Characterizing the glioma cell of origin in vivo using MADM, a mouse genetic mosa

描述（申请人提供）：胶质瘤是最常见的恶性脑肿瘤。尽管癌症医学取得了广泛的进步，但这种毁灭性的疾病仍然无法治愈。最近有报道称，胶质瘤中含有一群肿瘤干细胞，这些干细胞可以在培养中形成自我再生的肿瘤球，并在移植到免疫抑制的小鼠体内后重新启动胶质瘤。靶向这些肿瘤干细胞是治疗胶质瘤的一个令人兴奋的前景。然而，开发这种治疗策略所需的关键信息，包括胶质瘤干细胞的发育起源，仍然未知。为了解决肿瘤细胞起源问题，需要使用神经胶质瘤动物模型来分析早期恶性前肿瘤细胞。不幸的是，目前的小鼠模型不能为这类研究提供足够的体内分辨率。我们的实验室开发了一种新的小鼠胶质瘤模型，该模型基于一种称为MADM的新型遗传镶嵌系统（双标记镶嵌分析，Zong et al Cell 2005）。利用MADM，我们可以在正常小鼠体内产生罕见的、绿色荧光蛋白（GFP）标记的神经干细胞（NSCs），这些细胞在两种关键的肿瘤抑制基因p53和1型神经纤维瘤病（NF1）中是双零的。这种方法使我们能够在体内以单细胞分辨率分析胶质瘤形成的整个过程。我们的初步研究结果表明，尽管这些突变是在NSCs中特异性产生的，但由此产生的胶质瘤细胞表现出许多少突胶质细胞前体（OPCs）的细胞特征。在恶性肿瘤发生之前，OPCs是唯一在MADM小鼠中急剧过度扩张的细胞系。在胶质瘤肿块中，OPCs也是维持活跃细胞分裂的主要细胞类型。当我们纯化这些opc样胶质瘤细胞时，它们表现出显著的胶质瘤干细胞特征，包括形成可再生的肿瘤球，分化成多细胞系，并在移植到免疫抑制小鼠后重新启动胶质瘤。基于这些初步结果，我们将验证以下假设：1)OPCs是启动和更新胶质瘤形成的关键细胞类型；2)突变的OPCs可以去分化以获得干细胞特性；3)靶向OPCs或其干细胞特性将是治疗胶质瘤的有效策略。我们提出的研究将有助于对胶质瘤的发育过程有价值的基本认识。肿瘤起始细胞的鉴定应为设计治疗策略提供依据。从概念上讲，我们提出的工作探索了肿瘤起始的未知领域，并为改进小鼠模型以了解人类癌症的机制提供了重要的基础。

DESCRIPTION (provided by applicant): Glioma is the most common type of malignant brain tumor. Despite widespread advances in cancer medicine, this devastating disease remains incurable. Recently, it was reported that gliomas contain a population of tumor stem cells that can form self renewable tumor spheres in culture and re- initiate gliomas after transplantation into immuno-suppressed mice. Targeting these tumor stem cells is an exciting prospect towards a glioma cure. However, critical information required to develop such therapeutic strategies, including the developmental origin of the glioma stem cells, remains unknown. To address the tumor cell of origin problem, it requires the use of glioma animal models that allow the analysis of early-stage pre-malignant tumor cells. Unfortunately, current mouse models cannot provide adequate in vivo resolution for such studies. Our laboratory has developed a new mouse glioma model based on a novel genetic mosaic system termed MADM (Mosaic Analysis with Double Markers, Zong et al Cell 2005). Using MADM, we can generate rare, green fluorescent protein (GFP)-labeled neural stem cells (NSCs) that are double null for two key tumor suppressor genes, p53 and Neurofibromatosis Type 1 (NF1), within an otherwise normal mouse. This approach allows us to analyze the entire course of gliomagenesis with single-cell resolution in vivo. Our preliminary findings show that, although the mutations are generated specifically in NSCs, resulting glioma cells manifest many cellular features of oligodendrocyte precursors (OPCs). Prior to malignancy, OPCs are the only cell lineage that drastically over-expands in the MADM mice. In the glioma tumor mass, OPCs are also the predominant cell type that maintains active cell divisions. When we purify these OPC-like glioma cells they manifest salient glioma stem cell features, including forming renewable tumor spheres, differentiating into multiple cell lineages, and reinitiating gliomas after being transplanted into immuno-suppressed mice. Based on these preliminary results, we will test the following hypothesis: 1) OPCs are the key cell type that initiates and renews gliomagenesis; 2) mutant OPCs can de-differentiate to acquire stem cell properties; and 3) targeting OPCs or their stem-cell characteristics will be effective treatment strategies for gliomas. Our proposed studies will lead to valuable basic understanding of the developmental process of gliomas. The identification of tumor-initiating cells should provide a basis for designing rationale treatment strategies for the cure. Conceptually, our proposed work explores the uncharted territory of tumor initiation, and provides critical groundwork for the refinement of mouse models for mechanistic understanding of human cancers.