Exosome Analysis in Prostate Cancer: How They Can Improve Biomarkers' Performance

Abstract

Exosomes are extracellular nanovesicles (EV), that is, carriers of different biomolecules such as lipids, proteins, nucleic acids. Their composition and the fact that their release dramatically increases in cases of tumorigenesis open up different scenarios on their possible application to research into new biomarkers. The first purpose of the present review was to specifically analyze and compare different methodologies available for the use of exosomes in prostate cancer (PC). The most widely applied methodologies include ultracentrifugation techniques, size-based techniques, immunoaffinity capture-based techniques (mainly ELISA), and precipitation. To optimize the acquisition of exosomes from the reference sample, more techniques can be applied in sequence for a single extraction, thereby determining an increase in labor time and costs. The second purpose was to describe clinical results obtained with the analysis of PSA-expressing exosomes in PC; this provides an incredibly accurate method of discriminating between healthy patients and those with prostate disease. Specifically, the IC-ELISA alone method achieved 98.57% sensitivity and 80.28% specificity in discriminating prostate cancer (PC) from benign prostatic hyperplasia (BPH). An immunocapture-based ELISA assay was performed to quantify and characterize carbonic anhydrase (CA) IX expression in exosomes. The results revealed that CA IX positive exosomes were 25-fold higher in plasma samples from PC patients than in those from healthy controls. The analysis of PC-linked exosomes represents a promising diagnostic model that can effectively distinguish patients with PC from those with non-malignant prostatic disease. However, the use of exosome analysis in clinical practice is currently limited by several issues, including a lack of standardization in the analytical process and high costs, which are still too high for large-scale use.

Keywords: biomarker; exosome; prostate cancer.

Figure 1

Exosome isolation and characterization in prostate cancer cases. (1) PSA exosome extraction. From blood samples, after centrifugation, plasma is obtained. The protocol includes nanoparticle tracking analysis (NTA) for the quality control of plasmatic samples after ultracentrifugation; afterwards, both nanoscale flow cytometry and an immunocapture-based ELISA are used for the extracellular vesicles’ characterization and quantification. In both the analyses, an antibody specific for a typical exosome antigen (CD81) is exploited to identify exosomes within the pool of extracellular vesicles, and an antibody for PSA is used for the detection of plasmatic exosomes expressing PSA. (2) Carbonic anhydrase IX exosome extraction. Human plasma samples are collected from EDTA-treated whole blood. Nanoparticle tracking analysis (NTA) is used for size distribution and concentration measurements of exosome samples in liquid suspension. A Western blot analysis is performed. An ELISA for CA is performed, and then the exosomal pH is evaluated using nanoscale flow cytometry. Intracellular acidity is analyzed via confocal microscopy using fluorescent tracers. Figure created with BioRender.com, accessed on 10 May 2023.