The Research On Effect Of Tumor Microenvironment On Lymph Node Metastasis Of Colon Cancer By CC Chemokine Receptor 7-Mediated Mechanism

Part I Effect of CCL21/CCR7 on MMP-9 expression in the stable transfected SW480 cell line with the CCR7 shRNA vector in vitroObjective To investigate effect of CCL21/CCR7 on MMP-9 expression in the stable transfected SW480 cell line with CCR7 shRNA vector (SW480/CCR7-) and negative control vector (SW480/control)Methods SW480/CCR7-and SW480/control cells were treated with Ong/ml and 100ng/ml CCL21 for 24 hours. Levels of MMP-9 protein and MMP-9 activity were analyzed by enzyme linked immunosorbent assay and gelatin zymography.Results 100ng/ml CCL21 could significantly regulate MMP-9 protein levels and activity in SW480/control (P<0.05).The effect of CCL21 was not obvious in SW480/CCR7- cells (P>0.05).CCR7 gene silencing blocked MMP-9 production induced by CCL21.Conclusion CCL21/CCR7 could induce production of MMP-9 in human colon cancer cell line SW480. Part II Effect of CCR7 gene silencing on MMP-9 expression and lymph node metastasis of colon cancer in vivoObjective To investigate effect of CCR7 gene silencing on MMP-9 expression in cancerous tissues, the growth and lymph node metastasis of colon cancer and the survival of nude mice in a xenografted mouse model.Methods The mice were divided into SW480/CCR7" group treated with SW480/CCR7-cells and SW480/control group treated with SW480/control cells. The growth of colon cancer was observed by using whole body fluorescence imaging. Levels of MMP-9 protein and activity in xenografted tissues were analyzed by enzyme linked immunosorbent assay and gelatin zymography. Lymph node metastasis was detected by immunohistochemistry analysis. Kaplan-Meier survival analysis evaluated the survival of nude mice.Results The MMP-9 protein levels and activity in SW480/control group cancerous tissues were significantly higher than SW480/CCR7- group (P<0.05).The xenografts in SW480/control group had greater tumor size and more lymph node metastases than SW480/CCR7- group (P<0.05).The mice in SW480/control group had shorter survival as compared with SW480/CCR7" group (P<0.05).Conclusion Silencing expression of CCR7 could inhibit MMP-9 production and lymph node metastasis of colon cancer. Partâ…¢Effect of human bone marrow mesenchmal stem cells on the proliferation and invasion of colon cancer cell line SW480Objective To culture and identify human bone marrow mesenchymal stem cells (hBMSCs); to detect expression of CCR7 in hBMSCs; to evaluat effect of hBMSCs on the proliferation and invasion of colon cancer cell line SW480.Methods hBMSCs were primary cultured by density gradient centrifugation and adherent from human bone marrow. The cell surface molecular markers were identified by flow cytometry. Differentiation culture of hBMSCs for mesenchymal lineage was achieved when cells were treated with adipogenic differentiation medium and osteogenic differentiation medium for two weeks. Expression of CCR7 was analyzed by immunofluorescence and western blotting in hBMSCs. Effect of hBMSCs on the proliferation and invasion of colon cancer cell line SW480 was evaluated by MTT and invasion assayResults hBMSCs were sucessfully cultured. Cells surface molecular markers were CD34", CD45-, CD44+and CD90+. They had the ability of adipogenic differentiation and osteogenic differentiation. CCR7 expressed in hBMSCs. The conditioned medium of hBMSCs could stimulate the growth of SW480 cells (P<0.05). Co-culture of hBMSCs and SW480 could stimulate tumor cells invasion (P<0.05).Conclusion Human bone marrow mesenchymal stem cells had expression of CCR7 protein. They could stimulate the proliferation and invasion of SW480 cells. Part IV Effect of human bone marrow mesenchymal stem cells on the lymph node metastasis of colon cancer by CCR7-mediated mechanism in vivoObjective To evaluate effect of human bone marrow mesenchymal stem cells on the lymph node metastasis of colon cancer; to discuss the role of CCR7 in this process of lymph node metastasis which hBMSCs interacted with colon cancer cells.Method The mice were divided into SW480/CCR7-, SW480/control, SW480/CCR7-+hBMSCs and SW480/control+hBMSCs group. The growth of colon cancer was observed by using whole body fluorescence imaging. Lymph node metastasis was detected by immunohistochemistry analysis. Kaplan-Meier survival analysis evaluated the survival of nude mice.Results The mice in SW480/control+hBMSCs group had greater tumor size, more lymph node metastases than SW480/control group (P<0.05). The mice in SW480/CCR7-+hBMSCs group had greater tumor size, more lymph node metastases and shorter survival than SW480/CCR7- group (P<0.05).Conclusion The human bone marrow mesenchymal stem cells could stimulate cancer cells growth and lymph node metastasis by CCR7-mediated molecular mechanism.