Exploring the Therapeutic Potential of Stem Cell Biology in Gliomas

我们已经进行了实验，以提高我们对TSCs中非调控分化途径的分子机制的理解：干细胞自我更新和分化之间的微妙平衡受到各种细胞内在和外在因素的控制，这些因素对正常组织的动态平衡至关重要。尽管GSCs和正常干细胞在表型和功能上有广泛的相似性，但GSCs的分化潜能并不完全正常。阐明在正常干细胞和GSCs中均可操作的分化途径对于全面了解肿瘤的发生至关重要，并可能导致新的治疗靶点。我们还鉴定了一组来自人类原发GBM来源的GSCs的非调控分化途径。阐明其潜在的分子机制将为预测分化治疗方法的敏感性提供重要线索。TSCs在分化诱导剂方面的特性进一步揭示了传统的胶质瘤细胞系在血清中生长的局限性。例如，维甲酸处理和CNTF暴露可以有效地诱导大多数GBM肿瘤起始细胞(TICs)分化，但不能诱导传统细胞系分化。这促使我们质疑之前在细胞系中研究的许多潜在的肿瘤抑制基因和/或细胞抑制基因是否没有被识别。鉴于从生物信息学方法和实验室积累的干细胞培养技术专业知识中鉴定出的胶质母细胞瘤干细胞中潜在的GSG和癌基因的数量不断增加，我们建立了筛选系统来研究这些基因在干细胞培养中的功能。此外，我们在Keystone项目之一上取得了重大进展，即了解我们的胶质瘤TSC和正常神经干细胞(NSC)之间的基因组和分子信号的异同。我们对7个不同的GBM来源的TSC和正常胚胎NSC系在增殖和分化条件下进行了非常大规模的研究，并获得了高通量的mRNA和microRNAs图谱。自2014年11月Gilbert博士启动新的胶质瘤干细胞翻译研究项目以来，我们又创造了6个胶质瘤来源的TSC，引入了3个IDH突变的TSC，并随后对这些GSC中的遗传学和信号通路进行了计算分析，使这些细胞系能够用于探索胶质瘤干细胞生物学的治疗潜力，如代谢变化和免疫反应。此外，通过合作和材料转移协议，我们获得了重要的细胞系，为实验室提供了出色的资源。NOB实验室现在总共有62个人类肿瘤株。作为该项目的一部分，这些细胞系中的每一个都进行了广泛的分子表征，包括完整的外显子组测序、甲基组分析和RNA测序。这些数据正被上传到数据库系统中，以便为NOB的所有研究小组分析和选择用于临床前研究的最佳模型系统。

We have performed experiments to improve our understanding of the molecular mechanisms of deregulated differentiation pathways in TSCs: The delicate balance between stem cell self-renewal and differentiation is controlled by various cell intrinsic and extrinsic factors that are critical for normal tissue homeostasis. Despite extensive phenotypic and functional similarities between GSCs and normal stem cells, the differentiation potentials of GSCs are not entirely normal. Elucidation of the differentiation pathways that are operative in both normal stem cells and GSCs will be critical for fully understanding tumorigenesis and will likely lead to novel therapeutic targets. We have also identified a set of deregulated differentiation pathways in GSCs derived from human primary GBM. Elucidation of underlying molecular mechanism will provide important clues for predicting sensitivity of differentiation therapeutic approach. Characterization of TSCs in aspect of differentiation-inducing agents further revealed the limitations of traditional glioma cell lines grown in serum. For example, retinoic acid treatment and CNTF exposure potently induce differentiation in most GBM tumor initiate cells (TICs) but not of traditional cell lines. This prompted us to question whether many of potential tumor suppressors and/or cytostatic genes previously studied in cell lines, were not recognized. Given the ever-increasing number of potential GSGs and oncogenes in glioblastoma TSCs identified from bioinformatics approaches and technical expertise of stem cell culture accumulated in the laboratories, we have set up screening systems to study the function of these genes in stem cell cultures. In addition, we have made significant progress on one of keystone projects that is to understand the genomic and molecular signaling similarities and differences between our glioma TSCs and normal neural stem cells (NSC). We have performed a very large scale study of 7 different GBM-derived TSCs and normal embryonic NSC lines under both proliferative and differentiating conditions and derived high-throughput mRNA and microRNAs profiling. Since November of 2014, when Dr Gilbert initiated the new glioma stem cell translational study project, we have created 6 more glioma-derived TSCs, introduced 3 IDH- mutated TSCs, and subsequently performed the computational analyses for characterization of the genetics and signaling pathways in these GSCs, enabling these cell lines to be used to explore the therapeutic potential of glioma stem cell biology, such as metabolic changes and immunological reactions. Additionally, we have through collaborations and Material Transfer Agreements obtained important cell lines that provide an outstanding resource for the laboratory. In total, there are now 62 human tumor lines in the NOB Laboratory. As part of this project, each of these cell lines has been extensively molecularly characterized including whole exome sequencing, analysis of the methylome and RNA sequencing. These data are being uploaded into a database system to enable analysis and selection of optimal model systems for preclinical studies for all of the research groups in the NOB.