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 ( = 58; 42 with Gleason score 6-7, 16 with Gleason score ≥8) and metastatic prostate cancer ( = 11) compared with patients with benign prostatic hyperplasia ( = 15) and healthy individuals ( = 15) using ELISA, without prior laborious exosome isolation. ANOVA showed different CLDN3 plasma levels in these groups ( = 0.004). CLDN3 levels were higher in patients with Gleason ≥8 tumors compared with patients with benign prostatic hyperplasia ( = 0.012) and Gleason 6-7 tumors ( = 0.029). In patients with localized tumors CLDN3 levels predicted a Gleason score ≥ 8 (AUC = 0.705; = 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.

Challenges and Opportunities in Clinical Applications of Blood-Based Proteomics in Cancer

“…However, the validation of this small study including longitudinal follow-ups of clinical outcomes is needed. Other proteomic approaches, including the use of cell lines or animal models for discovery, followed by validation in clinical specimens, have demonstrated early promise for discriminating between cancer and healthy patients, or as markers of [ 206 , 207 , 208 ], but validation cohorts will be needed for confirming the value of serum/plasma proteomic-based biomarkers in clinical settings.…”

Spondyloarthritis patients with and without intestinal symptoms – searching for discriminating biomarkers

“…Accumulating evidence shows that the dysregulation of the claudin circulating pool occurs in different pathological conditions, suggesting that proteins of this family might represent potential blood-borne biomarkers of diagnostic and/or prognostic value [39][40][41]. For example, the presence of CLDN3 in exosomes released by prostate cancer cells and elevated plasma CLDN3 concentrations in men suffering from prostate cancer have recently been reported [42]. Moreover, plasma CLDN3 has been proposed as a putative prognostic biomarker associated with more aggressive prostate cancer, predictive of disease progression.…”

Promise and Implementation of Proteomic Prostate Cancer Biomarkers

“…Claudin 3 was identified as a potential biomarker through proteome analysis of exosomes extracted from conditioned media of PC3 (malignant) and PNT1A (benign) cell lines [ 81 ]. Plasma levels of Claudin 3 were subsequently determined using ELISA (n = 58 localized PC, n = 11 metastatic PC, n = 15 BPH, n = 15 healthy individuals), with increased level detected in GS ≥ 8 compared to BPH and GS ≥ 8 vs. GS6-7.…”