 1.M2 TAMs mediates CXCL1-CXCR2 axis to participate in the regulation of stable HIF-1αexpression through STAT3 and AKT signaling pathway,which promotes the malignant biological behavior of esophageal squamous cell carcinoma cells.2.The overexpression of CXCL1 and CXCR2 in esophageal squamous cell carcinoma predicts the poor prognosis of tumor patients.