Project Summary Tumors are a heterogenous population of cancer cells, infiltrating host cells, secreted factors and extracellular matrix proteins–together comprising the tumor microenvironment. The tumor microenvironment plays profound roles in supporting tumor initiation, survival, progression and metastasis. We have discovered that tumor endothelial cells provide an “instructive” signal to cancer cells that drives metastatic progression. We found Slit2, an axon guidance molecule, to be upregulated in endothelial cells within metastatic tumors. Endothelial-specific deletion of Slit2 strongly inhibited metastasis. In a surprising discovery, we found that endothelial-Slit2 deletion also impairs tumor innervation. Nerves are only starting to be recognized as key players within the tumor stroma, with recent evidence demonstrating their requirement for tumor initiation of gastric and prostate cancers. In breast cancer, although there is some preliminary evidence that innervation correlates with tumor aggressiveness, nerve dependence for metastasis has not been explored. In this Project, we will define the role of neurons within the stroma of primary breast tumors and understand how neurons regulate metastasis. Using isogenic cell lines of differential metastatic potential, retrograde tracing and imaging studies, we will first identify neuronal sub-populations that regulate breast cancer metastasis. We will use 3D co-culture systems and in vivo denervation experiments to identify the role of neurons in tumor invasion, proliferation, local and systemic dissemination, distant seeding, and metastatic outgrowth. We will also evaluate the role of tumor innervation in therapeutic resistance in breast cancer. Next, we will use unbiased sequencing approaches to define tumor- trophic factors secreted by neurons and delineate downstream signaling axes in cancer cells. Finally, we will characterize the role of endothelial cells in guiding neurons into metastatic tumors and determine if the pro- metastatic effects of neurons are partly mediated by immune cells. The proposed inter-disciplinary research plan combines molecular, genetic, biochemical, imaging and computational approaches to establish an integrated model for neural regulation of metastatic progression by breast cancer. Collectively, these studies will define the cellular and molecular interactions between endothelial cells, neurons, and immune cells within the tumor microenvironment–an overarching goal of our proposed MetNet research center. These studies have tremendous potential for impact as that they stand to identify novel therapeutic targets that perturb neural-tumoral signaling axes driving metastasis.