The Effect Of Tumor Associated Macrophages On Recurrence And Metastasis Of Gastric Cancer And Its Mechanism

The aim of this study is to analyze cancer stem cell markers and the expression of tumor-associated macrophages in gastric carcinoma tissues,to investigate the relationship between TAMs and cancer stem cells,and study the role of tumor microenvironment in the recurrence and metastasis of gastric cancer.Through a series of clinical and in vitro experiments,we can get a deeper understanding of the role of tumor-associated macrophages in promoting the formation of tumor stem cells,to further explore the role of recurrence and metastasis of gastric cancer and related mechanisms,so as to provide a new strategy for the treatment of gastric cancer.tumor associated macrophages,This paper is divided into three parts:1.Investigation of the infiltration of TAMs on CSCs in gastric cancer tissue and its clinical significance;2.The gastric cancer stemcells were isolated and identified by cloning and suspension culture of AGS cell;3.Effects of macrophages on human gastric carcinoma cell line EMT and its mechanism.Material and methods:1.This study included consecutive 236 patients with histologically confirmed primary GC.TAMs marker CD 163 and CSCs-related proteins were detected by immunohistochemistry(IHC)in GC tissues and their prognostic values were all investigated.2.Tumor spheres rich in gastric cancer stem cells are cultured in the stem cell medium,i.e.serum free DMEM/F12 medium containing EGF and bFGF.Colony formation analysis tumor spheres forming the ability of colony.Gastric cancer stem cells are identificated by immunofluorescent staining.CCK8 method tests the proliferative effect of tumor spheres.The migration ability of tumor spheres is detected by cell scratch and transwell.3.Human gastric cancer cell line AGS and macrophages were co-cultured,and the concentrations of IL-6 system were detected by using ELISA method;exogenous IL-6 effects on tumor stem cell markers were observed using Western Blot;effect of exogenous IL-6 on the invasion of gastric cancer cells were observed by Transwell invasion assay;application of Western Blot detection of exogenous IL-6 on gastric cancer cell epithelial mesenchymal transformation and cancer stem like phenotype by activate STAT3 signaling pathway in the cells.Results:1.High expression of CD 163+ TAMs was found in patents with aggressive characteristics,especially for patents with recurrence.There existed a significant correlation between high expression of CD 163 and CSCs-related markers in GC tissues.In patients with recurrence,high-expression of CD 163 TAMs was an independent worse prognostic factor.2.We observed the morphology and differentiation heterogeneity of gastric cancer cell line AGS cell colonies.AGS cells were plated in a low density,and its clonogenic efficiency was 39.5%.According to morphology phenotype,colonies were classified into three types:tight colonies,intermediate colonies and loose colonies,which took a percentage of 15.1%,65.6%and 19.3%respectively.Immunofluorescent staining was employed to assay expression of cancer stem cell marker CD44 by different colonies,and the tight colonies showed the strongest staining for CD44.Holoelone clones could differentiate into the gastrointestinal epithelium and express the cancer stem cell marker.Holoelone cloning not only forms a Holoclone clone similar to that of the original clone,but also can form Merodone and Paraclone clones,indicating that the Holoclone derived cells have the ability of self-renewal.3.Human macrophages and gastric cancer cell line AGS were co-cultured,and the IL-6 concentration was significantly higher than that of human gastric cancer cell line AGS system(P<0.001);exogenous IL-6 can promote the migration and invasion ability of gastric cancer cells.Exogenous IL-6 can activate CD44 through STAT3 pathway,so that cell transformation to cancer stem cell like phenotype,and epithelial mesenchymal transition.Conclusion:1.High infiltration of TAMs was related to aggressive behavior,associated with aberrant expression of CSC markers,and an independent worse prognostic factor in GC.Targeting TAMs may be a potential treatment strategy for GC,including patients with recurrence.2.AGS cell colonies possess heterogeneity relating to morphology and differentiation,and can be classified into tight colonies,intermediate colonies and loose colonies morphologically.Cells derived from Holoclones exhibit the characteristics of cancer stem cells such as self-renew and multi-differentiation.3.The effect of macrophages on the phenotype of cancer stem cells through IL-6/STAT3 signaling pathway.