Elucidating transcriptomic and functional heterogeneity of microglia in low-grade glioma and glioma-associated epilepsy.

项目摘要/摘要 低级别胶质瘤(LGG)是一种疾病，尽管有现代的治疗标准，但经常复发或进展到高度恶性- 级别胶质瘤，如胶质母细胞瘤(GBM)。大多数LGG患者在出现症状时就有癫痫发作，并有复发。 越来越多的研究癫痫样活动如何影响胶质瘤微环境的工作表明， 这种胶质瘤相关癫痫(GAE)通过直接的神经元-神经胶质瘤促进了胶质瘤的增殖和侵袭。 发信号。然而，GAE可能驱动胶质瘤的其他机制仍不清楚。与以前的工作不同 领域，这项建议集中于研究GAE与转录和功能变化的相关性 小胶质细胞，一种已知在推动基底膜进展中起关键作用的细胞类型。众所周知，小胶质细胞表现为转录下调。 以突触为靶点的颞叶癫痫(TLE)的变化和吞噬细胞过度活动。然而，尽管井- 这种细胞类型在GBM和TLE中扮演的角色已得到证实，但在LGG和TLE中仍未完全探索 盖伊。有趣的是，研究表明，人类癫痫瘤周皮质中有树突丢失，树突丢失。 已被证明在许多疾病中由小胶质细胞介导，包括癫痫。反过来，它也表明， 吞噬神经元分子驱动抗炎信号，诱导一系列与小胶质细胞平行的细胞因子 特征性征象预示着GBM预后较差。这项提议将检验GAE与 突触吞噬过度的小胶质细胞群丰富。此外，它还将确定 突触的吞噬作用是否与提示促肿瘤的小胶质细胞信号上调有关 功能。在目标1中，我将使用单核测序和免疫组织化学或RNAScope来鉴定小胶质细胞 亚型在GAE和LGG中有不同的富集性，并在原位验证了我的发现。然后我将检查GAE的相关性 以及TCGA数据集中与临床结果相关的LGG相关小胶质细胞特征。在目标2中，我将使用Single Cell 光学表型和表达测序(SCOPE-SEQ)配对基于成像的单个小胶质细胞测量 吞噬能力与来自同一细胞的单细胞RNA测序数据。我会检查GAE是否与 随着小胶质细胞突触吞噬功能的异常升高，将识别表现为更大突触的小胶质细胞亚型 吞噬能力和研究突触的吞噬是否促使小胶质细胞信号上调 提示具有促肿瘤作用，或与那些被发现预测GBM临床结果较差的患者相似。这个项目将 阐明小胶质细胞吞噬作用和小胶质细胞转录组在LGG和GAE中是如何受到差异干扰的 可能揭示新的小胶质细胞治疗靶点，这些靶点可能被调节以减缓LGG的进展。

PROJECT SUMMARY/ABSTRACT Low-grade glioma (LGG) is a disease that, despite the modern standard of care, frequently recurs or progresses to high- grade glioma, such as glioblastoma (GBM). Most LGG patients suffer from seizures on presentation and with recurrence. A growing body of work investigating how epileptiform activity affects the glioma microenvironment has suggested that this glioma-associated epilepsy (GAE) drives increased glioma proliferation and invasion by way of direct neuron-glioma signaling. However, other mechanisms by which GAE may drive glioma remain unclear. In contrast to prior work in the field, this proposal focuses on investigating the correlation between GAE and transcriptomic and functional changes in microglia, a cell type known to be critical in driving GBM progression. Microglia are well known to exhibit transcriptomic changes and phagocytic overactivity targeting synapses in temporal lobe epilepsy (TLE). However, despite the well- validated roles this cell type plays in both GBM and TLE, they have remained incompletely explored in both LGG and GAE. Intriguingly, it has been shown that there is dendritic loss in human epileptic peritumoral cortex, and dendritic loss has been shown to be microglia-mediated in many diseases, including epilepsy. In turn, it has also been shown that phagocytosis of neuronal elements drives anti-inflammatory signaling, inducing a set of cytokines that parallel microglial signatures shown to predict poorer prognosis in GBM. This proposal will test the hypothesis that GAE is associated with enrichment of microglial populations exhibiting overactive phagocytosis of synapses. Moreover, it will determine whether phagocytosis of synapses correlates with upregulation of microglial signatures suggestive of pro-tumorigenic function. In aim 1, I will use single-nucleus sequencing and immunohistochemistry or RNAscope to identify microglial subtypes differentially enriched in GAE and LGG and validate my findings in situ. I will then examine correlation of GAE and LGG-associated microglial signatures with clinical outcomes in the TCGA dataset. In aim 2, I will use Single Cell Optical Phenotyping and Expression sequencing (SCOPE-seq) to pair imaging-based measurements of single microglial phagocytic capacity with single-cell RNA-sequencing data from the same cell. I will examine whether GAE is associated with aberrantly elevated microglial synaptic phagocytosis, will identify microglial subtypes that exhibit greater synaptic phagocytic capacity and study whether phagocytosis of synapses drives upregulation of microglial signatures that suggest pro-tumorigenic function or parallel those found to predict worse clinical outcomes in GBM. This project will elucidate how microglial phagocytosis and the microglial transcriptome are differentially perturbed in LGG and GAE and may reveal novel microglial therapeutic targets that may be modulated to slow LGG progression.