Gilbert, Caroline

AbstractBackgroundThe 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.Methods and PatientsWe 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.ResultsIsolated exosomes from patients with CRPC had a smaller hydrodynamic size than those isolated from localized patients with PCa, while AChE 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).ConclusionsOur 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.

ABSTRACT The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell‐released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV‐associated functional activities. Finally, a checklist is provided with summaries of key points.