Patients with Cholangiocarcinoma Present Specific RNA Profiles in Serum and Urine Extracellular Vesicles Mirroring the Tumor Expression: Novel Liquid Biopsy Biomarkers for Disease Diagnosis

J.p. Jimeno ,Esperanza González ,Alberto Sánchez-Campos ,Laura Izquierdo-Sánchez ,Tom H. Karlsen,Raúl Jimenez‐Agüero ,Luís Bujanda ,Álvaro Santos‐Laso ,Ana M. Aransay,Colm J. O’Rourke,Frank Lammert,Ainhoa Lapitz,José Luis Lavín ,Ander Arbelaiz,Cesar Ibarra,Jesús M. Bañales ,Adelaida La Casta,María J. Perugorria ,Marco Marzioni,Ioana Riaño,Marcin Krawczyk,Juan M Falcón-Pérez ,Jesper B. Andersen,Rocı́o I.r. Macı́as ,Pedro M. Rodrigues,José J.g. Marin

doi:10.3390/cells9030721

Abstract

Cholangiocarcinoma (CCA) comprises a group of heterogeneous biliary cancers with dismal prognosis. The etiologies of most CCAs are unknown, but primary sclerosing cholangitis (PSC) is a risk factor. Non-invasive diagnosis of CCA is challenging and accurate biomarkers are lacking. We aimed to characterize the transcriptomic profile of serum and urine extracellular vesicles (EVs) from patients with CCA, PSC, ulcerative colitis (UC), and healthy individuals. Serum and urine EVs were isolated by serial ultracentrifugations and characterized by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. EVs transcriptome was determined by Illumina gene expression array [messenger RNAs (mRNA) and non-coding RNAs (ncRNAs)]. Differential RNA profiles were found in serum and urine EVs from patients with CCA compared to control groups (disease and healthy), showing high diagnostic capacity. The comparison of the mRNA profiles of serum or urine EVs from patients with CCA with the transcriptome of tumor tissues from two cohorts of patients, CCA cells in vitro, and CCA cells-derived EVs, identified 105 and 39 commonly-altered transcripts, respectively. Gene ontology analysis indicated that most commonly-altered mRNAs participate in carcinogenic steps. Overall, patients with CCA present specific RNA profiles in EVs mirroring the tumor, and constituting novel promising liquid biopsy biomarkers.

Highlights

Cholangiocarcinomas (CCAs) are heterogeneous biliary malignancies characterized by dismal prognosis. the incidence and mortality rates of these cancers are rapidly increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies [1,2,3].According to their anatomical localization, CCAs are classified into intrahepatic, perihilar, or distal. the etiology of most CCAs is unknown
Clinical characteristics of patients and tumors are summarized in Supplementary Table S1. the diagnosis of primary sclerosing cholangitis (PSC) was based on the European Association for the Study of the Liver (EASL) guidelines [19] by demonstrating the presence of bile duct alterations using magnetic resonance cholangiopancreatography (MRCP) after excluding secondary causes of cholangitis. the diagnosis of ulcerative colitis (UC) was performed by combining endoscopic and histological studies, mainly colonoscopy in parallel with hematoxylin and eosin (H&E) staining, after excluding other potential diseases
The extracellular vesicles (EVs) protein markers CD63 and CD81 were highly enriched in the isolated EV fraction, when compared to total serum or normal human cholangiocyte (NHC) whole-cell extracts (WCE), while the endoplasmic reticulum marker 78 kDa glucose-regulated protein (GRP78) was completely absent in isolated serum and urine EVs but only found expressed in WCE from NHCs (Figure 1B), substantiating a proper isolation and a high purity of the obtained

Summary

Introduction

Cholangiocarcinomas (CCAs) are heterogeneous biliary malignancies characterized by dismal prognosis. the incidence and mortality rates of these cancers are rapidly increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies [1,2,3].According to their anatomical localization, CCAs are classified into intrahepatic (iCCA), perihilar (pCCA), or distal (dCCA). the etiology of most CCAs is unknown. The incidence and mortality rates of these cancers are rapidly increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies [1,2,3]. According to their anatomical localization, CCAs are classified into intrahepatic (iCCA), perihilar (pCCA), or distal (dCCA). CCA), a chronic cholestatic liver disease that is associated with autoimmune phenomena against the intra- and extrahepatic bile ducts [1,4,5]. Late diagnosis combined with the chemoresistant nature of these tumors [6] highly compromise the current therapeutic options, mainly based on surgery, significantly impacting on patient’s welfare and outcome [1,2]. the diagnosis of CCA is usually conducted by combining clinical, biochemical, radiological, and histological information

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