Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Isolation and characterization of urine microvesicles from prostate cancer patients: different approaches, different visions.

Author(s): García-Flores M, Sánchez-López CM, Ramírez-Calvo M, Fernández-Serra A, Marcilla A, López-Guerrero JA

Publication: BMC Urol, 2021, Vol. 21, Page 137

PubMed ID: 34579682 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the concentration, size, and protein and miRNA levels among EVs isolated from urine using ultracentrifugation, SEC, and the Exolute kit. Protein concentration and miRNA were also compared between EVs isolated from the urine of healthy patients and those with prostate cancer. Urine from six prostate cancer patients and four healthy men was collected in sterile urine containers and preserved with a protease inhibitor cocktail. Urine was centrifuged at 1000 x g for 10 min at 4°C and the supernatant was frozen at -80°C. Urine was thawed at 4°C, cellular debris was removed by centrifugation at 1000 x g for 15 min at room temperature followed by 3000 x g for 15 min. Tamm Horsfall protein was removed by addition of sodium chloride, centrifugation at 16000 x g for 20 min at 4°C and filtration through a 22 µm filter. Urine was aliquoted and EVs were isolated by ultracentrifugation (100,000 x g for 2 h at 4°C), size exclusion chromatography (using Sepharose-CL2B stacked syringe and concentrated using Amicon Ultra-4 Centrifugal Filter Devices), or the ExoLutE Urine Kit. Protein levels were quantified using a spectrophotometer. Particle size distribution was evaluated using nanoparticle tracking analysis (NTA) and transmission electron microscopy.  CD-9 and CD-63 were quantified using a luminescent assay. miRNA profiles were evaluated using the HTG EdgeSeq miRNA Whole Transcriptome Assay.

   Summary of Findings:

   Protein concentrations were comparable between EVs isolated using ultracentrifugation and SEC (7.57±8.2 μg/mL and 7.468±3.77 μg/mL, respectively), but protein concentrations were higher when isolated with the Exolute kit (25.99±19.25 μg/mL).  Ultracentrifugation isolated more particles per mL of urine than the Exolute kit (4.13±3 x 10¹¹ particles/mL versus 1.78±1.03 x 10¹¹ particles/mL, P=0.021) or SEC (4.13±3 x 10¹¹ particles/mL versus 8.1±3.54 x 10¹¹ particles/mL, P=0.0011). Particle size distributions (evaluated by NTA) were similar between EVs isolated by ultracentrifugation and the Exolute kit (160.28±18.51 nm and 167.84±32.19, respectively), but particles isolated by SEC were larger (194.1±16.4 nm, P=0.0026). TEM analysis showed the three EV isolation methods produced similar size distributions, although EVs isolated using SEC displayed a wider distribution (80.2-86.3 nm for exolute; 84–91.5 nm for ultracentrifugation, and 78.7–105.7 nm for SEC). TEM revealed a “dense smudge” in the background when EVs were isolated using the Exolute kit.  Purity (assessed by the ratio of particles to proteins) was highest when EV isolation was by ultracentrifugation (5.97±6.24 x 10¹⁰), followed by SEC (2.01±0.97 x 10¹⁰) and the Exolute kit (0.44±0.28 x 10¹⁰).  EV isolation by SEC resulted in the highest concentrations of CD-9 and CD-63 (9.7 ± 9.1 x 10⁵), followed by ultracentrifugation (6.7 ±8.9 x 10⁵) and the Exolute kit (1.1 ± 1.3 x 10⁵). The median sum of normalized miRNA counts was also highest when EVs were isolated using SEC (16,814) followed by ultracentrifugation (16,260) and the Exolute kit (16,137). Importantly, PCA plots of miRNA expression showed that specimens grouped by EV isolation method rather than patient. Nevertheless, EVs isolated by the three methods still displayed strongly correlated miRNA expression; the strongest correlations were observed between ultracentrifugation and SEC (R²=0.8) and this correlation was strongest for miRNA in the highest expression quartile.

   Protein concentrations and the CD9 and CD63 levels were comparable between EVs from healthy patients and those with prostate cancer, although patients diagnosed with prostate cancer had non-significantly higher protein and particle concentrations when EV isolation was by ultracentrifugation or SEC.  The number of miRNAs that were differentially expressed between healthy and prostate cancer patients was dependent upon EV isolation method: 21 miRNAs were differentially expressed when EV isolation was by SEC, three miRNAs were differentially expressed when EV isolation was with ultracentrifugation, and no miRNAs were differentially expressed when EVs were isolated with the Exolute kit.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing
   Cell count/volume	Light scattering
   Protein	Immunoassay
   Protein	Spectrophotometry
   Cell count/volume	Electron microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Healthy Prostate cancer
   Analyte Extraction and Purification	Analyte isolation method	Exolute Urine kit Ultracentrifugation SEC
   Electron microscopy Specific	Technology platform	NTA

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