The long-term objective of this research proposal is to i) develop a single-cell isolation technology by coupling a NanoVelcro Chip with Laser MicroDissection (LMD) techniques to enable highly efficient enumeration and specific isolation of viable/preservative-free circulating melanoma cells (CMCs) from blood, and ii) to demonstrate the feasibility of performing molecular and functional analyses of the isolated single CMCs. In collaboration with the UCLA melanoma team, we will validate the clinical utility of the proposed single-CMC molecular assays for dynamically monitoring disease progression, treatment outcomes and drug resistance in melanoma patients treated with BRAF inhibiters (BRAFi). Our team at UCLA has demonstrated a highly efficient, inexpensive circulating tumor cell (CTC) assay capable of enriching, identifying and isolating CTCs in whole-blood samples collected from patients with different solid tumors. First, we pioneered a unique concept of NanoVelcro cell-affinity substrates, by which capture agent (antibodies or aptamers) -coated nanostructured surfaces were utilized to immobilize CTCs in a stationary device setting. Second, by integrating the NanoVelcro substrate with an overlaid microfluidic component that can generate vertical flows, further improved CTC capture efficiency (>85%) has been achieved as a result of the enhanced collisions between CTCs and the substrate. Side-by-side analytical validation studies using both artificial and patient CTC samples suggested that the sensitivity of NanoVelcro CTC Assay outperformed that of CellSearchTM. CTCs and CMCs are cancer cells that break away from either the primary tumor or metastatic sites and circulate in the peripheral blood. Enumeration of CTCs/CMCs has established clinical utility in patients with metastatic solids tumors, in whom the CTC/CMCs number becomes an independent and accurate predictor for a patient's response to chemotherapy, disease free/overall survival. It is conceivable that a minimally invasive blood-based diagnostic technique could allow repeated characterization of CTCs/CMCs, providing insight into tumor biology during the critical window where intervention could actually make the difference. Currently, FDA- cleared CellSearchTM Assay is costly and inefficient in capturing CTCs/CMCs without contamination of surrounding white blood cells, thus the diagnostic values of CTCs/CMCs are not fully utilized. Herein, we will explore the combined use of new NanoVelcro Assay and LMD technique for isolating viable/preservative-free single CMCs from blood samples collected from melanoma patients over the course of BRAFi treatment. We will then subject the isolated CMCs for molecular and functional analysis. We envision the variation of CMC number and resulting CMC-based molecular signatures can be used to better investigate and monitor evolution of resistance mechanisms during BRAFi treatment, and to guide development of next- generation kinase inhibitor-based melanoma treatments.