阻断负调节分子CTLA-4- - 增强ALDH+黑色素瘤干细胞疫苗抗瘤效应的新策略

In our prior work, we evaluated the systemic immune responses elicited by ALDH+ melanoma tumour stem cells (MTSCs)-based DC vaccination. The results showed that MTSCs-based vaccination significantly inhibited tumor growth and lung metastasis compared with conventional vaccine. It is interesting to note that MTSCs-based vaccination significantly decreased MTSC population in tumor. However, it has recently been identified that MTSCs can evade antitumor immunity. Thus, the immunoevasive characteristic of MTSCs should be a consideration in the design of targeting of MTSCs elimination experiments so as to improve the quality and applicability of results. CTLA-4 is a key negative regulator of T cell activation. The immunotherapy of melanoma makes significant progress due to anti-CTLA-4, FDA-approved immunotherapy for advanced metastatic melanoma in March 2011. This raises the possibility that MTSCs might possess the phenotypic and functional characteristics of CTLA-4 to modulate tumor-directed immune inhibition. To confirm this hypothesis, we have designed this program. The expression of CTLA-4 on ALDH+MTSCs and whether CTLA-4 involved in moduation of MTSCs mediated T cell activation will be monitored. In vivo, mice will be challenged with D5 melanoma and vaccinated with anti-CTLA-4 in addition to ALDH+MTSCs-DC vaccination. The anti-tumor effect of combination immunotherapy will be investigated. We will focus on the effect of combination immunotherapy on eradicating MTSCs. To further explore the mechanisms involved in eradicating MTSCs, tumor microenvironment immune response and host immune response will be evaluated in the combination immunotherapy. In tumor microenvironment, we will concentrate on the change of tumor-infiltrating lymphocyte populations, the mechanisms of lymphocytes infiltrating tumor, and the efficacy of tumor specific cytotoxic T-lymphocytes (CTL) mediated target-MTSC killing. The answer to the above question is of great importance to develop targeting cancer stem cells via combining immunotherapy.

我们前期研究发现基于ALDH+黑色素瘤干细胞（MTSCs）抗原的DC疫苗显著增强抗肿瘤免疫，并能靶向杀伤MTSCs。MTSCs具有"免疫豁免"效应，起始肿瘤的发生与进展；如何克服其免疫逃逸是彻底清除MTSCs而治愈肿瘤的关键。CTLA-4是一种负性免疫调节分子。2011年FDA批准CTLA-4抗体用于治疗黑色素瘤，提示了削弱免疫抑制途径在增强抗肿瘤免疫中起重要作用。本研究拟在前期工作基础上深入研究ALDH+MTSCs表达CTLA-4的特点及其在MTSCs调节T细胞活化中的作用，明确CTLA-4与MTSCs诱导免疫逃逸的关系；从整体水平和干细胞水平研究阻断CTLA-4介导的免疫抑制对MTSCs-DC疫苗诱导抗肿瘤效应和靶向杀伤MTSCs效能的影响，并从肿瘤微环境和全身免疫状态两个方面揭示阻断CTLA-4增强疫苗清除MTSCs的作用机制；为探索高效的靶向MTSCs的免疫治疗提供新策略。

6.我们前期研究发现基于ALDH+黑色素瘤干细胞（MTSCs）抗原的DC疫苗显著增强抗肿瘤免疫，并能靶向杀伤MTSCs。肿瘤免疫治疗领域中取得重大进展的是免疫调节点抑制剂的研发。目前FDA已批准的用于治疗黑色素瘤免疫调节点抑制剂包括CTLA-4，PD-1/PD-L1的抑制剂。本研究拟在前期工作基础上深入研究MTSCs表达CTLA-4的特点及其在MTSCs自我更新中的作用；探讨CTLA-4抑制剂联合MTSCs-DC疫苗诱导抗肿瘤效应和靶向杀伤MTSCs的效能及其机制。结果显示：MTSCs表达CTLA-4约0.7%~1.6%；CTLA-4抑制剂显著抑制黑色素瘤细胞增殖，并呈剂量依赖性及时间依赖性；CTLA-4抑制剂同时促进了黑色素瘤细胞调亡，其机制与激活bax/bcl-2,caspse-3有关；在干细胞水平，CTLA-4抑制剂显著抑制ALDH mRNA的表达及ALDH的蛋白水平；CTLA-4促进黑色素瘤细胞集落形成，并抑制集落细胞凋亡；动物实验表明：CTLA-4抑制剂显著抑制肿瘤生长及降低宿主体内残留的MTSCs；CTLA-4抑制剂联合MTSCs-DC疫苗比单用MTSCs-DC疫苗在整体水平上未显示抑制肿瘤生长的优势，但CTLA-4及PD-L1抑制剂联合MTSCs-DC疫苗显著增强了疫苗抗肿瘤效应及降低宿主体内残留的MTSCs。进一步研究发现：CTLA-4及PD-L1抑制剂联合MTSCs-DC疫苗显著增强宿主体内抗原特异性CTL细胞毒效应。综上，我们目前研究提示了黑色素瘤细胞内源性CTLA-4在维持肿瘤细胞及肿瘤干细胞存活中的重要作用；由于肿瘤细胞免疫逃逸机制复杂，同时阻断抑制性免疫通路CTLA-4、PD-L1联合MTSCs-DC疫苗显著增强了疫苗抗肿瘤效应，为探索有效的联合免疫治疗以提高黑色素瘤治疗疗效提供理论基础。