Detection of extracellular vesicles in plasma and urine of prostate cancer patients by flow cytometry and surface plasmon resonance imaging

Abstract

Large (> 1 μm) tumor-derived extracellular vesicles (tdEVs) enriched from the cell fraction of centrifuged whole blood are prognostic in metastatic castration-resistant prostate cancer (mCRPC) patients. However, the highest concentration of tdEVs is expected in the cell-free plasma fraction. In this pilot study, we determine whether mCRPC patients can be discriminated from healthy controls based on detection of tdEVs (< 1μm, EpCAM +) and/or other EVs, in cell-free plasma and/or urine. The presence of marker+ EVs in plasma and urine samples from mCRPC patients (n = 5) and healthy controls (n = 5) was determined by flow cytometry (FCM) and surface plasmon resonance imaging (SPRi) using an antibody panel and lactadherin. For FCM, the concentrations of marker positive (+) particles and EVs (refractive index <1.42) were determined. Only the lactadherin + particle and EV concentration in plasma measured by FCM differed significantly between patients and controls (p = 0.017). All other markers did not result in signals exceeding the background on both FCM and SPRi, or did not differ significantly between patients and controls. In conclusion, no difference was found between patients and controls based on the detection of tdEVs. For FCM, the measured sample volumes are too small to detect tdEVs. For SPRi, the concentration of tdEVs is probably too low to be detected. Thus, to detect tdEVs in cell-free plasma and/or urine, EV enrichment and/or concentration is required. Furthermore, we recommend testing other markers and/or a combination of markers to discriminate mCRPC patients from healthy controls.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles

“…This is mostly used in kidney-related research, and is of particular importance in physiological studies Blijdorp et al, 2021). (Gori et al, 2020;Musante et al, 2017;Rikkert et al, 2020)…”

“…Moreover, the yield of specific uEVs in these capture approaches could be a concern. Capture based assays often use the (so called) general and abundant EV surface markers CD9, CD63, and CD81 for capture, for example, time‐resolved fluoroimmunoassay (TR‐FIA), surface plasmon resonance imaging (SPRi), ExoView® (Daaboul et al., 2016; Duijvesz et al., 2015; Rikkert et al., 2020). However, it has become increasingly apparent that only fractions of EVs carry these ‘general’ EV markers, and that expression of these markers is largely dependent on the cells of origin (Kowal et al., 2016; Salih et al., 2016).…”

Clinical significance of STEAP1 extracellular vesicles in prostate cancer

“…Nanoscale flow cytometry can be used to detect and quantify nanosized particles between~80 nm and 1 µm at single event resolution [15,19,24]. EVs derived from cell culture, plasma, and serum have been successfully analysed using various nanoscale flow cytometry platforms [15,[25][26][27][28]. Here, SEC isolated fractions 1-5 were analysed for STEAP1 by nanoscale flow cytometry.…”

Extracellular vesicles in urological malignancies

“…Both benign and malignant prostate cells release EVs which have been detected in plasma and urine with significant diagnostic potential [ 138 , 139 ]. The term prostasome has been used to describe EVs released by the prostate and these likely represent a mixed cohort of EVs derived from numerous pathways [ 43 , [140] , [141] , [142] , [143] , [144] ].…”