Quality Assessment and Comparison of Plasma-Derived Extracellular Vesicles Separated by Three Commercial Kits for Prostate Cancer Diagnosis

Bairen Pang,1,2 Ying Zhu,1– 3 Jie Ni,1,2 Juanfang Ruan,4,5 James Thompson,1,6,7 David Malouf,2,6 Joseph Bucci,1,2 Peter Graham,1,2 Yong Li1,2,8 1St George and Sutherland Clinical School, UNSW Sydney, NSW 2052, Australia; 2Cancer Care Centre, St. George Hospital, Sydney, NSW 2217, Australia; 3School of Biomedical Engineering, University of Technology Sydney, NSW 2007, Australia; 4Electron Microscope Unit, UNSW Sydney, NSW 2052, Australia; 5School of Biotechnology and Biomolecular Sciences, UNSW Sydney, NSW 2052, Australia; 6Department of Urology, St George Hospital, Sydney, NSW 2217, Australia; 7Garvan Institute of Medical Research/APCRC, Sydney, UNSW 2010, Australia; 8School of Basic Medical Sciences, Zhengzhou University, Henan 450001, People’s Republic of ChinaCorrespondence: Ying Zhu; Yong LiResearch and Education Centre, St George Hospital, 4– 10 South St, Kogarah, NSW 2217, AustraliaEmail ying.zhu@unsw.edu.au; y.li@unsw.edu.auIntroduction: Current standard biomarkers in clinic are not specific enough for prostate cancer (PCa) diagnosis. Extracellular vesicles (EVs) are nano-scale vesicles released by most mammalian cells. EVs are promising biomarker source for PCa liquid biopsy due to its minimal invasive approach, rich information and improved accuracy compared to the clinical standard prostate-specific antigen (PSA). However, current EV separation methods cannot separate pure EVs and the quality characteristics from these methods remain largely unknown. In this study, we evaluated the quality characteristics of human plasma-derived EVs by comparing three clinical suitable separation kits.Methods: We combined EV separation by commercial kits with magnetic beads capture and flow cytometry analysis, and compared three kits including ExoQuick Ultra based on precipitation and qEV35 and qEV70 based on size exclusion chromatography (SEC).Results: Our results indicated that two SEC kits provided higher EV purity and lower protein contamination compared to ExoQuick Ultra precipitation and that qEV35 demonstrated a higher EV yield but lower EV purity compared to qEV70. Particle number correlated very well particularly with CD9/81/63 positive EVs for all three kits, which confirms that particle number can be used as the estimate for EV amount. At last, we found that several EV metrics including total EVs and PSA-specific EVs could not differentiate PCa patients from health controls.Conclusion: We provided a systematic workflow for the comparison of three separation kits as well as a general analysis process in clinical laboratories for EV-based cancer diagnosis. Better EV-associated cancer biomarkers need to be explored in the future study with a larger cohort.Keywords: extracellular vesicles, prostate cancer, lipoprotein, separation, protein biomarker, immunomagnetic beads