Pre-analytical handling conditions and protein marker recovery from urine extracellular vesicles for bladder cancer diagnosis

Abstract

Extracellular vesicles (EVs) contain a variety of biomolecules and provide information about the cells that produce them. EVs from cancer cells found in urine can be used as biomarkers to detect cancer, enabling early diagnosis and treatment. The potential of alpha-2-macroglobulin (A2M) and clusterin (CLU) as novel diagnostic urinary EV (uEV) biomarkers for bladder cancer (BC) was demonstrated previously. To validate the diagnostic value of these proteins in uEVs in a large BC cohort, urine handling conditions before uEV isolation should be optimized during sample transportation from medical centers. In this study, we analyzed the uEV protein quantity, EV particle number, and uEV-A2M/CLU after urine storage at 20°C and 4°C for 0-6 days, each. A2M and CLU levels in uEVs were relatively stable when stored at 4°C for a maximum of three days and at 20°C for up to 24 h, with minimal impact on analysis results. Interestingly, pre-processing to remove debris and cells by centrifugation and filtration of urine did not show any beneficial effects on the preservation of protein biomarkers of uEVs during storage. Here, the importance of optimizing shipping conditions to minimize the impact of pre-analytical handling on the uEVs protein biomarkers was emphasized. These findings provide insights for the development of clinical protocols that use uEVs for diagnostic purposes.

Fig 1. Schematic diagram of the study design.

The study involved splitting urine samples into two halves, one of which underwent pre-processing, while the other did not. Both halves were then divided into seven vials and stored at either 4°C or room temperature (20°C) for six days. One vial from each half was transferred and stored at –70°C daily for one week. Finally, EVs were isolated using Exodisc and subjected to analysis. Created by Biorender.com.

Fig 2. Analysis of the protein concentration of uEVs from urine samples stored under different conditions.

The uEVs were isolated from each stored urine sample, and their protein concentrations were measured using a micro bicinchoninic acid assay. Data are shown as the mean ± SD, n = 3, *P < 0.05, **P < 0.01.

Fig 3. Western blot analysis for protein markers of uEVs.

CD63, CD9 and calnexin of uEVs stored under different conditions were analyzed using western blotting. Total proteins from each uEV sample were presented by staining on SDS-PAGE gels.

Fig 4. Analysis of the particle numbers of uEVs from urine samples stored at different conditions.

Using ZetaView analysis, the number of EVs was analyzed from each stored aliquot. Data are shown as mean ± SD, n = 3, *P < 0.05.

Fig 5. Analysis of A2M in uEVs from urine samples stored at different conditions.

Equal amounts (0.5 μg/well) of isolated uEVs from urine samples stored under different conditions were used in an A2M ELISA. The resulting absorbance values at 450 nm were presented. Data are shown as mean ± SD, n = 2, *P < 0.05, **P < 0.01.

Fig 6. Analysis of CLU in uEVs from urine samples stored at different conditions.

Equal amounts (0.5 μg/well) of isolated uEVs from urine samples stored under different conditions were used in an CLU ELISA. The resulting absorbance values at 450 nm were presented. Data are shown as mean ± SD, n = 2, *P < 0.05, **P < 0.01.