Serum extracellular vesicles contain protein biomarkers for ... : Hepatology (original) (raw)

Original Articles: HEPATOBILIARY MALIGNANCIES

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

Arbelaiz, Ander1; Azkargorta, Mikel2,3; Krawczyk, Marcin4,5; Santos‐Laso, Alvaro1; Lapitz, Ainhoa1; Perugorria, Maria J.1,3,6; Erice, Oihane1; Gonzalez, Esperanza7; Jimenez‐Agüero, Raul1; Lacasta, Adelaida1; Ibarra, Cesar8; Sanchez‐Campos, Alberto8; Jimeno, Juan P.9; Lammert, Frank4; Milkiewicz, Piotr10,11; Marzioni, Marco12; Macias, Rocio I.R.3,13; Marin, Jose J.G.3,13; Patel, Tushar14; Gores, Gregory J.15; Martinez, Ibon16; Elortza, Félix2,3; Falcon‐Perez, Juan M.3,6,7; Bujanda, Luis1,3; Banales, Jesus M.*,1,3,6

1Department of Liver and Gastrointestinal DiseasesBiodonostia Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU)San SebastianSpain

2Proteomics PlatformCIC bioGUNE, CIBERehd, ProteoRed‐ISCIII, Bizkaia Science and Technology ParkDerioSpain

3National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd)Carlos III National Institute of HealthMadridSpain

4Department of Medicine IISaarland University Medical Center, Saarland UniversityHomburgGermany

5Laboratory of Metabolic Liver DiseasesDepartment of General, Transplant and Liver Surgery, Medical University of WarsawWarsawPoland

6IKERBASQUEBasque Foundation for ScienceBilbaoSpain

7Metabolomics UnitCIC bioGUNE, CIBERehdDerioSpain

8Hospital of CrucesBilbaoSpain

9Complejo Hospitalario de NavarraPamplonaSpain

10Liver and Internal Medicine UnitDepartment of General, Transplant and Liver Surgery, Medical University of WarsawWarsawPoland

11Translational Medicine GroupPomeranian Medical University in SzczecinSzczecinPoland

12Department of GastroenterologyUniversità Politecnica delle MarcheAnconaItaly

13Experimental Hepatology and Drug Targeting (HEVEFARM)Biomedical Research Institute of Salamanca (IBSAL), University of SalamancaSalamancaSpain

14Department of Cancer BiologyMayo ClinicJacksonvilleFL

15Division of Gastroenterology and HepatologyMayo ClinicRochesterMN

16OWL MetabolomicsDerioSpain

* ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO:
Jesus M. Banales, Ph.D.
Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute
Donostia University Hospital
Paseo del Dr. Begiristain s/n, E‐20014
San Sebastian, Spain
E‐mail: [email protected]
Tel: +34‐943006067

Abstract

Cholangiocarcinoma (CCA) includes a heterogeneous group of biliary cancers with poor prognosis. Several conditions, such as primary sclerosing cholangitis (PSC), are risk factors. Noninvasive differential diagnosis between intrahepatic CCA and hepatocellular carcinoma (HCC) is sometimes difficult. Accurate noninvasive biomarkers for PSC, CCA, and HCC are not available. In the search for 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, serum EV concentration was found to be higher in HCC than in all the other groups. Round morphology (by transmission electron microscopy), size (∼180 nm diameter by nanoparticle tracking analysis), and markers (clusters of differentiation 9, 63, and 81 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 receiver operating characteristic curve of 0.878 for CCA versus control, 0.905 for CCA stage I‐II versus control, 0.789 for PSC versus control, 0.806 for noncirhottic PSC versus control, 0.796 for CCA versus PSC, 0.956 for CCA stage I‐II versus PSC, 0.904 for HCC versus control, and 0.894 for intrahepatic CCA versus HCC. 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: Proteomic signatures found in serum EV of CCA, PSC, and HCC patients show potential usefulness as diagnostic tools. (Hepatology 2017;66:1125‐1143).