Defining invasion dynamics of GBM reveals axon guidance genes drive invasion

项目摘要 成人多形性胶质母细胞瘤（GBM）是最常见和致命的恶性脑癌。肿瘤 细胞是浸润性的，渗透周围组织，为复发提供种子，并逃避治疗。 GBM的细胞和分子异质性进一步使其治疗复杂化。然而，肿瘤细胞如何 信号在早期肿瘤发生、进展和迁移期间发生变化，以及它们在不同时间点如何变化。 大脑中的位置仍然未知。此外，侵袭性表型的机制 对这种疾病的观察知之甚少。因此，本提案的总体目标是确定如何 肿瘤细胞随着时间的推移侵入周围组织，并确定驱动这一过程的机制。 最近的研究对入侵的GBM的前缘进行了采样，发现在毫米范围内有不同的种群。 这说明了在分析肿瘤细胞功能时微环境的重要性。映射 随着时间的推移，GBM通过大脑不同区域的演变，我们开发了一种内源性的 免疫活性小鼠模型，使我们能够在天然肿瘤背景下研究细胞侵袭。我们的初步 数据表明肿瘤细胞表达随肿瘤位置而变化，并与功能相关;然而，当 并且这些变化在疾病进展期间在何处出现仍然未知。因此，第一个目标是 拟议的研究是确定入侵的动力学和相关的基因和蛋白质表达的变化， GBM在我们的天然肿瘤模型的背景下。我们将使用肿瘤的3D绘图和RNA-Seq研究 在多个时间点肿瘤中的三个不同位置（原发性肿瘤、继发性肿瘤、胼胝体） 以确定GBM的入侵动力学。 在初步研究中，我们发现轴突导向基因在继发性肿瘤部位和体部富集 胼胝体由于它们在中枢神经系统内的迁移中的既定作用， 在癌症中，我们进行了轴突引导基因的条形码功能获得（GOF）筛选。我们发现EphA7 在原发性肿瘤外特异性富集，表明其有助于GBM迁移。当EphA7 尽管GBM患者的表达与预后较差相关，但其在侵袭中的作用尚不清楚。因此，我们的第二个 目的是通过对EphA7在GBM侵袭和迁移中的功能获得和丧失的研究， 在我们的天然肿瘤模型和人类患者来源的异种移植物中的背景下。 总的来说，本提案旨在扩展我们的初步数据，以生成完整的时间轴和3D地图 在肿瘤发生和侵袭之间发生的遗传事件和相应的表达变化 穿过大脑然后，我们将首先在我们的天然肿瘤模型中验证这些变化，然后突出显示 与体外和体内的人体系统相似。

PROJECT SUMMARY Adult glioblastoma multiforme (GBM) is the most common and deadly form of malignant brain cancer. Tumor cells are infiltrative, permeating surrounding tissue, providing the seeds for recurrence, and evading therapy. The cellular and molecular heterogeneity of GBM further complicate its treatment. However, how tumor cell signatures change during early tumorigenesis, progression, and migration and how they vary at different locations in the brain remain unknown. Additionally, the mechanisms responsible for the invasive phenotype observed in this disease are poorly understood. Therefore, the overarching goal of this proposal is to define how tumor cells invade surrounding tissues over time and identify the mechanism driving this process. Recent studies sampling the leading edges of invading GBM have found diverse populations within millimeters of one another, illustrating the importance of microenvironment when analyzing tumor cell function. To map the evolution of GBM through different regions of the brain over time, we developed an endogenous immunocompetent mouse model that allows us to study cell invasion in a native tumor context. Our preliminary data indicate that tumor cell expression varies by tumor location and correlates with function; however, when and where these changes arise during disease progression remains unknown. Therefore, the first aim of the proposed study is to define the dynamics of invasion and associated gene and protein expression changes in GBM within the context of our native tumor model. We will use 3D mapping of the tumor and RNA-Seq studies of three distinct locations (primary tumor, secondary tumor, corpus callosum) in the tumor at multiple time points in order to define the invasion dynamics of GBM. In preliminary studies, we found axon guidance genes to be enriched in the secondary tumor site and corpus callosum. Because of their established role in migration within the central nervous system and specifically in cancer, we performed a barcoded gain of function (GOF) screen of axon guidance genes. We found that EphA7 was specifically enriched outside of the primary tumor, suggesting it contributes to GBM migration. While EphA7 expression is associated with worse outcomes in GBM patients, its role in invasion is unknown. Thus, our second aim is to define the role of EphA7 in GBM invasion and migration through gain and loss of function studies in the context of our native tumor model and in human patient derived xenografts. Overall, this proposal aims to expand upon our preliminary data to generate a complete timeline and 3D map of the genetic events and corresponding expression changes that occur between tumor initiation and invasion through the brain. We will then validate those changes first in our native tumor model and then will highlight the parallels with human systems in vitro and in vivo.