Characterizing the Brain Metastatic Niche and Elucidating Its Functional Role during Brain Metastasis Outgrowth

Brain metastases are secondary tumors in the brain commonly derived from breast, lung, and melanoma cells that detach from their respective primary tumors and seed the brain parenchyma. The brain parenchyma represents a unique niche within which metastatic cells grow, and different constituents of the brain metastatic niche have been demonstrated to have contrasting effects on metastatic outgrowth. Here, leveraging recently developed techniques, we sought to (1) characterize spatial and pseudo-temporal aspects of key components of the metastatic niche, (2) compositionally and transcriptionally profile the myeloid immune component of the brain metastatic niche, and (3) elucidate the functional role of myeloid cells during brain metastasis and the molecular mechanism by which they regulate metastatic outgrowth. To characterize the spatial distribution of brain metastatic niche components, we pioneered a three-dimensional (3D) imaging and image analysis pipeline, which most notably permitted us to quantify morphometric features of brain metastasis-associated myeloid cells (Br.MAM), a family of innate immune cells positioned at the hub of the immune web. Myeloid cells displayed dramatic morphological changes in the presence of brain metastases that became increasingly pronounced nearing the metastatic lesion. Using a high dimensional canonical immune cell profiling technique, we demonstrated Br.MAM were compositionally diverse, inclusive to brain-resident microglia and subsets of bone marrow derived myeloid (BMDM) cells. Applying a multi-modal sequencing method that couples epitope profiling with transcriptome profiling in single cells, we showed microglia and BMDM undergo enormous transcriptional changes from the naive state to become highly inflammatory in response to brain metastasis. Intriguingly, depleting microglia during brain metastasis outgrowth reduced metastasis number, while inhibiting the infiltration of BMDM to metastatic lesions did not change metastasis number, implicating microglia as the main myeloid effector during brain metastasis. To elucidate a mechanism by which microglia regulate brain metastasis, we knocked out Cx3cr1, a highly downregulated gene in microglia during brain metastasis, to show the loss of this gene promotes brain metastasis. Cx3cr1 loss led to Cxcl10 upregulation, ultimately leading to an increased intra-metastatic accumulation of CD86+ microglia that displayed negative immune checkpoint genes, including Vsir (VISTA) and Cd274 (PD-L1). Targeting VISTA and PD-L1 by antibody blockade resulted in reduced brain metastasis number. Cumulatively, our work sheds light on spatial aspects of the metastatic niche and reveals an important role for microglia in promoting brain metastasis through promoting immune suppression in the metastatic niche.