Effect Of Tumor-associated Macrophages On STAT3 Activation And Promotion Of Invasion And Metastasis Of Esophageal Squamous Cell Carcinoma

Background and PurposeEsophageal cancer is one of the most predominant digestive malignancy,among which esophageal squamous cell carcinoma(ESCC)is the most common.China has the highest incidence and mortality rate of esophageal cancer in the world,and its mortality rate ranks the fourth among malignant tumors.Its five-year survival rate is only 10 to 25 percent.Tumor invasion and metastasis are the main causes of poor overall therapeutic effect and low long-term survival rate.Tumor microenvironment(TME)is refers to the internal environment of local tumor cell growth in tumor tissue,also known as tumor stroma.Macrophages in tumor stroma usually express the M2 phenotype,which is closely related to tumor,and are called tumor-associated macrophages(TAMs).It is the largest number of immune cells in the tumor microenvironment,can secrete a variety of cytokines,stimulate specific signaling pathways.So it plays an important role in promoting tumorinvasion,angiogenesis,matrixremodeling,metastasis,tumor immunosuppression,radiotherapy and chemotherapy resistance.Signal transduction and transcriptional activator 3(STAT3)are important transcription factors that mediate intercellular signal transduction and are involved in various physiological processes such as cell differentiation,immune response and embryonic development.STAT3 is a transcriptional activator and an oncogene.However,a large number of studies have shown that STAT3 continues to be activated in a variety of tumors,and is closely related to biological behaviors such as tumor cell proliferation,apoptosis,angiogenesis,infiltration and metastasis,immune escape,and epigenetic regulation.STAT3 is also an important link between cancer and inflammation,leading to pro-tumor and anti-tumor immune responses.Recent studies have found that the STAT3 pathway is one of the main pathways related to cytokine activation.In gastric cancer,liver cancer,lung cancer,breast cancer,myeloma and other polymorphic tumors,the invasion of TAMs is related to the activation of STAT3,and the amount of TAMs invasion in tumor stroma is closely related to the survival and prognosis of patients.However,until now,it is not clear whether TAMs plays a role in the activation of STAT3 and the promotion of the invasion and metastasis of esophageal squamous cell carcinoma.Part ? expression and significance of CD163,CD206,STAT3 and p-STAT3 in human esophageal squamous cell carcinoma tissueObjectiveTo investigate the correlation between TAMs and STAT3 activation and its effect on invasion and metastasis of esophageal squamous cell carcinoma.Methods1.Immunohistochemical SP assay was used to detect the expression of TAMs surface labeled molecules CD163 and CD206(respectively labeled TAMs)and STAT3 protein and its activated form p-STAT3 protein in 100 cases of human esophageal squamous cell carcinoma and adjacent normal tissues.2.Through the expression of CD163 and CD206 proteins,the correlation between the number of TAMs(Mean values of all the cases were taken as the standard,higher than mean values were considered as high expression,and lower than mean values were considered as low expression.mean value of CD163~+cells:16.62±5.34/HP;mean value of CD206~+cells:17.59±4.67/HP)and STAT3,p-STAT3was analyzed,as well as the relationship between TAMs,STAT3 and p-STAT3 and the clinicopathological characteristics.3.Compare the differences of TAMs labeling by CD163 and CD206.Results1.The number of CD163~+cells and CD206~+cells that are TAMs in the stroma of human esophageal squamous cell carcinoma was significantly higher than that in the adjacent normal tissue interstitium(P<0.01).The expression of STAT3 and p-STAT3in human esophageal squamous cell carcinoma was significantly higher than that in adjacent normal tissues(P<0.01).2.In the stroma of human esophageal squamous cell carcinoma,the number of CD163~+cells and CD206~+cells that are TAMs was positively correlated with tumor invasion depth(P<0.01),lymph node metastasis(P<0.01)and TNM stage(P<0.05),but not with gender,age and tumor histological grade(P>0.05).In human esophageal squamous cell carcinoma,the expression of STAT3 protein was positively correlated with tumor invasion depth(P<0.05),histological grade(P<0.05)and TNM stage(P<0.01),but not with gender,age or lymph node metastasis(P>0.05).p-STAT3protein expression was positively correlated with tumor invasion depth(P<0.05),lymph node metastasis(P<0.01)and TNM stage(P<0.01),but not with gender,age or tumor histological grade(P>0.05).3.The number of CD163~+cells and CD206~+cells that are TAMs in the stroma of human esophageal squamous cell