exRNA released by glioblastoma alters brain microenvironment

描述(由申请人提供)：我们的主要目标有两个：了解细胞外RNA(ExRNA)如何在肿瘤环境中调节正常细胞的表型，并产生新的实验模式，以阐明这种细胞间通信的潜在机制。具体地说，我们将重点了解胶质母细胞瘤细胞释放的exRNA如何调节肿瘤附近正常细胞的表型。我们的总体具体目标将包括：目标1-阐明GBM细胞外源RNA生物生成的基本分子和细胞机制，以及正常脑细胞的摄取和功能，使用现有和新兴技术来操纵这些过程。目的2-全面鉴定人GBM细胞的exRNA含量和细胞内RNA含量，以及正常脑细胞的细胞内RNA含量，并在培养的GBM细胞和GBM小鼠模型中评价exRNAs从GBM细胞到脑细胞的功能转移。目的3-评估外源RNA在脑细胞中的转移和去向，包括可视化外源RNA在EVS中的转移，监测mRNA翻译和miRNA功能，确定转座元件/癌基因可能的基因组整合，以及评估非编码外源RNA对基因组甲基化状态的影响。目的4-描述外显核糖核酸的组成、形成和释放动力学随基底膜基因型的变化，包括EGFR和PDGFRA信号通路的激活，这是人类基底膜肿瘤中最常见的两个遗传事件，以及放射线和药物治疗对基底膜外核糖核酸的影响。目的5-通过定制荧光和其他可视标记、载体、小鼠模型和试剂来开发外源RNA释放和摄取的调节器和报告程序，以广泛应用于监测外源RNA在培养和体内的释放、摄取和功能。这些目标将由一个共享的成像核心支持，该核心携带我们对细胞外小泡的活体成像以及它们与大脑中内源性细胞的相互作用。

DESCRIPTION (provided by applicant): Our broad term objective is two-fold: to understand how extracellular RNA (exRNA) modulates the phenotype of normal cells in the tumor environment and to generate new experimental modalities that can elucidate mechanisms underlying this form of communication among cells. Specifically we will focus on understanding how exRNA released from glioblastoma cells modulates the phenotype of normal cells in the vicinity of the tumor. Our overall specific aims will encompass: Aim 1 - Elucidation of basic molecular and cellular mechanisms of exRNA biogenesis by GBM cells, and uptake and function in normal brain cells, using existing and emerging technologies to manipulate these processes. Aim 2 - Full characterization of the exRNA content and intracellular RNA content of human GBM cells, as well as the intracellular RNA content of normal brain cells, and evaluation of functional transfer of exRNAs from GBM cells to brain cells in culture and in GBM mouse brain models. Aim 3 - Evaluation of transfer and fate of exRNA in brain cells, including visualizing RNA transfer in EVs, monitoring mRNA translation and miRNA functions, determining possible genomic integration of transposable elements/oncogenes, and evaluation of effects of non-coding exRNAs on status of genome methylation. Aim 4 - Description of the dependence of exRNA cargo composition, formation and release dynamics as a function of GBM genotype, including activation of EGFR and PDGFRa signaling pathways, the two most common genetic events in human GBM tumors, as well as changes in GBM exRNA in response to radiation and drug treatment. Aim 5 - Development of regulators and reporters of exRNA release and uptake by tailoring fluorescent and other visual labels, vectors, mouse models and reagents for broad applications in monitoring exRNA release, uptake and function in culture and in vivo. These aims will be supported by a shared imaging core carrying our intravital imaging of extracellular vesicles and their interaction with endogenous cells in the brain.