1 ABSTRACT – PROJECT 2 2 Brain metastases are highly aggressive, treatment resistant malignancies with debilitating neurological sequelae 3 and a grave prognosis. Efforts to understand and modulate the metastatic brain tumor microenvironment (TME) 4 have great therapeutic potential. Studies have suggested that polarization of macrophages (M1 or M2) may be 5 helpful or harmful in cancer progression (1, 2). Microglia, the brain’s myeloid cells, function in immune 6 surveillance and mediate the tumor-related inflammatory response, including within the context of the metastatic 7 brain tumor microniche (tumor-associated macrophages and tumor-associated microglia). We hypothesize 8 brain metastases are supported in part by native microglia (3), and that these interactions can be 9 manipulated for therapeutic benefit. The role of microglia in brain metastases has been difficult to investigate 10 given the relative inaccessibility of brain metastases samples, inability to isolate human tumor-associated 11 microglia or monitor their interactions within the TME, and lack of models that include or allow manipulation of 12 microglia while still reliably recapitulating the human disease. To overcome these critical hurdles, we employ the 13 following innovative methods: (i) single cell RNA-sequencing (scRNA-seq) on fresh, human brain metastases 14 and normal surrounding brain (Neuropathology Core)(4, 5); (ii) isolation and dynamic array sequencing of cell 15 free messenger RNA (cfRNA) from human cerebrospinal fluid (6, 7); (iii) generation of human and murine-derived 16 brain metastasis 3D air-liquid interface (ALI) organoids that include tumor-associated microglia/macrophages 17 (Toolkit Core); and (iv) repopulation of microglia with circulation derived microglia-like cells (CDMCs) with 18 greater than 90% efficiency throughout the entire brain. These four innovative methods allow us to investigate 19 the role of tumor-associated microglia in human brain metastases, model immune cell interactions, and 20 manipulate their function in an animal model of metastatic malignancy. Our multidisciplinary team is uniquely 21 positioned to test the following three aims: (1) Test the extent to which tumor-associated microglia create an 22 immunosuppressive TME supporting brain metastasis. In addition to relating microglia function to brain 23 metastasis local recurrence and distant progression, we will cross-reference our findings with cancer cell intrinsic 24 factors (Project 1) and systemic macrophage reactivity (Project 3); (2) Model tumor-associated microglia within 25 the brain metastatic TME using ALI organoids. We will model tumor and tumor-associated microglia/macrophage 26 interactions and determine the impact of immunotherapy on microglia/macrophage activation and phagocytosis, 27 T-cell activation states, and cell death. Our findings will be integrated with expanding knowledge of cell-intrinsic 28 factors (Project 1) and brain-periphery immune interact...