Melanoma Brain Metastasis (MBM) carries a dismal prognosis with a median overall survival of only 4-6 months. If patients develop leptomeningeal disease, the overall survival is even lower. The incidence of MBM has been reported to be up to 43% in clinical settings and up to 75% in the autopsy series. Although notions that Circulating Tumor Cells (CTCs) act as ?seeds? of intractable metastasis are established, there is no knowledge characterizing MBM-colonizing CTCs. Single-cell CTC transcriptional profiling has also demonstrated that CTCs isolated from patients are very distinct from cell lines that are widely used for drug discovery. This is even more compelling considering that significant discrepancies of biomarkers among CTCs and corresponding primary and metastatic tumors have been observed. Moreover, while the presence of CTCs in the cerebrospinal fluid (CSF) remains the gold standard, the sensitivity of cytology is only 50-56% at time of the first CSF analysis. Therefore, the development of effective therapy approaches - CTC-based tests - could have a tremendous clinical impact to treat MBM. We hypothesize that the neurotrophin receptor p75NTR and Heparanase (HPSE), two markers implicated in MBM models, are novel CTC biomarkers to predict clinical MBM and potential therapeutic targets to prevent MBM. The objective of this application is to demonstrate that the p75NTR/HPSE axis is diagnostic in clinical MBM; and that p75NTR and HPSE are novel therapeutic CTC targets to combat MBM. In aim 1, we will: a) isolate and characterize p75NTR/HPSE CTC subsets from blood and CSF (multiparametric flow cytometry and DEPArrayTM technologies among others), and compare the expression of p75NTR/HPSE combinations in CTCs of melanoma patients diagnosed either with or without MBM; b) directly link patient-isolated CTC subsets, possessing p75NTR/HPSE expression and combinations, to clinical MBM. In aim 2, we will assess effects of regulating functions of p75NTR/HPSE CTC subsets on MBM development by using small-molecule p75NTR and new HPSE inhibitors along with CTC xenografts; and complement these effects with regulatory p75NTR/HPSE gene expression (pINDUCER lentiviral toolkit). In aim 3, we will: a) determine roles of CTC-expressed Merlin as an important integrator of p75NTR/HPSE pathways altering CTC proliferation vs. growth arrest; b) delineate HPSE-induced, syndecan-mediated modulation of Merlin/Hippo signaling to affect CTC properties driving MBM. Uncovering MBM CTC phenotypes offers the opportunity to modify treatment by extending studies directly to human melanoma. This project lead by an inter-disciplinary and well-integrated team will study and validate new and specific CTC biomarkers responsible for CTC-induced MBM. It has high therapeutic impact and is paradigm- shifting. We are uniquely positioned to perform this study not only for having access to an extensive cohort of blood/CSF samples from melanoma patients but also for the extensive expertise interrogating the entire CTC spectrum by combining multiple and complementary CTC technologies unlike other groups in the country.