Project Summary Tumor hypoxia is linked to worse outcome of glioblastoma (GBM), the most malignant type of brain cancer. Hypoxic GBM cells are thought to resist therapy, and to have high migratory capacity to move away from hypoxic zones, infiltrating normal brain tissue. Aside from these cell-autonomous attributes, hypoxic tumor cells also contribute to GBM progression by exerting non-cell autonomous functions, in particular inducing angiogenesis and reprograming tumor microenvironment (TME) into an immunosuppressive and tumor-supporting state. To better understand the gene signatures of GBM cells in hypoxic niches and their interactions with TME cells, it is critical to conduct studies with in vivo models. However, earlier approaches to track hypoxic cells, such as pimonidazole or HRE-GFP have limited sensitivity and resolution. To facilitate improved in vivo tracking of hypoxic tumor cells during GBM progression, I will apply a genetic reporter with HRE (hypoxia response element promoter) driving expression of UnaG, a fluorescent protein that does not require oxygen for fluorophore maturation, thus offering superior sensitivity over GFP in hypoxic conditions. The UnaG mRNA transcript also allows me to distinguish hypoxic from normoxic cells in single cell (sc) RNA-seq studies. Indeed, my preliminary data with a patient-derived GBM stem cell line engineered with HRE-UnaG showed sensitive reporting of hypoxia in vivo in intracranial transplant experiments. Excitingly, using immunohistological analysis combined with scRNA-seq, I have collected preliminary evidence supporting the central hypothesis that GBM cells in hypoxic niches in vivo are relatively quiescent and display a shift to a mesenchymal state, both features that are linked to therapy resistance and migratory potency. I have also identified top differentially regulated genes in hypoxic GBM cells in vivo, with potential roles in promoting migration and enhancing proliferation of neighboring tumor cells. This proposal will expand upon my initial studies to further define in vivo GBM hypoxia gene signatures. A key focus in my first aim will be the interaction of hypoxic tumor cells with surrounding TME, in particular tumor associated macrophages (TAM) in hypoxic niches. To dissect the molecular mechanisms of hypoxia-driven GBM malignancy, I will conduct for my second aim functional analyses of two candidate genes, CXCR4 and NXPH4, which are highly upregulated in hypoxic GBM cells. I will test the hypothesis that CXCR4 promotes migration of hypoxic tumor cells, while NXPH4 may support the growth of neighboring GBM cells. Lastly, for my third aim, I will explore a therapeutic strategy to target the hypoxic population to evaluate its efficacy to sensitize GBM cells to irradiation therapy. I plan to carry out my studies with two patient-derived GBM stem cell lines of different transcriptional subtypes (mesenchymal and proneural), to assess how different GBM subtypes respond to hypoxia. In sum...