基于胶质母细胞瘤干细胞的高通量抗肿瘤药物筛选技术的建立和应用

Theory of tumor stem cell is getting more and more experimental supports and tumor stem cell is widely accepted as a main cause of tumor chemoresistance, radioresistance, tumor migration and recurrence. The tumor stem cell is the ideal target of therapy. Theoretically drugs against tumor stem cell should have better anti-tumor effect. However current anti-tumor drugs were screened out by using cancer cell lines. It is necessary to use tumor stem cell for anti-tumor drug screening. However there are not many efforts on screening with tumor stem cells. The main reason is lacking a good high-throughput screening system by using tumor stem cell. Glioblastoma stem cell is one of a few cancer stem cells that can be grown and passaged in vitro for long time both as neurosphere and adherent culture without obvious change in genetic and gene expression profile, which are the prerequisite for high-throughput screening. In this study，1) we will establish a glioblastoma stem cell-based high-throughput screening system by homologous integration of reporter gene EGFP into downstream of stem cell marker gene Nestin promoter. With this system,both cytotoxic and differentiation-inducing compounds can be screened simultaneously. The single cell information from high content screening technology can discriminate cytotoxicity and differentiation induced by compounds；2) Compound library, specifically natural compound and conventional drugs etc., will be screened; 3) In vivo anti-tumor effect and mechanism of selected candidate from screening will be elucidated. The screening system and compound candidate will undoubtedly promote development of anti-tumor stem cell drugs.

肿瘤干细胞的存在近年来获得了大量的实验证据支持，是肿瘤耐药、复发和转移的主要原因，理应是肿瘤治疗的主要靶细胞。目前的抗肿瘤药物都是用肿瘤细胞系筛选出来的，而抗肿瘤干细胞的药物将具有更好的肿瘤治疗效果，但目前缺乏针对肿瘤干细胞的药物，其重要原因是没有基于肿瘤干细胞的良好的高通量筛选技术。与其他肿瘤干细胞相比，胶质母细胞瘤干细胞可长期体外、贴壁培养，并且传代过程中遗传学、生物学特性等不易改变，具有用于高通量筛选的潜力。本项目拟遴选有代表性基因变异的胶质母细胞瘤干细胞株，将报告基因EGFP定点整合在胶质母细胞瘤干细胞分子标记Nestin启动子下游，利用细胞高内涵筛选系统，建立可同时筛选细胞毒性和分化诱导药物的技术；利用这一技术将对天然产物库及其他化合物库进行抗肿瘤药物筛选，获得针对肿瘤干细胞的先导化合物并对部分化合物抗肿瘤效果和机制进一步研究。

肿瘤干细胞的存在近年来获得了大量的实验证据支持，是肿瘤耐药、复发和转移的主要原因，理应是肿瘤治疗的主要靶细胞。目前的抗肿瘤药物都是用肿瘤细胞系筛选出来的，而抗肿瘤干细胞的药物将具有更好的肿瘤治疗效果，但目前缺乏针对肿瘤干细胞药物，其重要原因是没有基于肿瘤干细胞的良好的高通量筛选技术。与其他肿瘤干细胞相比，胶质母细胞瘤干细胞可长期体外、贴壁培养，并且传代过程中遗传学、生物学特性等不易改变，具有用于高通量筛选的潜力。本项目通过遴选有代表性基因变异的胶质母细胞瘤干细胞株，将报告基因EGFP定点整合在胶质母细胞瘤干细胞分子标记Nestin启动子下游，利用细胞高内涵筛选系统，建立可同时筛选细胞毒性和分化诱导药物的技术；利用这一技术对天然产物库、老药库和中药成分进行了筛选，发现了数种特异性抗肿瘤干细胞的药物，并对地高辛、3α-acetonyltabersonine、Isocostunolide和WADM-3等进行了体内外抗肿瘤实验和分子机制研究；目前已成功构建了小鼠、树鼩、食蟹猴胶质瘤模型和成体小鼠胰腺癌模型，后期将利用这些动物模型做进一步的化合物药效评价。上述研究结果中，抗肿瘤干细胞高通量筛选平台可筛选细胞毒性和诱导分化的抗肿瘤干细胞化合物，促进新型抗肿瘤药物研发。已建立的肿瘤动物模型可用于新药测试或肿瘤免疫与免疫治疗、循环肿瘤细胞和肿瘤早期诊断等研究，为治疗胶质母细胞瘤的药物发现及云南省特有动物植物资源开发做出了一定的贡献。