Database-augmented Mass Spectrometry Analysis of Exosomes Identifies Claudin 3 as a Putative Prostate Cancer Biomarker

Thomas Stefan Worst,Jost Von Hardenberg,Julia Christina Gross,Philipp Erben,Martina Schnölzer,Ingrid Hausser,Peter Bugert,Maurice Stephan Michel,Michael Boutros

doi:10.1074/mcp.m117.068577

Abstract

In prostate cancer and other malignancies sensitive and robust biomarkers are lacking or have relevant limitations. Prostate specific antigen (PSA), the only biomarker widely used in prostate cancer, is suffering from low specificity. Exosomes offer new perspectives in the discovery of blood-based biomarkers. Here we present a proof-of principle study for a proteomics-based identification pipeline, implementing existing data sources, to exemplarily identify exosome-based biomarker candidates in prostate cancer.Exosomes from malignant PC3 and benign PNT1A cells and from FBS-containing medium were isolated using sequential ultracentrifugation. Exosome and control samples were analyzed on an LTQ-Orbitrap XL mass spectrometer. Proteomic data is available via ProteomeXchange with identifier PXD003651. We developed a scoring scheme to rank 64 proteins exclusively found in PC3 exosomes, integrating data from four public databases and published mass spectrometry data sets. Among the top candidates, we focused on the tight junction protein claudin 3. Retests under serum-free conditions using immunoblotting and immunogold labeling confirmed the presence of claudin 3 on PC3 exosomes. Claudin 3 levels were determined in the blood plasma of patients with localized (n = 58; 42 with Gleason score 6-7, 16 with Gleason score ≥8) and metastatic prostate cancer (n = 11) compared with patients with benign prostatic hyperplasia (n = 15) and healthy individuals (n = 15) using ELISA, without prior laborious exosome isolation. ANOVA showed different CLDN3 plasma levels in these groups (p = 0.004). CLDN3 levels were higher in patients with Gleason ≥8 tumors compared with patients with benign prostatic hyperplasia (p = 0.012) and Gleason 6-7 tumors (p = 0.029). In patients with localized tumors CLDN3 levels predicted a Gleason score ≥ 8 (AUC = 0.705; p = 0.016) and did not correlate with serum PSA.By using the described workflow claudin 3 was identified and validated as a potential blood-based biomarker in prostate cancer. Furthermore this workflow could serve as a template to be used in other cancer entities.

Highlights

Exosomes from malignant PC3 and benign PNT1A cells and from FBS-containing medium were isolated using sequential ultracentrifugation
We developed a scoring scheme to rank 64 proteins exclusively found in PC3 exosomes, integrating data from four public databases and published mass spectrometry data sets
The great clinical need for minimally invasive biomarkers is currently driving biomarker research. Because of their known cancer-related functions, exosomes and the proteins contained inside them bear great hope for cancer biomarker research

Summary

Introduction

Exosomes from malignant PC3 and benign PNT1A cells and from FBS-containing medium were isolated using sequential ultracentrifugation. A custom-made scoring system incorporated information from four publicly available databases (Table I) and studies reporting proteomic mass spectrometry data sets of PCa tissue, identified from PubMed (date of search: December 12th 2016). Expression data from primary tumors and metastases Expression in blood plasma Protein conformation, association to membrane Expression in exosomes http://www.cbioportal.org/public-portal/ (date of access: January 26th 2017)

Results

Conclusion