靶向肾癌干细胞的特异性免疫治疗研究

There are few, if not none, reports about immunotherapy by targeting renal cell carcinoma (RCC) stem cells in literature. Based on our previous study, we propose to isolate the cancer stem cells (CSC) from RCC cell lines and primary cultured RCC cells by using aldehyde dehydrogenase (ALDH) as a new CSC biomarker in this study. The CSC nature of ALDH (high)-cell enriched RCC cell population will be characterized. We will then use the ALDH (high)-CSC lysate as a source of antigen to pulse dendritic cells (DC) to generate CSC-DC to induce autologous cytotoxic T lymphocyte (CTLs) which can specifically target RCC cancer stem cells. Using in vitro immune-cell model experiments and in vivo mouse model experiments, the anti-CSC immunity of CSC-DC - primed CTLs will be evaluated, such as the direct killing /lysis of CSCs by such CTLs; in vivo antitumor activity of the administrated CTLs, as well as the potential mechanisms involved. From this study, we will establish a new immunotherapy method by targeting RCC stem cells, which may significantly improve the therapeutic efficacy of RCC immunotherapy. Furthermore, the achievement of this study will provide more specific and effective adjuvant treatment for traditional clinical therapy of renal cancer. This method may show positive significance for stabilizing treatment effects, controlling tumor recurrence and metastasis, and improving prognosis of RCC patients after surgery, by selective destroying RCC stem cells.

肾癌干细胞靶向免疫治疗研究国内外尚未见有相关报道。本项目在前期研究基础上，采用乙醛脱氢酶（ALDH）为标志物，从肾癌细胞系和原代培养的肾癌细胞中分离肾癌干细胞，并对其肿瘤干细胞特性进行鉴定。利用分离鉴定的肾癌干细胞，提取抗原，负载树突状细胞（DC）后，诱导针对肾癌干细胞的特异性细胞毒T淋巴细胞（CTL）生成，并在体外（免疫细胞学实验）、体内（小鼠模型实验）验证其靶向杀伤肾癌干细胞的特异性，同时进行机制分析。通过本研究，我们以免疫特异性识别的特性，建立肾癌干细胞靶向性免疫治疗的新方法，从而有效提高肾癌免疫治疗的效果。本项目研究成果将为临床提供更特异、更有效的肿瘤辅助治疗手段，有望成为肾癌综合治疗的新方法，对于肾癌患者术后的巩固疗效、抑制复发和转移、改善预后具有积极意义。

肿瘤干细胞（CSC）与肿瘤的发生、发展密切相关，并且对传统治疗方法如化疗、放疗的敏感性较差，可能导致肿瘤复发。理想的治疗策略是选择性地靶向和破坏CSC，从而预防肿瘤复发。本研究的目的是评估树突状细胞（DC）负载CD105+肾癌干细胞相关抗原对肾癌的治疗作用，从体内外实验验证其作用及可能机制。. 我们在两种人RCC细胞系A498和SK-RC-39中分离得到CD105+细胞，发现CD105+肾癌干细胞干细胞相关基因，OCT-4A，SOXN，NAONG，ABCG2和C-MYC等的表达较CD105-组肾癌细胞显著增高。CD105+肾癌干细胞能够形成结构致密且体积较大的球囊。至少需要注射剂量为1×10E6的CD105-肾癌细胞才能可以在BALB/c雌裸鼠腋窝皮下成瘤。然而1×10E4的CD105+ 肾癌干细胞就能成瘤。并且CD105+肾癌细胞在裸鼠体内成瘤的速度要显著高于CD105-肾癌细胞。将分选得到的CD105+和CD105-肾癌细胞裂解物负载DC细胞，得到DC疫苗。CD105+CTLs对CD105+肾癌干细胞有比较强的特异性杀伤能力。体内实验表明CD105+CSC-CTLs组的肿瘤生长明显受到抑制，流式和免疫组化发现这组肿瘤内CD105+的细胞明显减少，说明CD105+CSC-CTLs能特异性杀伤CD105+的肾癌干细胞。只有负载CD105+肾癌干细胞裂解物抗原的DCs诱导的CTLs细胞（CD105+_CTLs）才能特异性的与MHC-CD105肽四聚体结合。CD105+CTLs，CD105-CTLs，unsorted-CTLs分泌的IFN-γ、TNF-α、GranzymeB、Perforin的量较Con-CTLs显著升高，但这三组之间没有显著性差异。负载CD105+肾癌干细胞裂解物抗原的DCs疫苗能够刺激B淋巴细胞产生更多的能与CD105+肾癌干细胞结合的特异性抗体。. 实验结果表明，负载CD105+ CSC裂解物并不会改变DC 本身的物性，但可以诱导抗CSC的特异性杀伤作用，并可产生特异性抗体，在体内可明显抑制小鼠肿瘤生长。我们的研究结果可以为一种新的肿瘤免疫治疗策略奠定基础。