Selective Differentiation of Glioblastoma Stem Cells

描述（由申请人提供）：肿瘤干细胞是从肿瘤（包括胶质母细胞瘤）中分离出来的细胞亚群，具有干细胞样特性和有效启动肿瘤形成的能力。胶质母细胞瘤是一种原发性恶性脑肿瘤，对所有形式的治疗基本上都是难治的，几乎都是致命的，中位生存期约为15个月。越来越多的证据表明，肿瘤干细胞在胶质母细胞瘤的进展、治疗抵抗和最终复发中起着重要作用。与大块肿瘤相比，胶质母细胞瘤干细胞（GSCs）具有自我更新、分化为更成熟、致瘤性更低的表型的能力，并产生异质细胞类型，这是胶质母细胞瘤的标志。我们已经分离并表征了一组GSC细胞系，这些细胞形成的肿瘤再现了在分离它们的患者肿瘤中观察到的组织病理学和异质性。迄今为止，对肿瘤干细胞的研究是基于我们对正常干细胞及其发育的理解。然而，关于肿瘤干细胞如何区别于正常干细胞的基本问题，以及如何利用这些差异来获得治疗益处，可能需要不同的方法。因此，我们建议在FDA批准和已知生物活性的无偏倚高通量筛选中发现诱导GSCs分化的化合物，而不是神经干细胞，这是一种以前没有描述过的方法。经过验证的“击中”应该揭示选择性参与GSC维持和分化的细胞途径，并且由于GSC分化损害致瘤性，导致针对GSC治疗胶质母细胞瘤的新药的开发。为了实现这一目标，我们将开发两种方法来筛选分化诱导剂：(1)在贴壁的GSC培养物中进行高含量的基于图像的免疫荧光检测，检测内源性的干性和分化标记，以及(2)在转导的GSC中进行基于启动子驱动的荧光素酶报告的荧光荧光检测。GSC报告系（来自(2)）提供了一种模型，可以在肿瘤进展过程中实时无创地跟踪GSC分化。这些方法可以直接转化为来自其他类型癌症的肿瘤干细胞，并应提供工具和试剂，将有利于该领域和胶质母细胞瘤患者。

DESCRIPTION (provided by applicant): Tumor stem cells are a subpopulation of cells isolated from tumors, including glioblastoma, that have stem-like properties and the ability to efficiently initiate tumor formation. Glioblastoma is a primary malignant brain tumor that has remained largely refractory to all forms of therapy and is almost uniformly lethal with a median survival of approximately 15 months. There is increasing evidence that tumor stem cells play an important role in glioblastoma progression, resistance to treatment, and ultimate recurrence. In contrast to the bulk tumor, glioblastoma stem cells (GSCs) have the ability to self-renew, differentiate into more mature and less tumorigenic phenotypes, and give rise to the heterogeneous cell types that are a hallmark of glioblastoma. We have isolated and characterized a panel of GSC lines, and these cells form tumors that recapitulate the histopathology and heterogeneity observed in the patients' tumors from which they were isolated. Studies of tumor stem cells have thus far been based on our understanding of normal stem cells and development. However, addressing fundamental questions about how tumor stem cells are distinct from normal stem cells, and how these differences can be exploited for therapeutic benefit are likely to require different approaches. Thus, we are proposing to discover compounds that induce differentiation of GSCs, but not neural stem cells, in unbiased high throughput screens of FDA approved and known bioactives, an approach that has not been previously described. The validated 'hits' should shed light on cellular pathways selectively involved in the maintenance and differentiation of GSCs, and because GSC differentiation impairs tumorigenicity, lead to the development of novel drugs targeting GSCs for the treatment of glioblastoma. To accomplish this, we will develop two assays to screen for differentiation inducers: (1) a high-content image-based immunofluorescence assay of endogenous markers of stemness and differentiation in adherent GSC cultures, and (2) a luminescence-based assay of promoter-driven luciferase reporters in transduced GSCs in sphere culture. The GSC reporter lines (from (2)) provide a model to non-invasively follow GSC differentiation during tumor progression in vivo in real time. These approaches are directly translatable to tumor stem cells from other types of cancer and should provide tools and reagents that will benefit the field and patients with glioblastoma.