| Objective:To observe the role of CD4~+Treg cells onthegrowth,invasion and metastasis in Lewis lung cancer mouse model.Explore the effects of CD4~+CD25~+T cells on the function of Lewis lung cancer cells in vitro.Whether regulatory T cells could influence the type conversion of macrophages through adenosine-adenosine receptor pathway and promote the migration and migration of Lewis lung cancer by regulatory T cells were discussed.The mechanism of invasion was preliminarily explored.Methods:1.Establish the subcutaneous transplantation model of Lewis lung cancer in mice,and observe the growth and survival rate of the tumor by adoptive transfer CD4~+CD25~+Treg cells.HE staining was used to observe the number of nodules that metastasized from tumors to distant lung.2.The effect of CD4~+CD25~+Treg cells on the proliferation of tumor cells was observed by co-culture of CD4~+CD25~+Treg cells and LLC in vitro CCK8 test.The effect of CD4~+CD25~+Treg cells on the migration and invasion of LLC cells was evaluated by Transwell experiment.3.The tissue localization of CD4~+CD25~+Treg cells and tumor-related macrophages,the expression of CD73 and CD39 molecules on the surface of tumor infiltrating CD4~+CD25~+FoxP3~+Treg cells and the interaction between CD4~+CD25~+FoxP3~+Treg cells and tumor-related macrophages were determined by immunofluorescence staining,in vivo CD4~+CD25~+Treg cell clearance test and flow cytometry.4.The role of adenosine/adenosine receptor pathway in the regulation of macrophage polarization by CD4~+CD25~+Treg cells was clarified,with or without intratumoral adenosine receptor blocking.5.The interaction between CD4~+CD25~+Treg cells and macrophages through adenosine/adenosine receptor pathway was verified by cell co-culture,flow cytometry and Q-PCR in vitro.Results:1.The results of flow cytometry showed that the number of CD4~+CD25~+FoxP3~+Treg cells in spleen of tumor-bearing mice was significantly higher than that of normal control group.CD4~+CD25~+Treg cells were injection via tail vein,and promote the growth of tumor and reduce the survival rate of mice bearing tumors.Moreover,the number of lung metastatic tumor nodules in tumor-bearing mice increased significantly after CD4~+CD25~+Treg cells were adoptive transfered.2.The results of co-culture of CD4~+CD25~+Treg cells and LLC showed that CD4~+CD25~+Treg cells did not directly promote the proliferation,migration and invasion of LLC.3.Immunofluorescence staining of tumor tissues in tumor-bearing mice showed that there were F4/80~+macrophages around CD4~+FoxP3~+Treg cells.The results showed that the number of tumor-related macrophages increased and the expression of CD80,CD86 and CD40 molecules on the cell surface increased after the removal of CD4~+CD25~+Treg cells by cyclophosphamide in vivo.4.Flow cytometry results of CD4~+CD25~+Treg cells in tumor tissues of tumor-bearing mice showed that CD4~+CD25~+Treg cells expressed high levels of CD73 and CD39molecules in tumor tissues of tumor-bearing mice.5.The results of adenosine-adenosine receptor pathway blocking experiment showed that adenosine receptor blocker inhibited the growth of Lewis lung cancer after intratumoral injection,and the results of flow cytometry showed that the expression levels of 1A/E,CD80 and CD86 on the surface of tumor-related macrophages increased after intratumoral injection of adenosine receptor blocker.6.The results of co-culture of CD4~+CD25~+Treg cells and peritoneal macrophages in vitro showed that CD4~+CD25~+Treg cells increased the expression levels of Arg1,MMP9 and VEGFa in macrophages;on the contrary,the expression levels of Arg1,MMP9 and VEGFa in macrophages were inhibited by blocking adenosine receptor.Conclusion:CD4+CD25+Treg cells promote the growth,invasion and metastasis of Lewis lung cancer;CD4+CD25+Treg cells promote the proliferation,migration and invasion of Lewis lung cancer by regulating tumor-related macrophages;CD4+CD25+Treg cells regulate the M2 polarization of tumor-infiltrating macrophages through adenosine-adenosine receptor pathway,thereby promoting the growth,invasion and metastasis of Lewis lung cancer. |
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