Abstract
Extracellular vesicles (EVs) have potential as minimally invasive biomarkers. However, the methods most commonly used for EV retrieval rely on ultracentrifugation, are time-consuming, and unrealistic to translate to standard-of-care. We sought a method suitable for EV separation from blood that could be used in patient care. Sera from breast cancer patients and age-matched controls (n = 27 patients; n = 36 controls) were analysed to compare 6 proposed EV separation methods. The EVs were then characterised on 8 parameters. The selected method was subsequently applied to independent cohorts of sera (n = 20 patients; n = 20 controls), as proof-of-principle, investigating EVs’ gremlin-1 cargo. Three independent runs with each method were very reproducible, within each given method. All isolates contained EVs, although they varied in quantity and purity. Methods that require ultracentrifugation were not superior for low volumes of sera typically available in routine standard-of-care. A CD63/CD81/CD9-coated immunobead-based method was most suitable based on EV markers' detection and minimal albumin and lipoprotein contamination. Applying this method to independent sera cohorts, EVs and their gremlin-1 cargo were at significantly higher amounts for breast cancer patients compared to controls. In conclusion, CD63/CD81/CD9-coated immunobeads may enable clinical utility of blood-based EVs as biomarkers.
Highlights
Extracellular vesicles (EVs) are nanosized lipid bilayer-surrounded particles released by cells into their surrounding biofluid [1]
Separation methods, from cancer and controls sera, and characterised on 8 parameters, no method is perfect at producing pure EVs from small volumes of sera
Additional to the fact that DIFF-Differential ultracentrifugation (UC) is unsuitable for routine clinical utility based on the need for ultracentrifugation, it did not produce adequate EVs from clinically relevant sera volumes to detect any EV markers investigated
Summary
Extracellular vesicles (EVs) are nanosized lipid bilayer-surrounded particles released by cells into their surrounding biofluid [1]. Blood-based EVs have potential as minimally invasive diagnostic, prognostic and/or predictive tools. Proteins used for breast cancer diagnosis include uPA and PAI-1, but these require invasive biopsies [2]. Serum biomarkers CA15–3, carcinoembryonic antigen, and tissue polypeptide antigen are not sensitive enough to detect early-stage breast cancer [3]. Jesneck et al [4] investigated if a panel of 98 serum freely-circulating biomarkers could decipher breast cancer from healthy controls, but found that they could not. A number of studies have investigated the potential of blood based EVs as biomarkers for breast cancer [5]
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