The Study Of Effects And Mechanisms Of IL-9 On The Tumor Immune Microenvironment Of Colon Cancer

Objective:To detect the expression levels of IL-9 in colon cancer tissues and paracancerous tissues,and analyze the correlation of IL-9 expressions with the clinicopathological characteristics and colon cancer patients’ prognosis.To study the anti-tumor effects and mechanisms of IL-9 on the tumor microenvironment of colon cancer by constructing tumor-bearing mice model.Methods:92 cases of colon cancer tissues and paracancerous tissues were served as clinical study data.Immunohistochemistry and quantitative Real-time polymerase chain reaction(qRT-PCR)were used to detect the expression levels of IL-9 in colon cancer tissues and paracancerous tissues.And then evaluated the expressive differences and analyzed the correlation of IL-9 expressions with the clinicopathological characteristics and colon cancer patients’ prognosis.The CT-26 cell lines which overexpressing IL-9 were constructed by lentivirus transfection.After amplification and culture in vitro,cell suspension was subcutaneously injected into BALB/C mice to construct the subcutaneous transplanted tumor model.The sizes of the tumor were measured and counted every two days.After the observation period,the tumor growth curve was drawn and the subcutaneous tumors were immediately resected when the mice were killed.One part of the tumors was used to detect the expression levels of IL-9 by immunohistochemistry.The other part of the tumors was made into single cell suspensions to detect the changements of tumor microenvironment,and learn the anti-tumor effects and potential mechanisms of IL-9 on the tumor microenvironment.In addition,the subcutaneous transplanted tumors were constructed again.This time the growth status of mice were observed,and the survival curve of mice was drawn after the observation period.Results:In the 92 cases of colon cancer patients,both the protein and mRNA expressions of IL-9 in colon cancer tissues were significantly lower than that in paracancerous tissues(P<0.001).The expression of IL-9 in colon cancer tissues was related with TNM stage,Ducks stage and lymph node metastasis(P=0.013,0.025,0.004),but not with gender,age,tumor size,differentiation degree and hepatic metastasis(P>0.05).The survival time of colon cancer patients with positive expression of IL-9 was longer than that of patients with negative expression(P=0.015).In vivo,compared with the other two control groups,the growth rate of subcutaneous transplanted tumors in CT26-IL-9 group was slower,the final tumors were smaller(P<0.05),and the survival time of mice was significantly prolonged(P=0.0018).At the same time,the infiltration of CD45+ TILs,CD4+T cells and CD8+T cells in the tumor microenvironment in CT26-IL-9 group were significantly higher than that in the other two groups.And the infiltration of CD4+/CD8+CD44+T cells,CD4+/CD8+IL-7Ra+T cells,CD8+Granzyme B+T cells and CD4+Foxp3+T cells in CT26-IL-9 group were also higher than that in the other two groups.Conclusions:The expression of IL-9 in colon cancer tissues was significantly lower comprared with that in paracancerous tissues,and was closely related to the colon cancer progress and patients’ prognosis,suggesting that IL-9 may have anti-tumor effect.IL-9 inhibited the growth of subcutaneous transplanted tumors and prolonged the survival time of mice.Furthermore,IL-9 was shown to increase anti-tumor immune responses,and exert anti-tumor effects by regulating the distribution of immune cell subsets in thetumor microenvironment.