Project Summary/Abstract Extracellular vesicles (EVs) are nanoscale vesicles containing a variety of proteins and nucleic acids that can be transferred to other cells. It is widely accepted that EVs released from a variety of cells into the circulation can home to different organs for distant intercellular communication. For instance, circulating tumor- derived EVs can contribute to metastasis formation through preparing a niche at pre-metastatic organs. However, the mechanisms by which circulating EVs interact with the different endothelial barriers to penetrate into distant organs remain poorly understood. Elucidating these mechanisms will provide novel insights into the currently unknown mechanisms of intercellular EV processing in endothelial barriers, which can then be generalized to a variety of other barriers, leading to broad clinical and scientific implications. This proposal aims to elucidate the molecular mechanisms by which circulating EVs interact with the unique vascular structure of the brain, the blood-brain barrier (BBB), within the context of breast cancer brain metastasis. Breast cancer is the most common cause of brain metastasis in women and is associated with a median survival time of only 10 months. Development of efficient diagnostics and therapeutics for brain metastasis has been hindered in part by the restrictive nature of the BBB. We have recently demonstrated that tumor-derived EVs can breach the BBB and have identified “transcytosis” as the mechanism underlying this process. Importantly, we have also demonstrated that through modulating the endocytic pathway in endothelial cells, tumor-derived EVs can circumvent the low rates of transcytosis at the BBB and facilitate their transcellular transport. This finding suggests a novel mechanism underlying the interaction of circulating EVs and the brain endothelium. In this proposal, we will identify the molecular mechanism(s) underlying EV-derived modulation of transcytosis and the clinical implications of these mechanisms for development of early diagnostics for brain metastasis. These studies will be pursued within the context of the following Specific Aims: 1. To determine the mechanism(s) by which breast cancer-derived EVs modulate the endocytic pathway in brain endothelial cells 2. To identify the EV cargoes that promote facilitation of transcytosis in brain endothelial cells 3. To determine the correlation between EV content and breast cancer brain metastasis Our proposed studies will identify novel mechanisms that mediate the interaction of circulating EVs with brain endothelial cells and in doing so, can unmask early steps in the process of brain metastasis formation and provide the foundation upon which to develop novel diagnostic approaches for early detection of brain metastasis.