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

追踪人胶质瘤干细胞的体内增殖史

基本信息

批准号：
8742022
负责人：
Roland Horst Friedel
金额：
$ 20.98万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8742022
关键词：
Address Affect Behavior Biological Assay Brain Brain Neoplasms Cell Culture Techniques Cell Line Cell division Cell surface Cells Central Neurocytoma Collaborations Development Diagnostic Differentiation Antigens Diffuse Doxycycline Engineering Excision Exhibits Fluorescence-Activated Cell Sorting Future Gene Expression Gene Expression Profiling Genetic Genetic Engineering Glioblastoma Glioma Green Fluorescent Proteins Growth Heterogeneity Histones Human Kinetics Label Laboratories Link Malignant Neoplasms Malignant neoplasm of brain Measures Memory Modeling Molecular Molecular Profiling Mus Nature Neurons Outcome Patients Pattern Physiologic pulse Population Prevalence Primary Brain Neoplasms Radiation Radiation therapy Recording of previous events Recurrence Relative (related person)Reporter Resistance Rodent SCID Mice Sampling Signal Pathway Signal Transduction Sorting - Cell Movement Source Spatial Distribution Specimen Staging Stem cells Subgroup Survival Rate System Testing Therapeutic Time Transplantation Tumor Markers Tumor Stem Cells Withdrawal Xenograft procedure base cancer stem cell chemotherapy clinically relevant design imprint in vivo innovation migration neoplastic cell nerve stem cell oligodendroglioma prospective public health relevance radiation resistance research study self-renewal spatial relationship stem cell biology stem cell niche therapy resistant tumor tumor growth tumorigenic

项目摘要

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.

描述（由申请人提供）：胶质瘤是最常见的原发性脑肿瘤。存活率很低，并且通常令人沮丧的结果突出了我们对胶质瘤细胞的定义特征及其对放疗或化疗的抵抗力的理解不足。胶质瘤中含有少量胶质瘤干细胞（GSC），能够进行不对称自我更新和多向分化。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肿瘤传播GCS的表面标志物，并且没有已知的治疗抗性标志物。我们建议检验静止状态赋予GSC亚群高致瘤潜能和治疗抗性的假设。为了验证这一假设，我们设计了一种创新的动力学分析，能够在体内跟踪神经胶质瘤的增殖历史，并允许将神经胶质瘤细胞分选为慢速和快速分裂的亚组。这是通过用多西环素诱导的组蛋白2B-GFP标记对人神经胶质瘤衍生的GSC（hGSC）进行基因工程改造来实现的。脉冲追踪研究将鉴定快速分裂的肿瘤细胞为GFP-，因为稀释，而静止细胞将保持GFP+。随后的干细胞培养将从这两组中选择肿瘤干细胞进行进一步研究。这种动力学分析有利于捕捉胶质瘤细胞的动态体内行为。在目的1中，我们将追踪三种hGSC系在异种移植物中在不同时间点的体内增殖行为。肿瘤生长后，将分析GFP高和GFP低亚组的比例、聚集模式和播散距离、神经干细胞或分化标志物的表达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，并将其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。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辐射抗性的假设。将检查XRT抗性神经胶质瘤细胞的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，在体内研究XRT-耐药胶质瘤细胞在XRT后的微环境中的增殖行为。总之，我们结合联合收割机创新的基因工程，人脑肿瘤，干细胞生物学，和分子研究，以了解在体内增殖的历史对胶质瘤干细胞的后续细胞行为的影响，以及其与致瘤潜力和治疗抗性的联系。我们的动力学研究的基础上的动态参数在体内细胞分裂将解决高级别胶质瘤的肿瘤内功能异质性和根本原因。