人胰腺癌细胞培养上清对树突状细胞TIM-3表达及其功能的影响

 目的: 研究人胰腺癌微环境对树突状细胞(DCs)T细胞免疫球蛋白及黏蛋白-3(TIM-3)表达及其功能的影响,初步探讨肿瘤微环境调节DCs上TIM-3表达的可能机制。方法: 流式细胞术检测肿瘤浸润树突状细胞(TIDC)以及癌旁组织、胰腺癌患者和健康人外周血诱导DCs上TIM-3的表达;观察人胰腺癌细胞培养液上清对健康人外周血单个核细胞经rhGM-CSF和rhIL-4诱导扩增制备DCs上TIM-3表达的影响;酶联免疫吸附法(ELISA)检测TIM-3⁺DCs组和对照组的DCs分别与凋亡胰腺癌细胞Capan-2共培养上清中细胞因子IFN-β和IL-12水平。结果: 胰腺癌组织中TIDC上TIM-3的表达明显高于癌旁组织及患者和健康人外周血的DCs(P<0.01)。人胰腺癌细胞株Canpan-2、SW1990和Panc-1的上清液较人皮肤成纤维细胞Hs27显著上调DCs上TIM-3表达(P<0.05),联合阻断VEGF、IL-10和PGE₂可明显降低Canpan-2细胞上清对DCs上TIM-3的上调作用(P<0.05)。TIM-3高表达DCs组较低表达组分泌的IL-12和IFN-β水平低,而阻断TIM-3后,IFN-β和IL-12水平均升高(P<0.01)。而这种升高趋势可在加入DNase和RNase后消失。结论: 人胰腺癌TIDC上TIM-3表达升高导致其固有免疫功能受损;肿瘤细胞分泌的VEGF、IL-10和PGE₂可能参与TIM-3的表达调控。

Effect of human pancreatic cancer cell supernatant on expression of TIM-3 and function of dendritic cells

AIM: To investigate the influence and mechanisms of human pancreatic cancer tumor microenvironments on T-cell immunoglobulin mucin-3(TIM-3) expression and function of dendritic cells(DCs). METHODS: Tumor-infiltrating dendritic cells(TIDC) and para-carcinoma tissue DCs were isolated by Ficoll-Hypaque density centrifugation from trypsinized pancreatic carcinoma tissues, and the peripheral blood mononuclear cells were isolated from pancreatic cancer patients or healthy people. The expression of TIM-3 on DCs was detected by flow cytometry. DCs isolated from healthy people peripheral blood mononuclear cells were induced by rhGM-CSF and IL-4. The expression of TIM-3 in the DCs treated with the culture supernatants of Capan-2, SW1990 and Panc-1 pancreatic cancer cells or human skin fibroblast(Hs27) cells for 48 h, and in the DCs treated with supernatants of Capan-2 cells, anti-VEGF-R2, anti-IL-10 and EP2 receptor blocking peptide were evaluated by flow cytometry. The releases of IFN-β and IL-12 in the culture supernatants of DCs pretreated with monoclonal antibody(mAb) to TIM-3 or DNase+RNase, followed by stimulation with apoptotic Capan-2 cells, were detected by ELISA. RESULTS: DCs in tumor microenvironments had higher expression of TIM-3 than the DCs from para-carcinoma tissues and pancreatic cancer patient or healthy people peripheral blood(P<0.01). TIM-3 expression in the DCs treated with the culture supernatants of Capan-2, SW1990 and Panc-1 pancreatic cancer cells for 48 h was much higher than that in Hs27 cells(P<0.05). Treatment with a combination of anti-VEGF-R2, anti-IL-10 and EP2 receptor blocking peptide largely diminished the upregulation of TIM-3 on the DCs mediated by Capan-2 cell supernatants(P<0.05). The concentrations of IFN-β and IL-12 in the DCs with high expression level of TIM-3 were lower than those in the DCs with low TIM-3 expression level. Treatment with mAb to TIM-3 resulted in much more IFN-β and IL-12 releases(P<0.01), but DNase+RNase made this effect disappear. CONCLUSION: TIM-3 serves as a negative regulator of DCs innate immune responses in the pancreatic cancer microenvironments. The secretion of soluble factors to tumor microenvironment by pancreatic cancer cells, including IL-10, VEGF and PGE₂, may contribute to the regulation of TIM-3 expression.