A simple immunoassay for extracellular vesicle liquid biopsy in microliters of non-processed plasma

Abstract

Background Extracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. However, in order to perform validation studies and screenings of patient samples, it is still necessary to develop general techniques permitting rapid handling of small amounts of biological samples from large numbers of donors. Results Here we describe a method that, using just a few microliters of patient’s plasma, identifies tumour markers exposed on EVs. Studying physico-chemical properties of EVs in solution, we demonstrate that they behave as stable colloidal suspensions and therefore, in immunocapture assays, many of them are unable to interact with a stationary functionalised surface. Using flocculation methods, like those used to destabilize colloids, we demonstrate that cationic polymers increase EV ζ-potential, diameter, and sedimentation coefficient and thus, allow a more efficient capture on antibody-coated surfaces by both ELISA and bead-assisted flow cytometry. These findings led to optimization of a protocol in microtiter plates allowing effective immunocapture of EVs, directly in plasma without previous ultracentrifugation or other EV enrichment. The method, easily adaptable to any laboratory, has been validated using plasma from lung cancer patients in which the epithelial cell marker EpCAM has been detected on EVs. Conclusions This optimized high throughput, easy to automate, technology allows screening of large numbers of patients to phenotype tumour markers in circulating EVs, breaking barriers for the validation of proposed EV biomarkers and the discovery of new ones. Graphical Abstract

MAPK inhibitors dynamically affect melanoma release of immune NKG2D-ligands, as soluble protein and extracellular vesicle-associated

“…As expected, levels of tetraspanins varied among different individuals, either patients with cancer or another condition. In any case, cancer patients presented, in general, higher CD63 + -CD81 + signal than non-cancer donors, in line with previous observations in lung cancer patients compared with healthy donor plasma ( Campos-Silva et al, 2022a ). Then, we compared EV-released MICA (combining MICA capture with tetraspanin detection) with soluble released ligands.…”

“…Next, we compared data of soluble NKG2D-ligands with EV-released. Importantly, in order to minimize EV subpopulation selection during sample processing for EV enrichment and to encourage easy translation in clinical settings in the future, direct detection by sandwich ELISA was performed ( Campos-Silva et al, 2021 ; Campos-Silva et al, 2022a ). To ensure the association of NKG2D-L to EVs, different combinations of capture and detection antibodies were used as depicted.…”