Proteome Analysis of Biliary Extracellular Vesicles by High-Precision Mass Spectrometry Reveals Potential Biomarkers for Cholangiocarcinoma and Primary Sclerosing Cholangitis

Abstract

Abstract:Background: Patients with primary sclerosing cholangitis (PSC) have a high risk of developing biliary cancer. Sufficient diagnostics do not yet exist to detect cholangiocarcinoma (CCA) in patients with PSC. This study aimed to explore biomarkers in extracellular vesicles (EVs) of bile of patients with CCA and PSC.Methods:  30 bile samples of PSC, CCA and bile stones (BS) patients were included retrospectively. Vesicles were enriched by differential centrifugation and filtering. EV characterization was performed with Western Blots for common exosome-markers and electron microscopy and concentration measured with Nanoparticle Tracking Analysis (NTA). Pooled samples were analyzed by liquid chromatography-mass spectrometry (LC-MS). Data analysis was carried out by MaxQuant (version 1.5.3.30) and R.Results:  The CCA-samples contained the largest number of proteins. After quantile-normalization and t-testing with an adjusted p-value (FDR) <0.01 for the difference between the two benign conditions and CCA, 22 proteins were identified to be enriched in CCA-patients.Among the most significant proteins both in comparison between CCA vs. benign groups and CCA vs. PSC were VPS4B, PPIA, PROM1/CD133, PROM2, CLIC1. PROM1 could also be shown to be enriched in EVs from CCA-bile in Western Blots and were identified on ELISA-level reproducibly. GO analysis showed the involvement of the enriched proteins in EVs, cell to cell communication and signal transduction.       Conclusion: Proteome analysis of biliary EVs reveals several proteins essential in cancer development highly enriched in CCA compared to PSC and BS patients. PROM1/CD133 as potential EV-biomarker could be validated in a clinically more feasible approach. Although the data of the NTA for small vesicles (<220nm) was not significant, results on EV biomarker level indicate higher concentrations of EVs in bile of CCA patients.

Note:
Funding declaration: Supported by a grant from “Deutsche Forschungsgemeinschaft (DFG)” to Rupp C.

Conflict of Interests: Simon Hennes, Constantin Ahlmann-Eltze, Renata Blatnik, Katja Nitschke, Thomas Stefan Worst, Christian Rupp declare no conflict of interest.

Ethical Approval: The study was approved by the Ethics Committees of the Heidelberg University Hospital (Heidelberg, Germany) (S-043/2011). Patients provided written, informed consent for their participation in the study.

Keywords: Primary sclerosing cholangitischolestasis chronic liver diseaseinflammationbiliary tractproteome