MOLECULAR DISSECTION OF SEIZURE MICROENVIRONMENT IN MALIGNANT GLIOMA

胶质母细胞瘤细胞触发药物抗性癫痫发作，可能促进肿瘤生长并降低肿瘤的质量。 剩余的生命。明确胶质瘤生长与其神经元之间的关系 微环境，并确定基因组生物标志物和机制，可能指向更好的预后， 治疗脑癌耐药性癫痫，我们正在分析新一代的基因定义的 在小鼠中工程化的CRISPR/子宫内电穿孔先天性胶质母细胞瘤（GBM）肿瘤模型。分子 胶质母细胞瘤细胞和周围神经元和未转化星形胶质细胞的病理生理学将被 在三种遗传小鼠品系的肿瘤发展的一系列阶段进行比较：野生型，癫痫倾向， 抗癫痫。初步数据显示，癫痫样脑电图尖峰是一个非常早期和可靠的临床前 在这些小鼠中，GBM扩张的特征先于其他神经功能缺损，随后迅速 几周之内就会逐渐发作死亡皮质星形胶质细胞的转录组学分析揭示了 一个富含促突触发生基因的亚群的扩展可能会驱动过度兴奋，这是一种新的 癫痫发生机制在具体目标1中，我们将系统地定义最早出现的 在具有典型GBM的野生型小鼠中的皮质过度兴奋，并将其进展与体内和 侵袭性肿瘤细胞定位的神经病理学成像，体外电生理学，以及 肿瘤周围神经元中的关键癫痫致病级联，包括受损的谷氨酸再摄取， GABA门控氯梯度和突触密度。在具体目标2中，我们将把这些发现与 肿瘤周围的转化型和野生型星形胶质细胞的详细的FACS分类转录组学特征 区域来检验新的假设，即肿瘤周围的过度兴奋部分是由星形胶质细胞亚型驱动的， 破坏突触E/I稳态。在Specific Aim 3中，我们将在WT brain中使用这种基准方法， 比较生长，电生理和分子病理学特征的相同肿瘤产生在一个 一个过度兴奋的大脑，带有一个单一的基因缺失（Kcna 1），大大降低了癫痫发作的阈值 并且缩短寿命，并且在单基因缺失菌株（MapT/tau）中，其提高皮层癫痫发作阈值， 为了检查宿主神经元兴奋性对肿瘤扩展的贡献，我们 该方法为广泛探索个性化肿瘤/宿主相互作用的发育生物学奠定了基础 在特定神经胶质细胞谱系中用新型人类肿瘤突变改造的小鼠中。

Glioblastoma cells trigger pharmacoresistant seizures that may promote tumor growth and diminish the quality of remaining life. To define the relationship between growth of glial tumors and their neuronal microenvironment, and to identify genomic biomarkers and mechanisms that may point to better prognosis and treatment of drug resistant epilepsy in brain cancer, we are analyzing a new generation of genetically defined CRISPR/in utero electroporation inborn glioblastoma (GBM) tumor models engineered in mice. The molecular pathophysiology of glioblastoma cells and surrounding neurons and untransformed astrocytes will be compared at serial stages of tumor development in three genetic mouse strains: wild type, seizure prone, and seizure resistant. Preliminary data reveal that epileptiform EEG spiking is a very early and reliable preclinical signature of GBM expansion preceding other neurological deficits in these mice, followed by rapidly progressive seizures and death within weeks. Transcriptomic analysis of cortical astrocytes reveals the expansion of a subgroup enriched in pro-synaptogenic genes that may drive hyperexcitability, a novel mechanism of epileptogenesis. In Specific Aim 1 we will systematically define the earliest appearance of cortical hyperexcitability in wild type mice with a prototypical GBM and correlate its progression with in vivo and neuropathological imaging of invasive tumor cell location, in vitro electrophysiology, and molecular markers of key epilepsy pathogenic cascades in peritumoral neurons, including impaired glutamate reuptake, altered GABA gated-chloride gradients, and synaptic densities. In Specific Aim 2 we will correlate these findings with detailed FACS-sorted transcriptomic profiles of both transformed and wild type astrocytes in the peritumoral region to test the novel hypothesis that peritumoral hyperexcitability is driven in part by astrocytic subtypes that disrupt synaptic E/I homeostasis. In Specific Aim 3, we will use this benchmark approach in WT brain to compare growth, electrophysiological and molecular pathological profiles of the same tumor generated in a hyperexcitable brain bearing a single gene deletion (Kcna1) that dramatically lowers the threshold for seizures and shortens lifespan, and in a monogenic deletion strain (MapT/tau) that raises cortical seizure threshold and prolongs life, in order to examine the contribution of host neuronal excitability to tumor expansion. Our approach sets the stage to broadly explore the developmental biology of personalized tumor/host interactions in mice engineered with novel human tumor mutations in specified glial cell lineages.