驱化因子CCL5介导骨髓间充质干细胞增加肾透明细胞癌细胞侵袭迁移能力

摘要

目的:明确骨髓间充质干细胞(BM-MSCs)对肾透明细胞癌侵袭迁移能力的影响,并通过体内、体外研究阐明阻断趋化因子CCL5信号对于抑制肾细胞癌侵袭进展效应评价.方法:利用体外细胞共培养模型,验证BM-MSCs细胞对于肾癌细胞系侵袭迁移能力的影响,利用细胞因子芯片筛选潜在差异性表达的趋化因子,在患者肿瘤组织及对应正常肾脏组织中确认该差异性细胞因子的表达情况,最终通过体外细胞侵袭、转移模型及体内小鼠成瘤实验,利用CCL5中和抗体特异性阻断该信号通路,验证该信号通路作用的特异性.结果:骨髓间充质干细胞可以显著增加肾癌细胞系(OS-RC-2、SW839)体外侵袭、迁移能力(P＜0.05);蛋白印迹法提示高侵袭性相关蛋白表达MMP9、MMP2提高(P＜0.05),并增加细胞EMT过程;细胞因子芯片筛选后发现细胞共培养后CCL5信号特异性高表达(P＜0.05);Western blot蛋白印迹法显示肿瘤组织中CCL5蛋白表达水平较正常肾脏组织提高(P＜0.05);体外细胞侵袭、迁移实验结果提示,CCL5特异性中和抗体阻断后能够显著抑制BM-MSCs导致的肾细胞癌侵袭、迁移能力(P＜0.05);体内小鼠成瘤实验发现CCL5特异性中和抗体阻断能够显著降低小鼠总体生存时间(P＜0.05).结论:BM-MSCs能够特异性通过CCL5信号增加肾透明细胞癌侵袭、迁移能力,特异性阻断CCL5信号后能够显著抑制肾癌细胞侵袭、迁移及延长荷瘤小鼠的生存时间,CCL5是潜在的肾透明细胞癌治疗新靶点.%Objective:The concept of detrimental contribution of the circulating bone marrow derived mesenchymal stem cells (BM-MSCs) in selective cancer progression is emerging, their potential roles in renal cell carcinoma (RCC) remained unclear.In this study,we evaluated the potential roles of CCL5 signals in BM-MSCs induced RCC progression.Methods:Transwell assay was performed for the migration and invasive ability detection before and after BM-MSCs treatment.The potential signal pathway was screened by cytokine superarray in the condition medium.Then we further confirmed candidate cytokine expression in the RCC and non-tumor tissue by Western blot assay.Finally, the interruption effect of cytokine specific antibody was applied by invasion, migration and in vivo mice transplanted tumor model.Results:The in vitro invasion assay study demonstrated that the recruited BM-MSCs might lead to increased RCC metastatic and migrative potential (P ＜ 0.05), and also upregulating the metastatic and migrative related proteins expression (MMP9, MMP2, Vimentin).The increased metastatic potential was due to alteration of CCL5 signaling (P ＜ O.05).Interruption of such signaling using CCL5 antibody led to suppress BM-MSCs-induced migration, invasion ability within RCC (P ＜ 0.05), and also increase the survival rate in mice model (P ＜0.05).Conclusion:Together, these results suggest the important role of BM-MSCs as a component in RCC microenvironment to promote metastasis and CCL5 may be a new potential target for metastatic RCC treatment by blocking BM -MSCs/RCC interaction.