Abstract The tumor microenvironment plays an essential role in the process of metastasis. However, the role of the peripheral nervous system, a crucial component of the tumor microenvironment, in cancer progression remains poorly understood. In breast cancer, nerve presence has been correlated with more invasive disease and worse prognosis. Yet, there is still a lack of detailed investigation into the specific types of nerves that are present in breast cancer as well as the mechanism by which nerve-cancer crosstalk contributes to breast cancer metastasis. We identified sensory nerves as more abundant in triple-negative human breast cancer tumors relative to healthy breast by mining gene expression datasets and immunostaining. Interestingly, the sensory nerve bundle structure in normal breast is disrupted in breast tumor, resulting in discrete nerve fibers throughout the tumor. To investigate the role of these sensory nerves in cancer progression, we cocultured primary sensory neurons from dorsal root ganglia of mice and human triple-negative breast cancer cells (MDA-MB-231). We found that cancer cells tend to attach and move along nerve fibers. These cells also have higher migration speed and proliferation rate when compared to both monoculture and conditioned media control. We then set out to investigate whether sensory nerves impact gene expression in tumors using RNA sequencing. By utilizing published algorithms, we were able to separate the transcriptome of human cancer cells and mouse neurons in coculture. Differential analysis shows that cancer cells in coculture upregulate genes related to cell migration, adhesion, and proliferation pathways, which further validates our experimental findings. We identified the Sema5A-PlexinB3 ligand-receptor pair as a potential mechanism of nerve-cancer crosstalk, where neurons provide Sema5A that interacts with upregulated PlexinB3 receptors on cancer cells. Knockdown of Plexin-B3 in tumor cells reduced the effect of sensory nerves on breast cancer cell migration. In vivo experiments injecting sensory nerves and tumor cells into the mammary gland of mice are ongoing. Overall, these studies demonstrate that sensory nerve density is high in TNBC and that sensory nerves promote tumor cell migration and proliferation via direct cell-cell interactions. We also show for the first time that nerves can induce gene expression changes in cancer cells. In the long-term, these findings could lead to novel treatments for metastatic disease that target nerve-cancer crosstalk. Citation Format: Thanh T. Le, Madeleine J. Oudin. Sensory nerves enhance breast cancer migration through PlexinB3 signaling [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO047.