小分子化合物诱导肺癌肿瘤干细胞形成及用其制备DC疫苗治疗肺癌的研究

While many studies have demonstrated that tumor stem cells exhibit strong resistance to radiation and chemotherapy, immunotherapy has shown great promise as an effective approach to killing cancer stem cells. However, developing a method to quickly obtain enough tumor stem cells to prepare tumor vaccine is still a key bottom neck challenge in the pursuit of targeted cancer immunotherapy. Recently, the development of Chemical Genetics has led to the discovery that inducing somatic cell reprogramming can successfully generate induced pluripotent stem cells (iPS). This finding provided the theoretical basis and practical experience to explore inducible cancer stem cells (iCSC) using small-molecule compounds. Our study in Prostate demonstrated that LNCaP prostate cancer cells can become cancer stem-like cells by over-expressing SOX2, while our study in Stem Cells found that proteins derived via repeated freezing and thawing from cancer stem cells can be used as the antigen to prepare dendritic cell (DC) vaccine. Three time vaccinations, which contained vaccine with tumor associated antigen derived from cancer stem cells could produce specific CTL, significantly improved the survival rate in brain tumor animal model. In this study, we seek to optimize the combination of small-molecule compounds in vitro to induce tumor cell reprogramming and to produce a significant number of iCSC. Then we will explore the mechanism by which iCSC form and study their immunological characteristics, as well as the efficacy of iCSC DC cancer vaccine in a novel EGFRL858R transgenic spontaneous lung cancer mouse model.

肿瘤干细胞具有很强的抗化疗和抗放疗的特性，免疫细胞治疗被认为是清除肿瘤干细胞的有效途径，但如何获得肿瘤干细胞用于制备疫苗，是肿瘤干细胞靶向免疫治疗的瓶颈问题。近年来化学遗传学的快速发展，特别是发现几种小分子化合物的组合能够诱导体细胞重编程进而成功地获得iPS，为我们利用小分子化合物诱导肿瘤干细胞提供了理论基础和实践经验。申请人前期在Prostate上发表的研究结果表明通过过表达SOX2使前列腺肿瘤细胞LNCaP变成具有肿瘤干细胞的特性。我们在Stem Cells上发表的结果表明利用肿瘤干细胞反复冻融液作抗原负载DC疫苗，三次免疫后 在脑瘤动物模型体内产生肿瘤干细胞特异性的CTL并延长动物的生存期。本课题将采用小分子化合物的优化组合体外诱导肿瘤细胞重编程，获得诱导肿瘤干细胞（iCSC），重点研究iCSC的形成机制及免疫学特性并在EGFRL858R自发性肺癌动物模型上探讨iCSC疫苗的治疗效果

肿瘤干细胞参与肿瘤的发生、发展、耐药与复发，具有很强的抗化疗和抗放疗的特性，免疫细胞治疗被认为是清除肿瘤干细胞的有效途径，但如何获得肿瘤干细胞用于制备肿瘤疫苗，从而特异性地针对肿瘤干细胞开展靶向性免疫治疗是当前急需解决的一个重大科学问题。有研究表明一些小分子化合物可以使小鼠的体细胞转化为多能干细胞，那么是否可以通过类似的方法将普通肿瘤细胞诱导为肿瘤干细胞呢？本项目筛选出最佳的小分子化合物组合，在体外诱导肺癌细胞重编程获得诱导性肿瘤干细胞（iCSC），从而深入研究相关机制及其免疫学特性。我们主要开展了以下四个方面的工作：1）利用小分子化合物组合体外诱导肺癌细胞重编程获得诱导性肿瘤干细胞；2）分析小分子化合物诱导肿瘤干细胞形成的分子机制；3）探讨诱导性肿瘤干细胞的免疫学特征；4）初步检测诱导性肿瘤干细胞作为免疫原的抗肿瘤效果。通过这些系统性的研究，我们首先确定了联合应用不同浓度的五种小分子化合物（VPA、CHIR99021、E-616452、Tranylcypromine、DZnep）可以将肺癌细胞A549、H460、LLC诱导成具有干性特征的肿瘤细胞，其表面干性标志物CD133以及干性基因OCT4、SOX2均显著上调。以A549-iCSC为研究对象，进一步分析发现：其耐药性增强，且发生EMT、侵袭能力增强；细胞中的NOTCH信号通路活性升高；诱导后细胞高表达部分肿瘤抗原和细胞因子，用其细胞裂解液致敏的DC细胞可以促进T细胞分泌TNF-α和IFN-γ及其细胞毒性作用。用LLC-iCSC细胞免疫实验小鼠增强抗体反应和T细胞的免疫活性。本项目所取得的结果将为肿瘤干细胞的靶向性免疫治疗提供一定的理论基础和实验依据。