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.

Fig. 1.

Data sources implemented into the scoring. Data from four publicly accessible databases was integrated.

Fig. 2.

Exosome quality control. A, Immunoblotting revealed exosomal samples to be positive for ALIX, TSG101 and HSC70 but to be negative for Calnexin. B, TEM showed cup-shaped vesicles. C, Isolated exosomes from PC3 (left) and PNT1A cells (right) had a typical size distribution using NTA. (C = cells, E = exosomes).

Fig. 3.

Data set comparison. A, Venn diagrams show a high overlap of identified proteins between replicates of PC3 and PNT1A exosomes. B, Comparison with medium control resulted in a reduction of data sets with 188 proteins remaining in the PC3 data set and 227 in the PNT1A data set. C, 64 proteins were exclusively found in PC3 exosomes, 103 in PNT1A exosomes.

Fig. 4.

In vitro validation experiments. Retests using serum-free medium confirmed a similar profile of exosome size (upper = PC3, lower = PNT1A) (A) and morphology with positive signal for CD63 (arrows) (B). C, Immunoblotting showed a strong enrichment of CLDN3 on PC3 exosomes, but not on PNT1A exosomes. D, CLDN3 (arrowheads) could also be localized on PC3 exosomes generated under serum-free conditions using immunogold labeling. (C = cells, E = exosomes).

Fig. 5.

Validation in human blood plasma samples. A, ANOVA indicated significant differences in the CLDN3 levels in the different patient groups. Post hoc inter-group comparison showed a significantly higher expression of CLDN3 in Gleason 8–9 localized tumors compared with Gleason 6–7 localized tumors and BPH. B, ROC curve analysis revealed CLDN3 to significantly predict Gleason score 8 or higher in patients with localized PCa.