This project evaluates the efficacy of using biomarkers carried by tumor-derived exosomes and other organellederived extracellular vesicles to differentiate patients with pancreatic cancer from patients with benign pancreatic disease. We will use high conductance dielectrophoresis-based technology to simultaneously recover different types of these cancer-derived nanoparticles from individual samples of volume limited patient plasma. This will enable access to volume restricted early stage cancer samples. This research will generate new knowledge supporting the development of a nanoparticle-based biomarker panel for early and late stage pancreatic cancer detection and will support the rationale to bring nanoparticle-based diagnostics to the clinical setting. Pancreatic cysts are coincidently discovered with increasing frequency as the use of high resolution CT and MRI imaging increases, with up to 9.3% of patients having an asymptomatic cyst. An invasive endoscopic ultrasound guided fine needle aspiration biopsy (EUS/FNA) is traditionally used to determine if the cyst is pancreatic ductal adenocarcinoma (PDAC). This has a high financial cost and associated health risk where 1/100 patients will develop acute pancreatitis and 1/10 of these patients will die. Currently 60-76% of patients that undergo EUS/FNA do not have pancreatic cancer. This signifies the unmet clinical need to develop a blood test to stratify patients with pancreatic cysts into categories of high-probability and low-probability for having PDAC, where high-probability would benefit from the EUS/FNA. Currently, no blood based PDAC biomarkers exist. Tumorderived nanoparticles offer a new source of potential PDAC related biomarkers. The challenge is that traditional nanoparticle recovery methods for each nanoparticle type require plasma volumes that are too large to be supported with currently available PDAC patient plasma samples. Our preliminary data now suggest that high conductance dielectrophoresis (DEP) techniques can recover sufficient amounts of nanoparticle derived biomarkers to detect precancerous lesions as well as differentiate early and late stage PDAC from controls and requires only 30 µl of plasma. Each Aim will translate our DEP validation of individual biomarkers into a panel that will be evaluated in a blinded cohort study consisting of patients with PDAC and benign pancreatic disease. We focus Aim 1 on particles actively released by tumors, and Aim 2 on different cellular organelle fragments. Aim 3 evaluates these biomarkers, and five from our preliminary data, on stage 1 and 2 PDAC samples. The significance of this research is that the knowledge generated will lead to potential validation of a panel of nanoparticle-associated biomarkers capable of differentiating PDAC from benign pancreatic disease. The research proposed here may ultimately support the rationale for the clinical development of a nanoparticle-based diagnostic blood test to identify patients with pancreatic cysts that would benefit from the EUS/FNA procedure.