Isolation and characterization of pancreatic cancer-derived small extracellular vesicles as a novel liquid biopsy approach

Background: A major challenge in managing pancreatic cancer (PanC) is late detection and the inability to identify non-responders early. Existing liquid biopsy approaches offer limited capacity to rapidly and noninvasively track the tumor's evolving molecular landscape, which is essential for overcoming therapy resistance and guiding targeted treatment. Small extracellular vesicles (sEV; <200 nm) circulate widely and carry cargo reflective of their cells of origin. Using our established methods for isolating tissue-specific sEV, we identified pancreas-specific surface markers, isolated circulating pancreas derived sEV (sEV Pancreas ), and evaluated their potential as a liquid biopsy platform. Methods: We analyzed fresh-frozen PanC tissues (n=12) along with matched healthy samples. sEV were isolated from tissues, subjected to surface protein shaving, and analyzed using LC-MS/MS based on published methods. Proteomic data, combined with the Human Protein Atlas, were used to identify sEV Pancreas . They were isolated from archived plasma samples of PanC patients (n=10) and healthy individuals (n=5) using biotin-tagged antibodies and streptavidin-coated magnetic beads. Isolated sEV/sEV Pancreas were characterized for size and concentration by nanoparticle tracking analysis (NTA), and for various biomarkers' expression using nano-flow cytometry, RT-PCR, RNA sequencing, and digital PCR. Results: Prolyl 4-hydroxylase subunit beta (P4HB) and annexin A4 (ANXA4) were identified as pancreas-specific sEV surface markers. Nano-flow cytometry confirmed significantly higher levels of P4HB- and ANXA4-positive sEV in PanC plasma compared to healthy controls (p<0.01). Using these markers, we isolated sEV Pancreas from blood plasma samples of PanC patients and controls. NTA confirmed that the isolated vesicles were <200 nm in both groups. Notably, sEV Pancreas from PanC patients exhibited significantly higher expression of the PanC biomarker cholecystokinin A receptor (p<0.01) and lower levels of miR-320-5p, a microRNA associated with poor prognosis. RNA sequencing of sEV Pancreas revealed several upregulated (e.g., endosulfine-alpha, MUC12) and downregulated (e.g., DYNC1I2, POM121C) genes in the PanC group. Finally, KRAS copy number and mutation status was reliably assessed in sEV Pancreas , highlighting the potential for KRAS mutational profiling.: we reliably characterized wild type and mutated KRAS status (G12D and G12V). Conclusions. We demonstrate that pancreas-derived sEV can be selectively isolated from blood using newly identified pancreas-specific markers. These sEV Pancreas harbor distinct molecular signatures and reliably capture KRAS copy number and mutations, supporting their potential as a rapid, minimally invasive liquid biopsy platform. These findings lay the groundwork for sEV-based assays for treatment monitoring and response assessment.