Tracking the in vivo proliferative history of human glioma-derived stem cells

项目摘要 胶质瘤是最常见的原发脑肿瘤。存活率很低，而且往往令人沮丧 结果突显了我们对胶质瘤细胞的定义特征和它们对 放射治疗或化疗。胶质瘤包含少量胶质瘤干细胞(GSCs)，能够 不对称的自我更新和多谱系分化。GSCs的临床相关特征包括高 致瘤能力和治疗耐药性。以前的研究未能确定确定的细胞表面 肿瘤传播性GCSs的标志物，以及没有已知的治疗耐药性的标志物。我们打算测试一下 一种假说认为，静止能赋予GSC亚群高致瘤能力和治疗能力-- 抵抗。为了验证这一假设，我们设计了一种创新的动力学分析，能够在体内追踪 胶质瘤细胞的增殖史，并允许将胶质瘤细胞分为慢分裂和快分裂 子组。这是通过对人类胶质瘤来源的GSCs(HGSCs)进行基因工程来实现的 多西环素诱导的组蛋白2B-GFP标记。一项脉冲追逐研究将识别快速分裂的肿瘤细胞 GFP-由于稀释，而静止细胞将保持GFP+。随后的干细胞培养将选择 来自这两组的肿瘤干细胞以供进一步研究。这样的动力学分析是有利的 捕捉胶质瘤细胞在体内的动态行为。在目标1中，我们将跟踪三个HGSC株系在体内的情况 不同时间点异种移植的增殖行为。肿瘤生长后，GFP高和GFP低 将分析亚群的比例、聚集模式、传播距离、表达 神经干细胞或分化标记物，以及与已知神经干细胞生态位的空间关系。在目标2中， 我们将对GFP High和GFP low亚群进行分类，并将它们置于神经干细胞培养条件下进行分离 神经胶质瘤。GSCfast和GSClow将在自我更新能力和差异化方面进行比较 潜力，以及它们的迁移和形成肿瘤的能力。基因表达谱研究将 确定独特的分子特征。GSCs还将经历另外两代作为胶质沥青的评估 体内先前的增殖史是否会留下影响未来细胞行为的“增殖记忆”。 在目标3中，我们将应用放射疗法(XRT)来测试静止使GSCs与 耐辐射。对XRT耐药的胶质瘤细胞将进行GFP标记检测，然后进行分离 用于分子表征。这一范式也为研究体内的增殖行为提供了机会。 XRT后微环境中对XRT耐药的胶质瘤细胞。总而言之，我们结合了创新的基因 工程学、人脑肿瘤、干细胞生物学和分子研究，以了解体内的影响 胶质瘤干细胞后续细胞行为的增殖史及其与肿瘤发生的关系 效力和治疗抵抗力。基于体内细胞分裂动力学参数的动力学研究 将解决高级别胶质瘤的瘤内功能异质性及其潜在原因。

Project Summary Gliomas are the most frequent form of primary brain tumors. Survival rates are low, and the often dismal outcome highlights our poor understanding of the defining features of glioma cells and their resistance to radiation or chemotherapy. Glioma contains a small population of glioma stem cells (GSCs) capable of asymmetric self-renewal and multi-lineage differentiation. The clinically relevant features of GSCs include high tumorigenic potency and therapeutic resistance. Previous studies failed to identify definitive cell surface markers of tumor-propagating GCSs, and no markers are known for therapeutic resistance. We propose to test the hypothesis that quiescence confers to a subgroup of GSCs high tumorigenic potency and therapy- resistance. To test this hypothesis, we designed an innovative kinetic analysis that enables in vivo tracking of the proliferative history of glioma cells and that allows sorting glioma cells into slow- and fast-dividing subgroups. This is achieved by genetic engineering of human glioma-derived GSCs (hGSCs) with a doxycycline-inducible Histone2B-GFP label. A pulse-and-chase study will identify fast-dividing tumor cells as GFP- due to dilution, whereas quiescent cells will remain GFP+. Subsequent stem cell cultures will then select tumor stem cells from these two groups for further studies. Such a kinetic analysis is advantageous in capturing the dynamic in vivo behaviors of glioma cells. In Aim 1, we will track three hGSC lines for their in vivo proliferative behaviors in xenotransplants at different time points. After tumor growth, GFPhigh and GFPlow subgroups will be analyzed for their proportion, aggregation patterns, dissemination distance, expression of neural stem cell or differentiation markers, and spatial relationship to known neural stem cell niches. In Aim 2, we will sort the GFPhigh and GFPlow subgroups and subject them to neural stem cell culture conditions to isolate gliomaspheres. The GSCfast and GSCslow will be compared for their self-renewal capacity and differentiation potential, as well as their migratory and tumor-forming capabilities. Gene expression profiling studies will identify unique molecular features. GSCs will also undergo two more passages as gliomaspheres to assess whether prior in vivo proliferation history leaves a "proliferation memory" that impacts future cellular behaviors. In Aim 3, we will apply radiation therapy (XRT) to test the hypothesis that quiescence confers GSCs with radiation-resistance. XRT-resistant glioma cells will be examined for their GFP labeling, and then be isolated for molecular characterization. This paradigm also offers an opportunity to study in vivo proliferative behaviors of XRT-resistant glioma cells in the post-XRT microenvironment. In summary, we combine innovative genetic engineering, human brain tumors, stem cell biology, and molecular studies to understand the impact of in vivo proliferative history on subsequent cellular behaviors of glioma stem cells as well as its link to tumorigenic potency and therapeutic resistance. Our kinetic studies based on a dynamic parameter of in vivo cell division will address the intratumoral functional heterogeneity of high-grade glioma and the underlying causes.