Pancreatic cancer is currently the fourth leading cause of cancer related death in the USA with a 5 year survival rate of <5%. The poor prognosis results from the biologically aggressive nature of this disease combined with late clinical presentation. The majority of potentially curable patients present with unresectable locally advanced disease where the standard of care includes a combination of chemotherapy and radiation therapy. A major challenge in the management of pancreatic cancer is the early assessment of treatment response and early detection of metastatic disease. Novel biomarkers of early treatment response are greatly needed to improve the management of patients with this disease. Further, malignant tumors are comprised of a small subset of distinct cancer stem cells (CSC) which have great proliferative potential. CSCs regenerate a tumor and may be more resistance to standard therapy. Thus the therapies used to treat this disease must address the unique survival mechanisms of the cancer stem cell population. It thus becomes essential to find methods for monitoring the presence of cancer stem cells during and after therapy. In the proposed work, we will develop methods for detection of markers in microvesicles called exosomes. These vesicles are secreted into the circulation of cancer patients and reflect the tumor protein content potentially allowing us to detect cancer stem cells from patient serum. We will thus develop methods for extraction of exosomes from serum and for developing a targeted mass spec based assay for the detection of a large number of potential CSC markers in serum in a multiplexed fashion. This exosome assay will be used to monitor changes in cancer stem cell markers during a course of treatment with chemotherapy and radiation therapy. Patients with locally advanced pancreatic cancer will undergo serial blood draws prior to, during, and after treatment with chemotherapy followed by chemoradiation therapy. Changes seen in exosome markers will be correlated with overall survival, distant metastasis free survival, local control, and radiographic response. This work will establish the potential use of these markers for monitoring changes in cancer stem cell content after treatment and relate the results to clinical outcome.