(PQ5) Assessing the contribution of mitochondrial heterogeneity to gliomagenesis using single cell approaches

这个修订后的建议解决了线粒体异质性如何影响 肿瘤发生或进展（NCI PQ 5）。我们将使用单细胞定量测量来表征 线粒体网络（参与线粒体动力学和细胞凋亡的蛋白质）在 多形性胶质母细胞瘤（GBM）。我们假设，胶质瘤干细胞样细胞（GSC）的亚群， 线粒体分裂的增加将显示自我更新能力的增加和细胞凋亡的增加。 对常规化疗的抵抗力。我们的预测是，所有这些属性的组合使得 这群细胞高度致瘤。假设GSC是肿瘤复发的基础-这是 95%的患者不可避免，使其成为GBM中的一个重要问题。这些单细胞研究可以 揭示了胶质母细胞瘤传播细胞的新特性，这些特性被基于人群的 方法。所提出的方法将在单细胞水平的信息集成到整体的复杂性 肿瘤的环境。我们的目标是了解线粒体基因组异质性的分子基础， 可能是对治疗的反应差异的基础的动力学/功能。 在目标1中，我们将使用线粒体活性和功能（线粒体动力学）的单细胞测量 和线粒体相关蛋白）与茎样标记物一起获得线粒体的总体概况 GSC的异质性，使用质谱细胞术。在此表型表征之后，我们将检查 使用脑内微环境中新鉴定的细胞亚群的位置识别 肿瘤类器官这种方法将适用于纵向测定，以跟踪肿瘤在体内的进展。 菜. 在目标2中，我们计划通过两种策略揭示GBM中线粒体状态的异质性。一是 将通过检查GBM肿瘤对常规化疗的敏感性来评估GBM肿瘤的启动状态， 使用动态BH 3分析。这将提供异质性的肿瘤间评估。二是 分离特定的细胞亚群（例如干细胞样/高或低线粒体分裂/融合）并评估它们的细胞亚群（例如干细胞样/高或低线粒体分裂/融合）。 对常规化疗和/或线粒体动力学机制抑制剂的敏感性。 这些目标的完成将在单细胞水平上提供关于 线粒体异质性与GBM中肿瘤发生和耐药性的关系。这些信息将提供 未来研究的基础，旨在改善目前的化疗方案，并合理化潜在的新的 治疗

This revised proposal addresses the provocative question of how mitochondrial heterogeneity influences tumorigenesis or progression (NCI PQ5). We will use single-cell, quantitative measurements to characterize the status of the mitochondrial network (proteins involved in mitochondrial dynamics and apoptosis) in glioblastoma multiforme (GBM). We hypothesize that the subpopulations of glioma stem-like cells (GSCs) with increased mitochondrial division will display increased self-renewal capacity and increased apoptotic resistance to routinely used chemotherapy. Our prediction is that the combination of all these properties makes this population of cells highly tumorigenic. GSCs are hypothesized to underlie tumor recurrence - which is inevitable in 95% of these patients, making it a significant problem in GBM. These single-cell studies could reveal new properties of the glioblastoma-propagating cells that have been overlooked by population-based approaches. The proposed approach will integrate information at the single-cell level into the overall complexity of the tumor environment. Our goal is to understand the molecular basis of the heterogeneity of mitochondrial dynamics/function that may underlie differences in the response to therapy. In Aim 1, we will use single-cell measurements of mitochondrial activity and function (mitochondrial dynamics and apoptotic-related proteins) together with stem-like markers to acquire a global profile of the mitochondrial heterogeneity in GSCs, using mass cytometry. Following this phenotypic characterization, we will examine the positional identity of the newly identified cell subpopulations within the tumor microenvironment using brain tumor organoids. This approach will be amenable to longitudinal assays for following tumor progression in the dish. In Aim 2, we plan to reveal the heterogeneity of the mitochondrial status in GBM by two strategies. First, we will assess the priming state of the GBM tumors by examining their sensitivity to conventional chemotherapy, using dynamic BH3 profiling. This will provide an inter-tumoral assessment of heterogeneity. Second, we will isolate specific subpopulations of cells (e.g. stem-like/high or low mitochondrial fission/fusion) and assess their sensitivity to conventional chemotherapy and/or inhibitors of the mitochondrial dynamics machinery. Completion of these Aims will provide valuable information at the single-cell level about the contribution of mitochondrial heterogeneity to tumorigenesis and drug resistance in GBM. This information will provide the foundation for future studies aimed to improve current chemotherapy regimens and to rationalize potential new therapies.