title: Database-augmented Mass Spectrometry Analysis of Exosomes Identifies Claudin 3 as a Putative Prostate Cancer Biomarker.
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.
authors.affiliation: From the ‡Department of Urology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany; thomas.worst@medma.uni-heidelberg.de.|§Department of Signaling and Functional Genomics, German Cancer Research Center, Heidelberg, Germany.
authors.forename: Thomas Stefan
authors.identifier: 0000-0001-5429-126X
authors.initials: TS
authors.lastname: Worst
authors.affiliation: From the ‡Department of Urology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany.|§Department of Signaling and Functional Genomics, German Cancer Research Center, Heidelberg, Germany.
authors.forename: Jost
authors.initials: J
authors.lastname: von Hardenberg
authors.affiliation: §Department of Signaling and Functional Genomics, German Cancer Research Center, Heidelberg, Germany.|¶Haematology and Oncology and Developmental Biochemistry, University Medical Center, Göttingen, Germany.
authors.forename: Julia Christina
authors.initials: JC
authors.lastname: Gross
authors.affiliation: From the ‡Department of Urology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Erben
authors.affiliation: ‖Genomics and Proteomics Core Facility, German Cancer Research Center, Heidelberg, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Schnölzer
authors.affiliation: **Institute of Pathology IPH, University Clinic Heidelberg and Electron Microscopy Core Facility, University of Heidelberg, Germany.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Hausser
authors.affiliation: ‡‡Institute of Transfusion Medicine and Immunology, Heidelberg University, Medical Faculty Mannheim, German Red Cross Blood Service Baden-Württemberg-Hessen, Mannheim, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Bugert
authors.affiliation: From the ‡Department of Urology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany.
authors.forename: Maurice Stephan
authors.initials: MS
authors.lastname: Michel
authors.affiliation: §Department of Signaling and Functional Genomics, German Cancer Research Center, Heidelberg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Boutros
mesh_terms: D000368:Aged; D014408:Biomarkers, Tumor / Q000097:blood / Q000378:metabolism*; D045744:Cell Line, Tumor; D062465:Claudin-3 / Q000097:blood / Q000378:metabolism*; D016208:Databases, Factual; D055354:Exosomes / Q000378:metabolism*; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D060787:Neoplasm Grading; D011470:Prostatic Hyperplasia / Q000097:blood / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000097:blood / Q000378:metabolism* / Q000473:pathology
chemical_list: D014408:Biomarkers, Tumor; C568122:CLDN3 protein, human; D062465:Claudin-3