carcinoma were all positively correlated with the expression of STAT3(P<0.05)and p-STAT3(P<0.01)in cancer cells.4.Among the 100 cases,there were 84 cases of high expression of CD163,82cases of high expression of CD206,77 cases of common high expression and 11cases of common low expression,with a consistent rate of 88%.Part ? Effect of tumor-associated macrophages on STAT3 activation and invasion of esophageal squamous cell carcinoma cellsObjectiveTo investigate the effect of TAMs on STAT3 activation and promoting invasion of esophageal squamous cell carcinoma cellsMethods1.Establishment of tumor-related macrophage modelHuman monocytes(THP-1)were induced and differentiated into tumor-associated macrophages(TAMs)by PMA combined with interleukin 4(IL-4)or interleukin 6(IL-6).The morphological changes of cells were observed by inverted microscope.Expression changes of molecular markers CD206 and CD163 on the surface of TAMs were detected by RT-PCR to verify whether TAMs was successfully induced.2.cell co-cultureExperimental cells were divided into three groups for cell culture:blank group:Esophageal squamous cell carcinoma cell EC9706 was isolated and cultured as blank group.co-culture group:CM(conditioned medium is the culture supernatant of TAMs in this paper)was co-cultured with esophageal squamous cell carcinoma cells EC-9706 for 48h.Co-culture plus blocker group:JAK2/STAT3 pathway specific blocker AG490was added to the co-culture group to block the STAT3 pathway.3.Effect of TAMs on STAT3 expression in human esophageal squamous cell carcinoma cellsThe expressions of STAT3 and p-STAT3 in cells of the blank group,co-culture group and co-culture group plus blocker group were detected by Western-Blotting and RT-PCR,respectively.It is to determine whether TAMs has an activation effect on STAT3.4.Effects of TAMs on invasion and migration of human esophageal squamous cell carcinoma cellsThe changes of cell invasion and migration ability in the blank group,co-culture group and co-culture group plus blocker group were detected by Transwell invasion test and scratch test,respectively,so as to determine whether TAMs can promote the invasion and migration of esophageal cancer cell lines through the STAT3 pathway.Results1.Test of tumor-related macrophage modelObservation of cell morphology by inverted microscope:After thp-1 induced differentiation,cell morphology changed significantly.The morphology of normal THP-1 cells is uniform and round,with good refractive property,and the cells grow in suspension.After directional induction and differentiation into TAMs,the cells were polygonal,with pseudopodia,increased in volume,and grew into clusters and adherent growth.Results of RT-PCR:After THP-1 was differentiated by PMA in combination with IL-4 or IL-6 respectively,the expression level of CD163mRNA and CD206mRNA that are the specific markers on TAMs cell surface were all significantly higher than that of THP-1(P<0.05),indicating that TAMs induction was successful;The expression level of TAMs CD163mRNA induced by PMA combined with IL-6 was significantly higher than that induced by PMA combined with IL-4(P<0.05),but there was no significant difference in the expression level of CD206mRNA(P>0.05).2.Effect of TAMs on STAT3 expression in human esophageal squamous cell carcinoma cellsResults of RT-PCR:Compared with the blank group,STAT3mRNA was significantly increased in the co-culture group(P<0.05);compared with the co-culture group,the expression of STAT3mRNA was decreased in co-culture plus blocker group(P<0.05).Results of western-blotting:Compared with the blank group,the expressions of STAT3 protein and p-STAT3 protein were all significantly increased in the co-culture group(P<0.05);compared with the co-culture group,there was no significant change in STAT3 protein(P>0.05),while the expression of p-STAT3 protein was significantly decreased in co-culture plus blocker group(P<0.05).3.Effects of TAMs on invasion and migration of human esophageal squamous cell carcinoma cellsResults of cell invasion test and scratch test:Compared with the blank group,the invasion ability and migration ability in the co-culture group were significantly enhanced(P<0.05);compared with the co-culture group,the invasion ability and migration ability in co-culture plus blocker group were significantly reduced(P<0.05).Conclusions1.In the stroma of esophageal squamous cell carcinoma,the increased number of tumor-related macrophages(TAMs)has an activation effect on STAT3 signal pathway,thereby promoting the invasion and metastasis of esophageal squamous cell carcinoma.2.PMA combined with IL-4 and IL-6 can induce the differentiation of human monocyte THP-1 into TAMs.Combined with IL-6-induced TAMs,CD163expression was higher.