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. 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 5 43), PSC (n 5 30), or HCC (n 5 29) patients and healthy individuals (control, n 5 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).

Exosome-based liquid biopsy in the management of hepatocellular carcinoma

“…However, depending on the starting material and downstream applications, other methods for the purification and enrichment of HCC-derived exosomes have also been used such as ultrafiltration, size exclusion chromatography (SEC) or the ExoQuick TC method [19,29] . Majority of HCC-derived exosomes have been identified by a combination of different methods including, round or cup shaped morphology by transmission electron microscopy, size of 50-100 nm in diameter by nanoparticle tracking analysis and exosomal surface profiling for markers such the tetraspanins (CD9, CD63, and CD81), heat shock proteins (HSP90 and HSP60), Alix and Tsg101 by immunoblot and flow cytometry [19,20,[29][30][31][32] . Studies have demonstrated high expression of glypican-3 (GPC-3) and AFP, traditional markers of HCC, within the HCC-derived exosomes, thereby confirming the hepatoma-based origin of these exosomes [31] .…”

“…In particular LG3BP could distinguish patients with HCC from CCA and PSC patients [30] . Together these studies suggest exosomal miRNAs, lncRNA and proteins may serve as novel diagnostic and prognostic biomarkers of HCC.…”

“…Serum exosomal miR-665 levels were significantly higher in HCC patients than those in healthy subjects. Additionally, exosomal miR-665 levels were elevated in larger tumours with local invasion Serum extracellular vesicles [30] and at an advanced clinical stage (stage III/IV) of HCC [40] . Another study found decreased expression of serum exosomal miR-638 in HCC patients [41] .…”

“…A significant reduction in miR-320a level was detected by miRNA sequencing of exosomes derived from cancer-associated stromal fibroblasts (CAFs) when compared to corresponding paracancer fibroblasts (PAFs) of 6 HCC patients [44] . By using nanoparticle tracking analysis, the serum exosome concentration in HCC patients was found to be higher than in cholangiocarcinoma (CCA) and primary sclerosing cholangitis (PSC) patients [30] . Long noncoding ribonucleic acid (lncRNA) X-inactive-specific transcript (Xist) was upregulated in peripheral blood of HCC patients [45] .…”

Utility of Claudin-3 in extracellular vesicles from human bile as biomarkers of cholangiocarcinoma

“…A relatively large numbers of EVs are produced by cancer cells where they are involved in the growth and differentiation of tumors 12 ; thus, they have been identified as novel biomarkers of cancer. Serum EV-derived proteins, such as aminopeptidase N (AMPN), pantetheinase (VNN1), and polymeric immunoglobulin receptor (PIGR), have www.nature.com/scientificreports/ been reported as biomarkers of CCA 13 . However, EVs in circulating body fluids, such as blood, reflect all pathophysiological changes that occur in the body.…”