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

项目摘要 神经胶质瘤是最常见的原发性脑肿瘤。 存活率很低， 结果强调了我们对胶质瘤细胞的定义特征及其对化疗的抗性的理解不足。 放疗或化疗。 神经胶质瘤含有一小群神经胶质瘤干细胞（GSC）， 不对称自我更新和多谱系分化。GSC的临床相关特征包括高 致瘤效力和治疗抗性。 以前的研究未能确定确定的细胞表面 肿瘤传播GCS的标志物，并且没有已知的治疗抗性标志物。我们建议测试 静止状态赋予GSC亚群高致瘤潜能和治疗的假设- 阻力为了验证这一假设，我们设计了一种创新的动力学分析， 神经胶质瘤细胞的增殖历史，并允许将神经胶质瘤细胞分选为慢分裂和快分裂 分组。 这是通过基因工程改造人神经胶质瘤衍生的GSC（hGSC）来实现的， 强力霉素诱导的组蛋白2B-GFP标记。 一项脉冲追踪研究将确定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 从这两组中提取肿瘤干细胞用于进一步研究。 这种动力学分析在以下方面是有利的： 捕捉胶质瘤细胞的动态体内行为。在目标1中，我们将追踪三种hGSC系的体内表达。 在不同时间点观察异种移植物的增殖行为。 肿瘤生长后，GFP高和GFP低 将分析亚组的比例、聚集模式、传播距离、 神经干细胞或分化标志物，以及与已知神经干细胞小生境的空间关系。在目标2中， 我们将对GFP高和GFP低亚组进行分类，并将它们置于神经干细胞培养条件下以分离 胶质球 将比较GSCfast和GSCslow的自我更新能力和差异化 潜力，以及它们的迁移和肿瘤形成能力。 基因表达谱研究将 识别独特的分子特征。 GSC还将作为胶质球再经历两次传代，以评估 先前的体内增殖历史是否留下了影响未来细胞行为的"增殖记忆"。 在目标3中，我们将应用放射治疗（XRT）来检验静止赋予GSC以下功能的假设： 抗辐射将检查XRT抗性胶质瘤细胞的GFP标记，然后分离 用于分子表征。这种模式也提供了一个机会，研究在体内增殖行为 XRT后微环境中的XRT抗性胶质瘤细胞。综上所述，我们联合收割机结合创新基因 工程，人脑肿瘤，干细胞生物学和分子研究，以了解体内的影响 增殖史对胶质瘤干细胞后续细胞行为的影响及其与致瘤性的关系 效力和治疗抗性。我们的动力学研究基于体内细胞分裂的动力学参数 将解决高级别胶质瘤的瘤内功能异质性和根本原因。

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.