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，对所有形式的治疗都很难治疗，并且几乎一致致命，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发挥重要作用。与大块肿瘤相比，胶质母细胞瘤干细胞（GSC）具有自我更新、分化成更成熟和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系，这些细胞形成的肿瘤概括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，肿瘤干细胞的研究是基于我们对正常干细胞和发育的理解。然而，解决肿瘤干细胞与正常干细胞的区别以及如何利用这些差异获得治疗益处的基本问题可能需要不同的方法。因此，我们提出在FDA批准的和已知的生物活性物质的无偏高通量筛选中发现诱导GSC而不是神经干细胞分化的化合物，这是一种以前没有描述过的方法。经验证的“命中”应该阐明选择性参与GSC维持和分化的细胞途径，并且由于GSC分化损害致瘤性，导致开发靶向GSC用于治疗胶质母细胞瘤的新型药物。为了实现这一点，我们将开发两种检测方法来筛选分化诱导剂：（1）贴壁GSC培养物中干细胞和分化的内源性标志物的高含量基于图像的免疫荧光检测，以及（2）球体培养物中转导GSC中启动子驱动的荧光素酶报告基因的基于发光的检测。GSC报告细胞系（来自（2））提供了一种模型，以在体内真实的时间内非侵入性地跟踪肿瘤进展期间的GSC分化。这些方法可直接转化为其他类型癌症的肿瘤干细胞，并应提供有益于该领域和胶质母细胞瘤患者的工具和试剂。 公共卫生相关性：胶质母细胞瘤是最常见的原发性恶性脑肿瘤，无论治疗方式如何，在短时间内总是致命的。肿瘤干细胞最近已经从胶质母细胞瘤肿瘤标本中分离出来，并且被认为是肿瘤进展、治疗抗性和最终复发的原因。我们的目标是开发和进行高通量筛选试验，以确定诱导胶质母细胞瘤干细胞分化的化合物和途径，以最终治疗胶质母细胞瘤患者。

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. PUBLIC HEALTH RELEVANCE: Glioblastoma, the most frequent primary malignant brain tumor, is invariably lethal within a short period of time irrespective of therapeutic modality. Tumor stem cells have recently been isolated from glioblastoma tumor specimens and are thought to be responsible for tumor progression, therapy resistance and ultimate recurrence. Our goal is to develop and perform high-throughput screening assays to identify compounds and pathways that induce differentiation of glioblastoma stem cells in order to ultimately treat glioblastoma patients.