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

描述(申请人提供)：胶质瘤是最常见的原发脑肿瘤。存活率很低，而且通常令人沮丧的结果突显了我们对胶质瘤细胞的定义特征及其对放射或化疗的抵抗力缺乏了解。胶质瘤含有少量胶质瘤干细胞(GSCs)，具有不对称自我更新和多向分化能力。GSCs的临床相关特征包括高致瘤性和治疗耐药性。以前的研究未能确定确定的细胞 肿瘤传播性GCSs的表面标记物，而没有已知的治疗耐药性标记物。我们建议检验这一假说，即静止期赋予GSCs亚群高致瘤性和治疗抵抗力。为了验证这一假设，我们设计了一种创新的动力学分析，能够在体内跟踪胶质瘤细胞的增殖历史，并允许将胶质瘤细胞分成缓慢和快速分裂的亚组。这是通过基因工程使人胶质瘤来源的GSCs(HGSCs)带有多西环素诱导的组蛋白2B-GFP标记实现的。一项脉冲式研究将确定快速分裂的肿瘤细胞由于稀释而为GFP-，而静止的细胞将保持GFP+。随后的干细胞培养将从这两组中挑选肿瘤干细胞进行进一步研究。这样的动力学分析有利于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系在不同时间点在异种移植中的体内增殖行为。在肿瘤生长后，将分析GFP高和GFP低亚群的比例、聚集模式和传播距离、神经干细胞或分化标记物的表达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分离胶质蛋白。将比较GSCfast和GSClow的自我更新能力和分化潜力，以及它们的迁移和肿瘤形成能力。基因表达谱研究将确定独特的分子特征。GSCs还将经历另外两代胶质细胞，以评估先前在体内的增殖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)来验证静止使GSC具有辐射抗性的假设。对XRT耐药的胶质瘤细胞将被检测其GFP标记，然后被分离出来进行分子鉴定。这一范式也为研究XRT后微环境中耐XRT的胶质瘤细胞的体内增殖行为提供了机会。综上所述，我们结合了创新的基因工程、人脑肿瘤、干细胞生物学和分子研究，以了解体内增殖史对胶质瘤干细胞后续细胞行为的影响，以及它与致瘤能力和治疗耐药性的联系。我们的动力学研究基于体内细胞分裂的动态参数，将解决高级别胶质瘤的瘤内功能异质性及其潜在原因。

DESCRIPTION (provided by applicant): 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 cels 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, and 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.