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

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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.