J. Zheng1, J. Hernandez1, A. Doussot1, L. Bojmar2, B. Costa-Silva2, E. VanBeek2, C. Zambirinis1, M. Tesic Mark3, H. Molina3, M. Gonen1, T. Kingham1, P. Allen1, M. D’Angelica1, R. DeMatteo1, D. Lyden1,2, W. Jarnagin1 1Memorial Sloan-Kettering Cancer Center,New York, NY, USA 2Weill Cornell Medical College,New York, NY, USA 3Rockefeller University,New York, NY, USA
Introduction: Exosomes have emerged as important vehicles for intercellular communication between cancer cells and their microenvironment and may facilitate tumor progression. Exosomes in pancreatic duct fluid have not been previously studied. We hypothesize that exosomes can be detected in the pancreatic duct fluid and that exosomal proteins may be useful as diagnostic and/or prognostic biomarkers in patients with pancreatic ductal adenocarcinoma (PDAC).
Methods: Pancreatic duct fluid was collected from 22 patients at the time of partial pancreatectomy for PDAC (n=13), intraductal papillary mucinous neoplasm (IPMN) (n=4) and other benign pancreatic diseases (n=5). Serial ultra centrifugation was used to isolate exosomes. Exosomes were quantified using the DS500 nanoparticle characterization system (NanoSight), and confirmed by transmission electron microscopy (TEM). Exosomal proteins were identified and analyzed using liquid chromatography-mass spectrometry (LC-MS). Expression levels of exosomal proteins were compared between patients with PDAC and benign diseases using Wilcoxon rank-sum test. Survival analyses among patients with PDAC were performed using Kaplan-Meier with log rank test.
Results: Exosomes were successfully isolated from the pancreatic duct fluid of all patients. The mean concentration of exosomes isolated was 5.2 +/- 1 x 108 particles/mL, with a mean vesicle size of 186 +/- 4nm. The presence of exosomes was confirmed for each patient using TEM. Characteristic exosome surface markers, including tetraspanins CD9 and CD63, were identified. No differences in either size or concentration of EVs were detected when patients were stratified by diagnosis, but significant differences in protein composition were found. Laminin β3, Laminin C2, Tenascin C, and matrix metalloproteinases 1 and 7 were significantly overexpressed among patients with PDAC as compared to patients with IPMN and benign diseases (Table). Similarly, CEACAM1, CEACAM5, and CEACAM6 were significantly overexpressed among patients with PDAC (p=<0.001, 0.025, and 0.030, respectively). Among patients with PDAC, patients having expression of all three CEACAMs (n=6) had significantly worse overall and recurrence-free survival compared to patients lacking any of the CEACAMs (n=7) (p=0.01 and 0.005, respectively).
Conclusion: Exosomes can be isolated from pancreatic duct fluid. Exosomal ECM-associated proteins and CEACAMs appear to possess diagnostic and prognostic utility for patients with PDAC. Further evaluation of the mechanistic significance of these proteins in exosomes is warranted.
