Mechanisms of Hedgehog signaling in glioblastoma

项目摘要 胶质母细胞瘤是致命的肿瘤，约占所有恶性原发性脑癌的一半。 胶质母细胞瘤干细胞存在于受肿瘤影响的可互换的细胞状态中 微环境，并且缺乏有关细胞机制的信息， 在胶质母细胞瘤细胞和肿瘤间质之间进行沟通。Hedgehog途径，它指导基因 对于发育和成体干细胞稳态至关重要的表达程序， 胶质母细胞瘤干细胞。奇怪的是，一项临床试验表明， 加速胶质母细胞瘤的生长。这些临床数据提供了一个机会，以重新评估如何 Hedgehog通路在癌症中发挥作用，并表明Hedgehog信号传导在胶质母细胞瘤干细胞中的作用。 细胞仍有待进一步研究。这项提案的重点是了解如何抑制刺猬途径 在肿瘤、细胞和分子水平上驱动胶质母细胞瘤。为了做到这一点，我们的建议包括一本小说， 胶质母细胞瘤的星形胶质细胞类器官模型，有助于肿瘤内细胞相互作用的实时成像 微环境当与CRISPR干扰、药理学和小鼠同基因和 患者来源的胶质母细胞瘤异种移植模型，我们的创新类器官系统将促进以前的 不可能的实验来了解胶质母细胞瘤的生物学。基于我们初步的类器官，在体内， 本申请中提供的单细胞RNA测序数据，我们假设癌细胞-外源性 肿瘤微环境中的Hedgehog信号通过抑制胶质母细胞瘤干细胞的更新和侵袭 诱导分化因子和重塑细胞外基质。我们将通过定义以下内容来检验这一假设： Hedgehog信号是如何通过胶质母细胞瘤微环境转导的;确定癌细胞 外源性Hedgehog信号通过诱导骨形态发生蛋白信号抑制胶质母细胞瘤干细胞; 并确定Hedgehog信号传导是否通过诱导调节胶质母细胞瘤细胞侵袭的基因， 通过细胞外基质迁移。这种方法的前提是，通过研究 Hedgehog信号在胶质母细胞瘤中的作用机制，我们将发现胶质母细胞瘤细胞 与肿瘤微环境沟通。事实上，我们对相互作用 胶质母细胞瘤细胞和肿瘤微环境之间的联系影响癌症的进展， 关于构成成人大脑中大多数细胞类型的星形胶质细胞如何影响胶质母细胞瘤侵袭 或肿瘤发生。虽然这项建议的目的是广泛地提高我们对 Hedgehog信号在胶质母细胞瘤中的作用机制，这项研究的长期目标是了解这些 这一过程足以考虑靶向治疗策略，以提高胶质母细胞瘤的生存率。因此，在本发明中， 这项工作将解释为什么Hedgehog通路抑制意外地加速胶质母细胞瘤的生长，阐明 靶向机制，并为胶质母细胞瘤患者的新临床试验提供临床前基础。

Project summary Glioblastomas are deadly tumors and account for approximately half of all malignant primary brain cancers. Glioblastoma stem cells exist in interchangeable cellular states that are influenced by the tumor microenvironment, and there is a paucity of information concerning the cellular mechanisms responsible for communicating between glioblastoma cells and the tumor stroma. The Hedgehog pathway, which directs gene expression programs that are essential for development and adult stem cell homeostasis, is critical for survival of glioblastoma stem cells. Paradoxically, a clinical trial suggested that pharmacologic inhibition of the Hedgehog pathway accelerates glioblastoma growth. These clinical data provide an opportunity to re-evaluate how the Hedgehog pathway functions in cancer, and suggest that the role of Hedgehog signaling in glioblastoma stem cells remains to be elucidated. This proposal focuses on understanding how inhibition of the Hedgehog pathway drives glioblastoma at the tumor, cellular, and molecular scale. To do so, our proposal incorporates a novel astrocyte organoid model of glioblastoma that facilitates live imaging of cellular interactions within the tumor microenvironment. When integrated with CRISPR interference, pharmacology, and mouse syngeneic and patient derived xenograft models of glioblastoma, our innovative organoid system will facilitate previously impossible experiments to understand glioblastoma biology. Based on our preliminary organoid, in vivo, and single cell RNA sequencing data presented in this application, we hypothesize that cancer cell-extrinsic Hedgehog signaling in the tumor microenvironment restrains glioblastoma stem cell-renewal and invasion by inducing differentiation factors and remodeling the extracellular matrix. We will test this hypothesis by defining how Hedgehog signals are transduced through the glioblastoma microenvironment; determining if cancer-cell extrinsic Hedgehog signaling restrains glioblastoma stem cells by inducing bone morphogenic protein signaling; and determining if Hedgehog signaling restrains glioblastoma cell invasion by inducing genes that regulate migration through the extracellular matrix. This approached is based on the premise that by studying the mechanisms of Hedgehog signaling in glioblastoma, we will discover new insights into how glioblastoma cells communicate with the tumor microenvironment. Indeed, we know surprisingly little about how interactions between glioblastoma cells and the tumor microenvironment influence cancer progression, and almost nothing about how astrocytes, which comprise the majority of cell types in the adult brain, influence glioblastoma invasion or tumorigenesis. Though the objective of this proposal is to broadly improve our understanding of the mechanisms of Hedgehog signaling in glioblastoma, a long-term goal of this research is to understand these processes well enough to consider targeted therapeutic strategies to improve survival from glioblastoma. Thus, this work will explain why Hedgehog pathway inhibition unexpectedly accelerates glioblastoma growth, elucidate targetable mechanisms, and provide the preclinical basis for new clinical trials in glioblastoma patients.