exRNA released by glioblastoma alters brain microenvironment

描述（由申请人提供）：我们的广义目标是双重的：了解细胞外RNA（exRNA）如何调节肿瘤环境中正常细胞的表型，并产生新的实验模式，可以阐明这种形式的细胞间通讯的机制。具体来说，我们将专注于了解从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细胞的exRNA生物合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 -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含量的完全表征，以及在培养物和GBM小鼠脑模型中评估exRNA从GBM细胞到脑细胞的功能性转移。目的3 -评估exRNA在脑细胞中的转移和命运，包括可视化EV中的RNA转移，监测mRNA翻译和miRNA功能，确定转座因子/癌基因的可能基因组整合，以及评估非编码exRNA对基因组甲基化状态的影响。目的4 -描述作为GBM基因型的函数的exRNA货物组成、形成和释放动力学的依赖性，包括EGFR和PDGFRa信号传导途径（人GBM肿瘤中两种最常见的遗传事件）的激活，以及GBM exRNA响应于辐射和药物治疗的变化。目的5 -通过定制荧光和其他视觉标记、载体、小鼠模型和试剂开发exRNA释放和摄取的调节剂和报告物，用于监测培养物和体内exRNA释放、摄取和功能的广泛应用。这些目标将得到一个共享的成像核心的支持，该成像核心携带我们的细胞外囊泡的活体成像及其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.