Characterizing the functional stem-like properties of primary and recurrent gliom

描述（由申请人提供）： 多形性胶质母细胞瘤（GBM）是最具侵袭性和最常见的恶性胶质瘤，平均总生存期仅为14.6个月。尽管积极的治疗包括手术切除，电离辐射，同时和维持化疗，疾病最终复发。确切的细胞和分子机制，使这些胶质瘤复发仍然是一个激烈的研究领域，但最近的证据表明，胶质瘤有一个相对较小的群体的干细胞样细胞，是耐辐射和化疗。我们已经观察到，当与替莫唑胺共培养时，原代神经胶质瘤培养物呈现出更多的干细胞样和侵袭性表型，并且在此提出在体内评估这些现象。为此，我们将用放射和/或化学疗法治疗鼠胶质瘤，并在复发时表征其干细胞样性质。我计划使用由RCAS-tva系统产生的PDGF驱动的胶质瘤的基因工程小鼠模型（GEMM）来开发复发性GBM的小鼠模型。在大约30%的人GBM中观察到异常的PDGF信号传导，并且用该系统产生的鼠神经胶质瘤与人类疾病的病理学和遗传特征非常相似。将使用各种测定来比较原发性和复发性鼠神经胶质瘤，以表征其功能性干细胞样特性。具体而言，将使用肿瘤球形成测定法，并通过免疫组织化学和免疫荧光检查干细胞样标志物的表达来比较胶质瘤。还将通过使用蛋白质印迹分析和定量真实的时间PCR检查干样标志物、通过进行侧群分析和通过测定原位肿瘤形成能力来定量比较原发性和复发性胶质瘤。此外，还将比较原发性和复发性胶质瘤，以评估已知可表达干细胞样表型的信号通路（如Notch和PI 3 K/AKT通路）活化的任何差异。总之，这些实验将表征复发性胶质瘤的功能性干细胞样特性，并探索可能赋予这种表型的途径。)

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is the most aggressive and most common malignant glioma with an average overall survival is only 14.6 months. Despite aggressive therapy consisting of surgical resection, ionizing radiation, and both concurrent and maintenance chemotherapy, the disease ultimately recurs. The exact cellular and molecular mechanisms that allow these gliomas to recur remains an area of intense research, but recent evidence suggest that gliomas have a relatively small population of stem-like cells that are resistant to irradiation and chemotherapy. We have observed primary glioma cultures take on a more stem-like and aggressive phenotype when co-cultured with temozolomide, and herein propose to evaluate these this phenomena in vivo. For this purpose, we will treat murine gliomas with either radiation and/or chemotherapy, and characterize their stem- like properties upon recurrence. I plan to develop a mouse model of recurrent GBM using a genetically engineered mouse model (GEMM) of PDGF-driven glioma generated by the RCAS-tva system. Aberrant PDGF signaling is seen in approximately 30% of human GBMs, and the murine gliomas generated with this system closely resemble both the pathology and genetic characteristics the human disease. Both primary and recurrent murine gliomas will be will be compared using a variety of assays to characterize their functional stem-like properties. Specifically, the gliomas will be compared using tumorsphere formation assays, and by examining the expression of stem-like markers with immunohistochemistry and immunofluorescence. Primary and recurrent gliomas will also be compared quantitatively by examining stem-like markers using western blot analysis and quantitative real time PCR, by performing side population analysis, and by assaying orthotopic tumor forming ability. Additionally, primary and recurrent gliomas will be compared to assess any differences in the activation of signaling pathways that are well known to convey a stem-like phenotype, such as the Notch and PI3K/AKT pathway. Together, these experiments will characterize the functional stem-like properties of recurrent gliomas, and explore the pathways that may confer this phenotype. )