Abstract The purpose of this study is to investigate localized delivery of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential therapeutic modality in breast cancer to alleviate tumor hypoxia and immunosuppression. A therapeutic that has the potential to target and reverse these factors of the tumor microenvironment (TME) would aid in reducing tumor aggressiveness, immune escape, metastasis, and importantly, could potentially be administered as a pre-treatment to reduce resistance to standard therapies such as chemotherapy and radiation. We have previously reported that 100 ng of intratumorally delivered recombinant GM-CSF 1) altered the macrophage profile from immunosuppressive M2-like to immunosupportive M1-like and 2) inhibited angiogenesis and induced near anoxic levels in the tumor due to the dose-dependent production of soluble VEGFR-1 from tumor macrophages that sequestered VEGF. Our current focus is to determine if a dosage window of GM-CSF exists that may normalize leaky and dysfunctional tumor vasculature and alleviate hypoxia in tumors by lesser sVEGFR-1 production, and simultaneously convert the immune cell profile from immunosuppressive to immunosupportive to promote cytotoxic T cell killing. After intratumoral injections with either GM-CSF or saline 3x per week for 2.5 weeks in an orthotopic PyMT murine breast cancer model, we utilized a soluble multifunctional trityl probe and Electron Paramagnetic Resonance Spectrometry to measure intratumoral oxygen and pH in vivo in real time. Our findings demonstrate that intratumoral injections of 15 ng GM-CSF rescued tumor oxygen by ~16% and raised pH ~0.2 units, while 5 ng enhanced oxygen even further by ~40% and raised pH ~0.4 units. Additionally, tumor-associated macrophages (TAMs) that were isolated from Stage IV MMTV-PyMT tumors and treated with 5 ng/ml GM-CSF 3x per week for 2 weeks demonstrated a reduction in IL-10 expression as determined by intracellular staining and flow cytometry. Further, as a result of these data, we have also formulated and characterized GM-CSF-containing PLGA/PEG-PLGA nanoparticles in vitro with average sizes under 200 nm for the purpose of systemic but localized delivery of dose-optimized recombinant GM-CSF to tumors. Nanoparticles yielded an entrapment efficiency of 87.9% +/- 4.2 S.D. and release of functional GM-CSF in vitro. Overall, we have demonstrated the potential of locally delivered recombinant GM-CSF to alleviate tumor hypoxia and acidity in vivo and reverse immunosuppressive TAM phenotype ex vivo, and we have formulated nanoparticles encapsulating recombinant murine GM-CSF as another modality which will be utilized for localized delivery of GM-CSF to breast tumors to achieve similar effects. Citation Format: Nicole Mihalik, Andrey Bobko, Valery Khramtsov, Benoit Driesschaert, Tim D. Eubank. Localized delivery of granulocyte-macrophage colony-stimulating factor to alleviate tumor hypoxia and immunosuppression in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2672.