阻断PD-L1/PD-1在肿瘤干细胞致敏的树突状细胞疫苗靶向肿瘤干细胞中的相关作用及机制

We previously described a strategy to target cancer stem cells (CSC) using CSC-dendritic cell (DC) vaccination. However, the efficacy of CSC targeted therapeutics may be greatest when they are deployed in the adjuvant setting. We have demonstrated SCC7 ALDHhigh-DC (CSC-DC) vaccination plus anti-PD-L1 administration significantly inhibited tumor relapse. In this study, we will check if anti-PD-L1 mAb could enhance the ability of T cells and B cells harvested from D5 ALDHhigh CSC–DC vaccinated animals to kill the ALDHhigh CSCs. And we will build a D5 melanoma model to verify that the therapeutic efficacy of CSC-DC vaccines could be significantly augmented by simultaneous administration of anti-PD-L1 mAb. The lungs and the tumors will be harvested at the end of the experiments to evaluate the level of tumor infiltrating lymphocytes and the percentage of ALDHhigh populations in tumors. This study also will test the expression of CTLA4 on T-lymphocytes in spleens and tumors from the mice with different treatment in the tumor model. This study is therefore undertaken to investigate the mechanisms underlying anti-PD-L1 mAb could strengthen the efficacy of ALDHhigh CSC-DC vaccine. Anti-PD-L1 mAb could increase the cellular and humoral immune responses induced by ALDHhigh CSC-DC vaccine and significantly reduce the percentage of ALDHhigh populations. In addition, the role of CTLA4 in boosting the efficacy of T cells target CSCs will be preliminary discussion in this study.

之前的实验研究已经证实ALDHhigh CSC-DC疫苗能够有效的靶向杀伤CSCs， ALDHhigh CSC-DC疫苗联合PD-L1单克隆抗体可以更加有效的抑制肿瘤复发。我们将检测PD-L1单克隆抗体是否能够增强ALDHhigh CSC-DC疫苗致敏的T淋巴细胞和B细胞靶向裂解CSCs的能力；建立ALDHhigh CSC-DC疫苗联合PD-L1单克隆抗体治疗动物模型，进一步验证PD-L1单克隆抗体能够增强ALDHhigh CSC-DC疫苗的靶向CSCs的抗肿瘤作用；并且收集各组小鼠的肿瘤组织，比较肿瘤浸润T淋巴细胞的数目水平和CSCs所占的比例；比较各组小鼠脾脏和肿瘤浸润T淋巴细胞上CTLA4的水平。本项目的实验目的是通过阐明PD-L1单克隆抗体通过增强ALDHhigh CSC-DC疫苗所诱导的体液免疫和细胞免疫，显著提高ALDHhigh CSC-DC疫苗靶向CSCs的能力。

目的：既往实验研究已证实ALDHhigh CSC-DC疫苗具有靶向杀伤CSCs的能力。本研究主要探讨PD-L1单克隆抗体是否可以显著提高ALDHhigh CSC-DC疫苗靶向CSCs的能力，进而更加有效的抑制乳腺癌肿瘤的肺转移；阐明PD-L1单克隆抗体对ALDHhigh CSC-DC疫苗抗肿瘤体液免疫及细胞免疫的影响，为肿瘤的免疫治疗提供新的方法。.方法：流式细胞术检测ALDHhigh CSCs及ALDHlow 非肿瘤干细胞上PD-L1的表达量；肿瘤负载小鼠脾脏T淋巴细胞和B淋巴细胞PD-1的表达量；体外LDH实验检测PD-L1单克隆抗体是否可增强CTL靶向杀伤CSCs的能力。建立ALDHhigh CSC-DC疫苗联合PD-L1单克隆抗体治疗乳腺癌肿瘤模型，比较各组小鼠肺转移的情况；检测各组肿瘤中CSCs所占的比例及CCR10的表达水平；利用抗体结合实验和LDH细胞毒性试验检测PD-L1单克隆抗体是否可以增强4T1 ALDHhigh CSC-DC疫苗所诱导的靶向CSCs的体液免疫和细胞免疫。.结果：ALDHhigh CSCs上PD-L1的表达量显著高于ALDHlow肿瘤细胞；肿瘤负载小鼠的脾脏B淋巴细胞和T淋巴细胞均表达PD-1。体外实验证实PD-L1单克隆抗体可增强致敏的CTL靶向杀伤CSCs的能力。在建立的动物模型中，ALDHhigh CSC-DC疫苗单独治疗可降低小鼠的肺转移，但ALDHhigh CSC-DC疫苗与PD-L1抗体联合治疗组小鼠肿瘤的肺转移的抑制效果尤为显著。联合治疗组小鼠肿瘤组织中CSCs的比例显著降低，肿瘤细胞上CCR10的表达量也显著降低。联合治疗组小鼠脾脏B细胞PD-1表达量显著减少，增殖活化后的B细胞靶向CSCs免疫作用显著增强，较单独治疗组有显著的统计学意义。联合治疗组T淋巴细胞PD-1表达量显著增加，但CTL靶向CSCs的能力显著增强。.结论：PD-L1单克隆抗体联合ALDHhigh CSC-DC疫苗可以通过减少肿瘤细胞上CCR10的表达量，增强机体靶向CSCs的体液免疫和细胞免疫，进而减少肿瘤组织中CSCs的比例，显著抑制小鼠的乳腺癌肺转移。但联合治疗组T细胞PD-1表达量显著增加与预期不符，仍需进一步研究探讨。

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