Abstract: Pancreatic cancer is currently the third leading cause of cancer related to death in the USA with a 5- year survival rate of <5%. Most 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. Novel markers of early treatment response are critically needed to make better informed decisions regarding the type and sequencing of therapies, given the high toxicity associated with these treatments. In our previous work we were able to identify 20+ exosomal protein markers from the serum of patients with locally advanced pancreatic cancer during therapeutic treatment by using exosome isolation methods and mass spectrometry analysis. However, such analysis for large clinical cohorts was limited by the reproducibility in exosome preparation for accurate quantification of exosomal proteins, which is a significant problem in the exosome omics research field. In the current proposal, we plan to develop the analytics of exosome isolation and characterization with a focus on rigor and reproducibility. We will develop a super-SILAC-based (Stable Isotope Labeling by/with Amino acids in Cell culture) exosome method as a universal internal standard (UIS). In principle, when the super-SILAC- based exosome is spiked into human serum for proteomic analysis it can follow the same experimental workflow as serum exosomes which could maximally reduce variations introduced by sample preparation and LC-MS (Liquid Chromatography-Mass Spec) analysis. Therefore, SILAC-labeled exosomes will be ideal with a dual function for biomarker analysis for reproducible sample preparation and acting as a UIS. Targeted LC-SRM and PRISM-SRM exosome analyses of markers from our prior studies will be used with the UIS to precisely monitor changes in proteins during a course of chemo-radiation therapy. Patients with locally advanced pancreatic cancer will undergo serial blood draws prior to, during, and after treatment with chemotherapy followed by chemo-radiation. We will then perform a targeted proteomics confirmation study based on exosome markers identified in our previous work that have a specific response to a course of chemo-radiation therapy. The marker value changes compared to baseline will be associated with the treatment outcomes (such as progression-free survival and overall survival) in patients. This work will establish the potential use and measurement reproducibility of these exosomal markers for monitoring changes in protein markers during and after treatment. The LC-SRM mass spec-based assay to be used herein has the distinct advantage of being multiplexed for over 20 markers simultaneously. The proposed work contains several novel aspects including: 1) the use of super- SILAC to address irreproducibility in accurate quantification of exosome proteins ...