Plasma extracellular vesicles as phenotypic biomarkers in prostate cancer patients.

Abstract

The development of phenotypic biomarkers to aid the selection of treatment for patients with castrate-resistant prostate cancer (CRPC) is an important priority. Plasma exosomes have excellent potential as real-time biomarkers to characterize the tumor because they are easily accessible in the blood and contain DNA, RNA, and protein from the parent cell. This study aims to investigate the characteristics of putative prostate-specific plasma extracellular vesicle (EV) markers and their relationship with clinical outcomes. We investigated plasma EVs in a total of 89 patients with prostate cancer (PCa) at different stages of disease progression. EVs were isolated using both precipitation and ultracentrifugation methods; physical characterization was performed using dynamic light scattering, acetylcholinesterase (AChE) activity, and velocity gradients. An immunocapture method was developed for the evaluation of prostate-specific membrane antigen (PSMA)-positive exosomes. Exosomal messenger RNA (mRNA) was quantified using droplet digital polymerase chain reaction for the expression of KLK3 and androgen receptor splice variant 7 (AR-V7) genes, which code prostate-specific antigen (PSA) and AR-V7, respectively. Serum sex steroids were measured using liquid chromatography-tandem mass spectroscopy. Isolated exosomes from patients with CRPC had a smaller hydrodynamic size than those isolated from localized patients with PCa, whileAChE activity showed no difference. Moreover, no differences were observed after initiation of androgen deprivation therapy in serial patient samples. Velocity gradients identified that PSMA-positive exosomes occupied a specific fraction of isolated EVs. A total of 35 patients with CRPC had mRNA analyzed from isolated plasma exosomes. Detectable exosomal KLK3 corresponded with higher concomitant serum PSA measurements, as expected (mean, 112.6 vs 26.61 ng/mL; P = .065). Furthermore, detectable levels of AR-V7 mRNA were associated with a shorter time to progression (median, 16.0 vs 28.0 months; P = .0499). Furthermore, detectable exosomal AR-V7 was significantly associated with testosterone levels below the lower limit of quantification (<0.1 nM). Our results suggest that exosomal AR-V7 is correlated with lower sex steroid levels in CRPC patients with a poorer prognosis. PSMA immunocapture does not appear sufficient to isolate PCa-specific exosomes.