Concentration and Composition of Circulating Adipocyte-Derived Extracellular Vesicles in Patients with Colonic Polyps and Colorectal Cancer

Abstract

Extracellular vesicles (EVs) are a heterogeneous population of membrane-bound nanoparticles (< 1 µm in size) secreted by various cell types. Most of EVs circulating in human blood are derived from platelets, leukocytes, erythrocytes, and endotheliocytes. The composition of circulating adipocyte-derived EVs under various pathological conditions has been virtually unknown. Small EVs were isolated by ultrafiltration and double ultracentrifugation from blood plasma of patients with colorectal cancer (CRC) and colonic polyps with obesity or metabolic syndrome. The composition of adipocyte-derived EVs was analyzed by immunoprecipitation combined with Western blotting and flow cytometry. EVs fractions (FABP4- and CD11b-immunoprecipitated EVs, as well as EVs contained in the supernatant after removal of CD11b-positive EVs) contained a complex of adipocyte markers (FABP4, PPAR-γ, perilipin 1). In CRC patients without obesity, monocyte/macrophage-derived EVs precipitated on CD11b-coated particles were characterized by a combined overexpression of FABP4 and perilipin 1, while such an overexpression was not typical for CRC patients with metabolic syndrome or obesity. The fraction of true adipocyte-derived EVs (supernatant after removal of CD11b-positive EVs) was characterized by the presence of a complex of adipocyte markers with a predominant expression of FABP4 in all patients both with metabolic syndrome/metabolically healthy obesity and without metabolic disorders. To correctly characterize circulating EVs in patients without obesity, it is necessary first to remove the fraction of CD11b-positive monocyte/macrophage-derived EVs from EV preparations by immunoprecipitation or similar methods, and then, after removal/sorption of precipitated EVs, to analyze the composition of adipocyte-derived EVs in the supernatant using a set of above-mentioned adipocyte markers. Moreover, in patients with metabolic disorders, given the insignificant FABP4 expression in CD11b-immunoprecipitated EVs, the pre-depletion of EV preparations does not appear to be that necessary.