The oncogenic functions of Neuropilin-1 in the glioblastoma tumor microenvironment

摘要 胶质母细胞瘤（GB）是成人中枢神经系统（CNS）最常见的原发性肿瘤。尽管 侵袭性手术、放疗和化疗，中位生存期只有15-20个月。在GB肿瘤中， 肿瘤中约30-50%的细胞是先天性骨髓免疫细胞，主要是巨噬细胞（MP） 和小胶质细胞（MG）。Tsirka实验室的研究表明，MP和MG通过以下方式增强GB增长： 促进血管生成和培养免疫抑制性肿瘤微环境（TME），部分通过 共同受体神经纤毛蛋白-1（NRP 1）的作用。我们最近的工作表明MG和MP的作用不同 神经胶质瘤神经胶质瘤相关MP和MG（GAM）上NRP 1的表达对其致癌活性至关重要。 髓样细胞中NRP 1的缺失会减弱血管生成并导致GB体积减少。先前的研究 已经表明，缺氧神经胶质瘤细胞上调干细胞相关基因，并发挥神经胶质瘤干细胞的功能， (GSC)，其被认为驱动GAM的免疫抑制和血管生成性质。NRP 1已经 对GAM对缺氧的反应至关重要我们研究小组的长期目标是确定 MG和MP的TME作用以及MP和MG可以通过其复极化以发挥抗-TME作用的方法。 而不是促进肿瘤生长。我的建议的总体目标是确定 MG和MP与缺氧区域和GSC相互作用以发挥致癌作用的机制。我 假设髓样细胞上NRP 1活化是促肿瘤发生信号传导的重要介质 缺氧胶质瘤细胞、GSC和GAM之间的差异。为了验证这一假设，我将追求两个具体目标：1） 确定缺氧胶质瘤细胞在GAM中激活的NRP 1通路;和2）确定GAM-NRP 1如何在GAM中激活。 信号传导影响GSC和GAM之间的相互作用。对于第一个目标，MP和MG将分别治疗 用来自在常氧或缺氧中培养的神经胶质瘤细胞的培养基，有或没有NRP 1抑制剂，并评估 促/抗炎和血管生成因子表达的差异， 磷酸蛋白质组学使用GB的免疫活性小鼠模型，我将研究GAM-NRP 1如何影响 GAM表型与常氧和低氧肿瘤区域的肿瘤细胞增殖。在第二个目标中， 将在培养物和体内研究GAM-NRP 1对GSC/GAM相互作用的贡献。拟议 这项研究预计将是重要的，因为它将确定MP和MG行为的作用， 进一步探讨GSC与GAM之间的相互作用。这也将有助于确定 调节NRP 1是否是促进GAM抗肿瘤输出的可行方法。

Abstract Glioblastoma (GB) is the most common primary tumor of the central nervous system (CNS) in adults. Despite aggressive surgery, radiation, and chemotherapy, median survival is only 15-20 months. In GB tumors, approximately 30-50% of the cells in the tumor are innate myeloid immune cells, primarily macrophages (MP) and microglia (MG). Research in the Tsirka laboratory has shown that MP and MG enhance GB growth by promoting angiogenesis and fostering an immunosuppressive tumor microenvironment (TME), partly through the action of the co-receptor Neuropilin-1 (NRP1). Our recent work has suggested distinct roles for MG and MP in glioma. Expression of NRP1 on glioma-associated MP and MG (GAM) is critical for their oncogenic activity. Deletion of NRP1 from myeloid cells blunts angiogenesis and results in decreased GB volume. Prior research has shown that hypoxic glioma cells upregulate stem cell-associated genes and function as glioma stem cells (GSC), which are thought to drive the immunosuppressive and angiogenic nature of GAM. NRP1 has been shown to be critical for the GAM responses to hypoxia. The long-term goal of our research group is to define the TME roles of MG and MP as well as methods through which MP and MG can be repolarized to exert anti- tumorigenic effects rather than facilitate tumor growth. The overall objective of my proposal is to determine the mechanisms by which MG and MP interact with hypoxic regions and GSC to exert oncogenic effects. I hypothesize that NRP1 activation on myeloid cells is an important mediator for pro-tumorigenic signaling between hypoxic glioma cells, GSC and GAM. To test this hypothesis, I will pursue two specific aims: 1) Determine the NRP1 pathways activated in GAM by hypoxic glioma cells; and 2) identify how GAM-NRP1 signaling affects the interaction between GSC and GAM. For the first aim, MP and MG will be separately treated with media from glioma cells cultured in normoxia or hypoxia, with or without a NRP1 inhibitor, and assessed for differences in the expression of pro/anti-inflammatory and angiogenesis factors identified through phosphoproteomics. Using immunocompetent mouse models of GB, I will investigate how GAM-NRP1 affects GAM phenotypes and tumor cell proliferation in normoxic and hypoxic tumor regions. In the second aim, the contribution of GAM-NRP1 to GSC/GAM interactions will be interrogated in culture and in vivo. The proposed research is expected to be significant because it will determine roles for MP and MG behavior in the context of hypoxic areas of glioma and further explore the interaction between GSC and GAM. It will also help determine whether modulation of NRP1 is a viable method for promoting anti-tumorigenic outputs for GAM.