Surface protein profiling of milk and serum extracellular vesicles unveils body fluid-specific signatures (original) (raw)
Cell-derived extracellular vesicles (EVs) are currently in the limelight as potential disease biomarkers. The promise of EV-based liquid biopsy resides in the identification of specific disease-associated EV signatures. Knowing the reference EV profile of a body fluid can facilitate the identification of such disease-associated EV-biomarkers. With this aim, we purified EVs from paired human milk and serum samples and used the MACSPlex bead-based flow-cytometry assay to capture EVs on bead-bound antibodies specific for a certain surface protein, followed by EV detection by the tetraspanins CD9, CD63, and CD81. Using this approach we identified body fluid-specific EV signatures, e.g. breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, as well as body fluid-specific markers associated to immune cells and stem cells. Interestingly, comparison of pan-tetraspanin detection (simultaneous CD9, CD63 and CD81 detection) and single tetraspanin detection (detection by CD9, CD63 or CD81) also unveiled body fluid-specific tetraspanin distributions on EVs. Moreover, certain EV surface proteins were associated with a specific tetraspanin distribution, which could be indicative of the biogenesis route of this EV subset. Altogether, the identified body fluid-specific EV profiles can contribute to study EV profile deviations in these fluids during disease processes. Extracellular vesicles (EVs) are submicron lipid bilayer-delimited particles naturally released by cells that act as mediators of inter-cellular communication by targeting biologically active molecules to adjacent and distant cells 1. Cells in body tissues communicate by releasing EVs into proximal body fluids, such as breast milk and blood 2. Circulating EVs can originate from cells present in the body fluids, cells lining the cavities of extruded fluids or from tissue-resident cells 2 , and for this reason they can carry body fluid-specific and tissue-specific signatures. Additionally, the molecular make-up of EVs can be affected by the status of their originating cells and, as such, EVs can be enriched or depleted for specific surface proteins, resulting in specific protein biomarker profiles associated with (patho)physiological conditions 3,4. Nowadays, EV-based biomarker discovery attracts a lot of attention for monitoring disease and health status. The promise of EV-based biomarkers resides in the unique combination of different EV molecules, resulting in a "combined" biomarker that outperforms single component-based biomarkers. To identify EV-based biomarkers of disease or disturbed homeostasis, knowing the "normal" molecular reference profile of EVs in different body fluids is of utmost importance. However, the discovery of such body fluid-specific EV profiles is complicated due to the colloidal properties of body fluids, containing non-EV particles with overlapping characteristics of EVs 5,6. For example, lipoproteins in blood and casein micelles in milk, which co-isolate to various degrees with EVs 7,8 , can act as confounders in (semi)-quantitative EV analyses. Hence for comparative analyses of EVs present in different body fluids, a tailored protocol for EV isolation might overcome these problems. In immunoassays developed for EV phenotyping, the tetraspanins CD9, CD63 and CD81 are commonly used as bona fide EV-associated markers for "total" EV detection. These tetraspanins have primary functions in EV formation, cargo selection/sorting and EV release and uptake 9. Via their extracellular domains, tetraspanins associate with other tetraspanins and surface proteins thereby forming "tetraspanin webs" resulting in membrane domains with a variety of surface protein profiles 10. Importantly, in recent years, it has been reported that