PROJECT SUMMARY: Breast cancer is the most common form of malignancy in women, accounting for almost one in three new female cancer diagnoses each year. The primary cause of breast cancer-related deaths is metastatic disease, where tumor cells disseminate to secondary sites in the body. Human breast tumors exhibit increased sensory nerve fiber density compared to healthy tissue, and increased innervation is associated with higher rates of distant organ metastasis. Notably, triple-negative breast cancer (TNBC), a highly metastatic molecular subtype, exhibits increased peripheral nerve innervation compared to other subtypes. Previous work in the Oudin lab demonstrated that sensory nerves promote the in vitro migration and in vivo metastasis of TNBC cells. The goal of this project is to uncover the mechanism driving increased sensory nerve innervation in TNBC, which could provide a novel target to reduce metastasis and improve patient survival. TRPV1 is a non-selective cation channel present on sensory nerves that drives neurite outgrowth and regeneration. Stimulation of TRPV1 activates the c-Jun transcription factor, which upregulates downstream genes involved in neurite outgrowth, such as interleukin 6 (IL6). Our preliminary transcriptomic analysis revealed that Il6 and other c-Jun target genes involved in neurite outgrowth are upregulated when primary sensory neurons were co-cultured with TNBC cells. I also found that TNBC cells promoted sensory neurite outgrowth and increased c-Jun expression in a TRPV1- dependent manner. I hypothesize that TNBC cell secretions stimulate TRPV1 on sensory nerves in adjacent healthy tissue, leading to c-Jun activation and an upregulation in genes associated with neurite outgrowth and regeneration, such as IL6, ultimately driving neurite outgrowth into the tumor microenvironment (TME). In three specific aims, I will test this hypothesis and further elucidate how TNBC cells recruit sensory nerves into the TME. In SA1, I will investigate the role of c-Jun and its downstream targets, such as Il6, in driving TNBC cell-mediated sensory neurite outgrowth in 2D. In SA2, I will utilize a 3D hydrogel model to determine the effects of TNBC cells on sensory neurite outgrowth and explore the role of TRPV1 in mediating outgrowth. To our knowledge, this innovative approach will be the first description of nerve-cancer interactions in a 3D system. In SA3, I will examine the role of TRPV1 in recruiting sensory nerves into mammary tumors in vivo. This will be completed by both genetic and pharmacologic inhibition of TRPV1 in a syngeneic mouse model of TNBC. Successful completion of these aims will provide valuable insights into the mechanism through which TNBC cells promote sensory innervation into the TME. As innervation drives TNBC cell metastasis, targeting this newly discovered mechanism could offer a promising clinical strategy to improve patient outcome. Aligned with this research proposal, a tailored training plan has been d...