Serum extracellular vesicles contain protein biomarkers for primary sclerosing cholangitis and cholangiocarcinoma

Abstract

Cholangiocarcinoma (CCA) includes a heterogeneous group of biliary cancers with poor prognosis. Several conditions such as primary sclerosing cholangitis (PSC) are risk factors. Non-invasive differential diagnosis between intrahepatic CCA (iCCA) and hepatocellular carcinoma (HCC) is sometimes difficult. Accurate non-invasive biomarkers for PSC, CCA or HCC are not available. In the search of novel biomarkers, serum extracellular vesicles (EV) were isolated from CCA (n=43), PSC (n=30) or HCC (n=29) patients, and healthy individuals (control, n=32), and their protein content was characterized. By using nanoparticle tracking analysis (NTA), serum EV concentration was found higher in HCC than all the other groups. Round morphology (by transmission electron microscopy), size (∼180 nm diameter by NTA) and markers (CD9, CD63 and CD81 by immunoblot) indicated that most serum EV were exosomes. Proteome profiles (by mass spectrometry) revealed multiple differentially expressed proteins among groups. Several of these proteins showed high diagnostic values with maximum area under the ROC curve (AUC) of 0.878 for CCA vs control, 0.905 for CCA stage I-I vs control, 0.789 for PSC vs control, 0.806 for PSC non-cirrhotic vs control, 0.796 for CCA vs PSC, 0.956 for CCA stage I-I vs PSC, 0.904 for HCC vs control, and 0.894 for iCCA vs HCC. The proteomic analysis of EV derived from CCA human cells in vitro revealed higher abundance of oncogenic proteins compared to EV released by normal human cholangiocytes. Orthotopic implant of CCA human cells in the liver of immunodeficient mice resulted in the release to serum of EV containing some similar human oncogenic proteins. Conclusion: novel proteomic signatures found in serum EV of CCA, PSC and HCC patients show potential usefulness as diagnostic tools. This article is protected by copyright. All rights reserved.

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