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）：神经胶质瘤是原发性脑肿瘤的最常见形式。存活率较低，并且通常令人沮丧的结果突出了我们对神经胶质瘤细胞定义特征的不良理解及其对放射线或化学疗法的抵抗力。神经胶质瘤含有少量的神经胶质瘤干细胞（GSC），具有不对称的自我更新和多局部分化。 GSC的临床相关特征包括高肿瘤效力和治疗性。先前的研究未能识别确定的细胞肿瘤传播GCS的表面标记和没有标记以治疗性抗性而闻名。我们建议测试以下假设，即静止属于GSC的高肿瘤效力和耐药性的亚组。为了检验这一假设，我们设计了一种创新的动力学分析，该分析可以在体内跟踪神经胶质瘤的增殖病史，并允许将神经胶质瘤细胞分类为缓慢和快速分散的亚组。这是通过具有强力霉素诱导的HASTONE2B-GFP标签的人神经胶质瘤衍生GSC（HGSC）的基因工程来实现的。一项脉搏和练习研究将由于稀释而识别出快速分散的肿瘤细胞为GFP，而静态细胞将保持GFP+。然后，随后的干细胞培养物将从这两组中选择肿瘤干细胞进行进一步研究。这种动力学分析在捕获神经胶质瘤细胞的动态体内行为方面是有利的。在AIM 1中，我们将在不同时间点跟踪三个HGSC线的体内增殖行为。肿瘤生长后，将分析GFPHIGH和GFPLOW亚组的比例，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壁ches的空间关系。在AIM 2中，我们将对GFPHIGH和GFPLOW亚组进行分类，并遵守神经干细胞培养条件以分离神经胶质细胞。 GSCFAST和GSCSLOW的自我更新能力和分化潜力以及迁移和形成肿瘤的能力将进行比较。基因表达分析研究将确定独特的分子特征。 GSC还将随着神经胶质细胞评估是否会影响未来细胞行为的“增殖记忆”，还将再经历两个段落。在AIM 3中，我们将采用放射治疗（XRT）来检验静止赋予GSC具有辐射抗性的假设。将检查耐XRT的胶质瘤细胞的GFP标记，然后分离以进行分子表征。该范式还提供了研究XRT后XRT微环境中耐XRT胶质瘤细胞体内增殖行为的机会。总而言之，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随后的细胞行为的影响，以及其与肿瘤效力和治疗耐药性的联系。我们基于体内细胞分裂的动态参数的动力学研究将解决高级神经胶质瘤和潜在原因的肿瘤内功能异质性。