The incidence of malignant melanoma is increasing steadily for the past three decades and it is among the leading causes of new cancer diagnoses in the US. Despite recent advances in targeted chemotherapy and immunotherapy for treatment of melanoma, there are no durable treatments for this cancer. Cutaneous melanoma can often be cured if diagnosed at early stages as a primary lesion in the skin. Survival rates decrease dramatically with diagnosis of increasing stage (Stage II and III) primary cancer. Even within a given stage of the early stage cancer, the survival rates are highly variable. Although majority of melanomas are diagnosed early and excised when presented in the skin, a significant number of patients diagnosed with early invasive skin lesions die from melanoma. Concerted efforts to identify and validate tissue-based biomarkers for prognosis have yielded mixed results. There is a need to better define prognostic markers that can reliably identify patients to assess, monitor and manage their risk of developing lethal metastatic disease. Circulating tumor cells (CTC) and/or tumor cell-derived macromolecules in peripheral blood are now considered reliable indicators of the presence of metastatic tumor. Enumerating the CTCs in patients’ blood sample is approved by US FDA for monitoring cancer before, during and after treatment. However, detection of the rare CTC in the blood before the appearance of metastatic disease remains a challenge due to the low sensitivity and lack of specificity of the currently available methods of CTC detection. We have developed a highly sensitive and specific biosensor to detect melanoma cells based on their cell surface proteins. This biosensor is readily adaptable to a multiplexed microfluidic device for sequential or parallel capture, detection and isolation of melanoma CTC based on melanoma-specific cell surface protein biomarkers. The overall goal of the proposed research is to develop a sensitive and selective method to capture and characterize CTC in patients to monitor and manage patients diagnosed with early stage melanoma. In this project, we propose to employ a multiplexed microfluidic biosensing device to detect, enumerate, isolate and characterize CTC in the blood of Veterans diagnosed with Stage I-III primary melanoma. The specific aims of this project are: Aim 1: Detection and capture of melanoma cells based on surface marker heterogeneity Using a panel of antibodies against melanoma cell surface antigens, we will optimize an immunosensor for sensitive detection of primary and metastatic melanoma cells. We will employ cultured human primary and metastatic melanoma cells spiked in normal blood and CTC in blood samples from Veterans with metastatic melanoma. Aim 2: Detection and characterization of CTC heterogeneity using multiplexed microfluidic immunosensor array We will integrate the immunosensor into a flow-through microfluidic platform for detection, capture and isolation of CTC in blood...