Genetic Mouse Models of Glioma

描述（申请人提供）：恶性星形细胞瘤是一种局部浸润性脑肿瘤，无法治愈，患者预后较差。尽管具有深远的治疗意义，但这些肿瘤的起源细胞的身份尚未得到严格确定。此外，多形性胶质母细胞瘤是脑肿瘤中最常见和最致命的一种，它既可以从低度星形细胞胶质瘤发展而来，也可以从头发生，其机制尚不清楚。我们之前报道了基于人类星形细胞瘤相关肿瘤抑制因子p53、Nf1和Pten条件失活的小鼠模型，其中通过体细胞杂合性丧失，突变小鼠产生组织学和分子上与人类星形细胞瘤相似的肿瘤，其外显率为100%。在目前的应用中，我们提出了旨在研究肿瘤起源细胞的实验，并扩展了我们对这些小鼠模型中肿瘤发生的分析。为此，Specific Aim 1严格验证了我们的假设，即胶质瘤起源于干细胞/祖细胞。我们的研究设计利用遗传和立体定向方法在干细胞生态位特异性地突变肿瘤抑制基因。我们还将使用遗传方法在我们的肿瘤模型中切除这些神经干细胞，然后分析其对肿瘤形成的影响。特异性目标2将利用我们培养新鲜肿瘤组织作为自我再生神经球的能力，进一步表征我们的Nf1， p53；Pten“新生”胶质瘤小鼠模型。我们将通过移植技术评估它们的生长和分化特性，以及它们的致瘤潜力。此外，使用干细胞特异性GFP转基因小鼠，我们将分析候选细胞表面标记的表达，目的是识别“签名标记”，这将使我们能够前瞻性地分离癌症干细胞。特异性目标3将采用微阵列分析来鉴定与新生和进展性胶质瘤相关的基因表达谱，使用来自Nf1、p53和p53的神经球。Pten和Nf1；P53肿瘤。我们还建议分析症状前小鼠的组织，以便深入了解肿瘤起始的早期分子事件。潜在的有趣基因将使用RNAi和过表达技术进行功能追踪。由于microrna最近与胶质瘤有关，我们也将使用微阵列分析来鉴定在我们的肿瘤模型中差异调节的microrna，并研究候选microrna在胶质瘤中的作用。我们的全渗透胶质瘤小鼠模型是临床相关的和强大的工具，用于识别和功能表征可能在人类胶质瘤治疗中易于处理的新基因和途径。公共卫生相关性：恶性星形细胞瘤是发生在成人中最常见的脑肿瘤类型，由于其浸润性和侵袭性，几乎是无法治愈的。我们的研究重点是利用小鼠脑肿瘤模型来识别和分析参与脑肿瘤发生和发展的基因。希望是通过识别这些基因，我们可以开始找到合适的药物靶点，并开发出能够消除或抑制这些破坏性肿瘤生长的疗法。

DESCRIPTION (provided by applicant): Malignant astrocytomas are brain tumors that are locally infiltrative and incurable, with poor prognosis for the patient. Despite profound therapeutic implications, the identity of the cell(s) of origin of these tumors has not been rigorously determined. In addition, glioblastoma multiforme, the most prevalent and deadly form of brain tumor, can either progress from lower grade astrocytic gliomas or arise de novo, the mechanisms by which are not well understood. We previously reported mouse models based on conditional inactivation of human astrocytoma-relevant tumor suppressors p53, Nf1, and Pten, wherein through somatic loss of heterozygosity, mutant mice develop tumors that histologically and molecularly resemble human astrocytomas with 100% penetrance. In the present application, we propose experiments designed to investigate the cell(s) of tumor origin and to extend our analyses of tumorigenesis in these mouse models. To this end, Specific Aim 1 rigorously tests our hypothesis that gliomas originate in stem/progenitor cells. Our research design utilizes genetic and stereotactic methods to mutate the tumor suppressor genes specifically in the stem cell niche. We will also use a genetic method to ablate these neural stem cells in our tumor models and then analyze the effect on tumor formation. Specific Aim 2 will exploit our ability to culture fresh tumor tissue as self-renewable neurospheres to further characterize our Nf1;p53;Pten "de novo" glioma mouse model. We will evaluate their growth and differentiation properties, as well as their tumorigenic potential via transplantation techniques. Additionally, using a stem cell-specific GFP transgenic mouse, we will analyze the expression of candidate cell surface markers with the aim of identifying "signature markers" that will allow us to prospectively isolate the cancer stem cells. Specific Aim 3 will employ microarray analyses to identify the gene expression profiles that correlate with de novo vs. progressive glioma, using neurospheres derived from Nf1;p53;Pten and Nf1;p53 tumors, respectively. We also propose to analyze tissue from pre-symptomatic mice in order to gain insight into the early molecular events of tumor initiation. Potentially interesting genes will be functionally pursued using RNAi and overexpression techniques. As microRNAs have recently been implicated in glioma, we will also use microarray analysis to identify microRNAs that are differentially regulated in our tumor models and also investigate the role of candidate microRNAs in glioma. Our fully penetrant glioma mouse models are clinically relevant and powerful tools for identifying and functionally characterizing novel genes and pathways that may be therapeutically tractable in human glioma. PUBLIC HEALTH RELEVANCE: Malignant astrocytomas are the most common type of brain tumors that occur in adults and due to their infiltrative and aggressive nature, are virtually incurable. The focus of our research is to use mouse brain tumor models to identify and analyze genes that are involved in brain tumor initiation and progression. The hope is that by identifying these genes, we might begin to find suitable drug targets and develop therapies that would eliminate or inhibit the growth of these devastating tumors.