Project Summary Triple negative breast cancer (TNBC) makes up to 15-20% of breast cancer diagnoses. While surgery and adjuvant chemotherapy are effective treatment options for early-stage disease, there are very few therapeutic options for advanced metastatic TNBC, and the 11% relative five-year survival rate highlights an urgent need to discover actionable targets. In the hematogenous metastatic cascade, a few aggressive tumor cells are capable of crossing the endothelial barrier at least twice: when entering systemic circulation (intravasation) and then again when exiting circulation to colonize distant organs (extravasation). High expression of JAGGED-1 (JAG1), a Notch ligand, is strongly associated with TNBC metastasis and consequent mortality. Our preliminary work in static culture suggests that JAG1 enhances TNBC binding to the endothelium and transendothelial migration (TEM), thereby promoting dissemination of tumor cells through vascular beds. We propose to study two distinct mechanisms for JAG1 in the metastatic cascade. In Aim 1, we will investigate how tumor JAG1 signals in trans to the endothelium to prime the vascular barrier for invasion. We will examine extravasation of our recently generated TNBC CRISPR JAG1 knockout clones and control cells using a microfluidics system that faithfully models capillary fluid shear forces and permits visualization of multiple critical steps of extravasation in vitro. We will also test the role of JAG1 in vivo by examining the rate of lung capillary extravasation and pulmonary seeding following tail vein injection of JAG1 knockout and control TNBC cells. In Aim 2, we will define the tumor cell- intrinsic transcriptional program regulated by JAG1. Our preliminary RNA sequencing data suggest that novel JAG1 targets promote cell surface interactions distinct from Notch targets. We will determine the metastatic importance of key JAG1 targets by restoring expression and testing for TEM phenotypes. In the training plan of the fellowship, I highlight an integrated scientific, clinical, and educational blueprint to enhance my personal research and doctoring skills as an aspiring physician-scientist in oncology.