Importance of between and within Subject Variability in Extracellular Vesicle Abundance and Cargo when Performing Biomarker Analyses

Abstract

Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human blood and present the intriguing potential for a ‘liquid biopsy’ to track disease and the effectiveness of interventions. Recently, we have further demonstrated the potential for EV derived biomarkers to account for variability in drug exposure. This study sought to evaluate the variability in abundance and cargo of global and liver-specific circulating sEV, within (diurnal) and between individuals in a cohort of healthy subjects (n = 10). We present normal ranges for EV concentration and size and expression of generic EV protein markers and the liver-specific asialoglycoprotein receptor 1 (ASGR1) in samples collected in the morning and afternoon. EV abundance and cargo was generally not affected by fasting, except CD9 which exhibited a statistically significant increase (p = 0.018). Diurnal variability was observed in the expression of CD81 and ASGR1, which significantly decreased (p = 0.011) and increased (p = 0.009), respectively. These results have potential implications for study sampling protocols and normalisation of biomarker data when considering the expression of sEV derived cargo as a biomarker strategy. Specifically, the novel finding that liver-specific EVs exhibit diurnal variability in healthy subjects should have broad implications in the study of drug metabolism and development of minimally invasive biomarkers for liver disease.

Role of Extracellular Vesicle-Derived Biomarkers in Drug Metabolism and Disposition

“…Prandial state has also been suggested to impact circulating EV (Witwer et al 2013). While this work demonstrated no effect of fasting on EV abundance, particle size and generic or liver specific markers (Newman et al 2021), others previously reported significant increases in fed compared to fasted samples (Mørk et al 2016). Differences may be attributed to EV isolation methods selected in these studies, as increased post-prandial lipoprotein levels can interfere with vesicle counting in less pure EV isolates.…”

“…The time of day at which samples are collected should be kept constant to control for circadian variations (Théry et al 2018;Witwer et al 2013;Coumans et al 2017). By way of example, a recent report from our group showed that while no change in total abundance and size of EVs occurred from morning to afternoon, levels of generic EV marker, CD81, was decreased and the hepatocyte specific marker, ASGR1, was increased (Newman et al 2021). The potential for variability in molecular cargo should thus be accounted for in sampling protocols, particularly in the context of liver derived markers such as DMEs.…”

“…To date the capacity to isolate tissue specific EVs from blood has only been demonstrated by a small number of research (Vella et al 2017;Park et al 2016;Goetzl et al 2017;Rodrigues et al 2021) and no commercial product is readily available. Selective isolation of liver derived EVs from blood has been demonstrated using an immunoprecipitation based approach that selectively captures EVs expressing asialoglycoprotein receptor 1 (ASGR1) (Newman et al 2021;Rodrigues et al 2021) (Figure 2). As with cell-based immunoprecipitation approaches, the selective isolation of ASGR1+ liver derived EVs requires the use of an antibody with an epitope against the extracellular facing domain (amino acids 62 to 291).…”

“…It Is necessary to demonstrate the absence of proteins from category 4 when claiming specific analysis of sEVs, and a category 5 protein must be analysed to document function activities. Traditionally each marker has been characterised independently by western blotting, however panel based liquid chromatography mass spectrometry assays have been reported (Newman et al 2021) to characterise panels of markers (e.g. CD9, CD81, TSG101, albumin and calnexin).…”

Extracellular vesicles in kidney transplantation: a state-of-the-art review

“…107,108 Conversely, a potential disadvantage of investigating EVs as biomarkers relates to the complexities of EV interactions with target cells and their poorly understood biogenesis pathways. 5,23 In addition, recent studies have detected diurnal patterns of expression in EV markers 109 and variability in EV content within participants related to physiological activities, such as exercise and sample collection conditions. 110 More studies are needed to disentangle this variability (e.g., by estimating the ratio of sample EVs to total protein in the sample), and to establish thresholds for specific pathologic states.…”