What is the blood concentration of extracellular vesicles? Implications for the use of extracellular vesicles as blood-borne biomarkers of cancer

Abstract

Circulating biomarkers have a great potential in diagnosing cancer diseases at early stages, where curative treatment is a realistic possibility. In the recent years, using extracellular vesicles (EVs) derived from blood as biomarkers has gained widespread popularity, mainly because they are thought to be easy to isolate and carry a vast variety of biological cargos that can be analyzed for biomarker purposes. However, our current knowledge on the plasma EV concentration in normophysiological states is sparse. Here, we provide the very first mean estimate of the plasma EV concentration based on values obtained from a thorough literature review. The different estimates obtained from the literature are correlated to the isolation techniques used to obtain them, illustrating how some methodologies may over-or underestimate the plasma EV concentration. We also show that the estimated plasma EV concentration (approximately 10 10 EVs per mL) defines EVs as a minority population compared to other colloidal particles of the systemic circulation, namely the lipoproteins, which are known contaminants in EV isolates and carry biomarker molecules themselves. Lastly, we introduce the possibility of regarding EVs and lipoproteins as a continuum of lipid-containing particles to which biomarker molecules can be associated. Using such a holistic approach, increased strength of plasma-derived cancer biomarkers may soon be revealed.

Cellular senescence contributes to age‐dependent changes in circulating extracellular vesicle cargo and function

“…However, the authors comment that estimates of the small EV populations, the most abundant EV sub‐type, were likely underestimated using these techniques. This is consistent with data from other groups which suggest that the isolation and quantification methods have a major impact on plasma EV concentration and purity (Johnsen, Gudbergsson, Andresen, & Simonsen, ). By comparing 38 articles published from 2013 to 2018 which isolated plasma EVs using a number of methods, Johnsen and colleagues estimate that the number of circulating plasma EVs in humans is ~1 × 10 10 particles/ml.…”

Pitfalls associated with lipophilic fluorophore staining of extracellular vesicles for uptake studies

“…Several studies have highlighted the high degree of lipoprotein contamination in ostensibly purified EV samples obtained from plasma [14,15]. The similarity in physical properties between EVs and different types of lipoproteins [16], and the ~6 orders of magnitude higher concentration of lipoproteins relative to EVs in human plasma [17] commonly lead to high lipoprotein contamination in EV isolates from plasma when using physical‐based isolation methods. Likewise, EV isolates from cell culture medium are also prone to lipoprotein contamination when serum (incl.…”

Unique Calibrators Derived from Fluorescence‐Activated Nanoparticle Sorting for Flow Cytometric Size Estimation of Artificial Vesicles: Possibilities and Limitations

“…Yet, EVs are a very heterogeneous population of biological nanoparticles when it comes to size and composition and therefore also refractive indexes. On that note, it should also be stressed that isolated plasma-derived EVs are highly prone to contamination with highly abundant lipoproteins (28,29). Recent work (12) shows that by taking the ratio between SSC and FSC (they used a high scatter sensitivity flow cytometer) in combination with Mie scattering theory, EV sizes in the range from 200 to 500 nm could be deduced.…”