Concentration and composition of circulating vesicles of adipocytic origin in patients with colon polyps and colorectal cancer

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Abstract

Extracellular vesicles (EVs) are a heterogeneous population of membrane particles less than 1 μm in size, secreted by various cell types. Most EVs circulating in human blood are particles of platelet, leukocyte, erythrocyte and endothelial origin. The composition of circulating adipocyte EVs in various pathological conditions has been virtually unknown. Small EVs from the blood plasma of patients with colorectal cancer (CRC) and colon polyps with obesity or metabolic syndrome were isolated by ultrafiltration with double ultracentrifugation. To study the composition of adipocyte EVs, immunoprecipitation in combination with Western blotting and flow cytometry were used. Vesicle 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 and perilipin 1). EVs of monocyte-macrophage origin precipitated on CD11b-coated particles in CRC patients without obesity were characterized by combined overexpression of FABP4 and perilipin 1, while such overexpression was not typical for CRC patients with metabolic syndrome or obesity. The fraction of truly adipocyte vesicles (supernatant after removal of CD11b-positive EVs) was characterized by the presence in all patients of a complex of adipocyte markers with predominant expression of FABP4 in both patients with metabolic syndrome/metabolically healthy obesity and patients without metabolic disorders. To correctly characterize circulating EVs of patients without obesity, it is first necessary to remove the fraction of CD11b-positive monocyte-macrophage EVs from EV preparations by immunoprecipitation or similar methods, and in the supernatant after removal/sorption of precipitated EVs, the composition of adipocyte vesicles can be studied using a set of markers (FABP4, PPAR-γ, perilipin 1, etc.). Moreover, in patients with metabolic disorders, taking into account the insignificant expression of FABP4 in CD11b-immunoprecipitated EVs, preliminary depletion of vesicle preparations is apparently not so necessary.

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About the authors

N. V. Yunusova

Siberian State Medical University, Department of Biochemistry and Molecular Biology with a course of clinical laboratory diagnostics

Email: svarovsky.d.a@gmail.com
Russian Federation, Tomsk

D. A. Svarovsky

Siberian State Medical University, Department of Biochemistry and Molecular Biology with a course of clinical laboratory diagnostics

Author for correspondence.
Email: svarovsky.d.a@gmail.com
Russian Federation, Tomsk

E. S. Kolegova

Cancer Research Institute, Tomsk National Research Medical Center

Email: svarovsky.d.a@gmail.com
Russian Federation, Tomsk

O. V. Cheremisina

Cancer Research Institute, Tomsk National Research Medical Center

Email: svarovsky.d.a@gmail.com
Russian Federation, Tomsk

D. N. Kostromitsky

Cancer Research Institute, Tomsk National Research Medical Center

Email: svarovsky.d.a@gmail.com
Russian Federation, Tomsk

I. V. Kondakova

Cancer Research Institute, Tomsk National Research Medical Center

Email: svarovsky.d.a@gmail.com
Russian Federation, Tomsk

E. A. Sidenko

Cancer Research Institute, Tomsk National Research Medical Center

Email: svarovsky.d.a@gmail.com
Russian Federation, Tomsk

A. Yu. Dobrodeev

Cancer Research Institute, Tomsk National Research Medical Center

Email: svarovsky.d.a@gmail.com
Russian Federation, Tomsk

A. E. Grigor’eva

Institute of Chemical Biology and Fundamental Medicine of Russian Academy of Science

Email: svarovsky.d.a@gmail.com
Russian Federation, Novosibirsk

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Supplementary files

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2. Fig. 1. Identification of isolated EVs. (a) TEM showed the presence of vesicles with typical morphology and the absence of vesicles larger than 220 nm. Insets show small EVs. Scale bars represent 100 nm. Electron microscopy, negative staining with uranyl acetate; (b) NTA assay data for EVs isolated from CRC and CP plasma. The abscissa axis represents particle size (nm), the ordinate axis represents particle concentration (particles/mL), and the minor axis represents cumulative frequency (%). (c) Expression of CD63, CD81, and CD24 on CD9-positive CRC and CP plasma sEVs. Representative mean fluorescence intensities (MFIs) are shown; each measurement was performed in triplicate. For isotype controls (right histogram), labeled anti-CD9-sEV particle-antibody complexes were incubated with mouse FITC IgG1 isotype control or FITC IgG2a isotype control. One representative isotype control is shown. As a negative control, nothing was added to CD9 antibody-labeled latex particles (left histogram). A negative control was also set up where coated particles were incubated with FITC-conjugated antibodies without the addition of vesicles (center histogram).

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3. Fig. 2. Concentration, size of EVs, and total protein level in fractions and supernatant during immunoprecipitation. (a) — EV concentration in preparations: 1 — before immunoprecipitation, 2 — in supernatant after immunoprecipitation of CD11b-positive EVs, 3 — in supernatant after sequential immunoprecipitation of CD11b-positive and FABP4-positive EVs in patients with CRC with metabolic disorders (CRC+MS) (dark boxes), in patients with PTC with metabolic disorders (CP+MS) (shaded boxes), and CRC patients without metabolic disorders (CRC without MS) (light boxes). * — differences are significant compared to EV concentration in patients with CP+MS and CRC without MS, p < 0.05. (b) — average vesicle size in patients in supernatants before and after immunoprecipitation; (c) — protein concentration in EV samples from patients in supernatants before and after immunoprecipitation, ** — differences are significant compared to protein concentration in vesicles from patients with CRC without MS, p < 0.05. (d) — NTA analysis data in circulating EV samples from patients before immunoprecipitation (left) and after immunoprecipitation of CD11b-positive EV (right). The abscissa axis represents particle size (nm), the ordinate axis represents particle concentration (particles/ml), and the auxiliary axis represents cumulative frequency (%).

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4. Fig. 3. Levels of FABP4, perilipin-1, and PPAR-ý in circulating EV fractions after EV immunoprecipitation on latex particles coated with antibodies to FABP4 (a) and to CD11b (b) and in the supernatant after removal of EV of monocyte-macrophage origin (c) in patients with CRC without metabolic disorders (light columns) and CRC with metabolic disorders (shaded columns) compared to patients with colon polyps with metabolic disorders (CP). RU — arbitrary units. Lanes 1, 2, 3 — CRC patients without metabolic disorders, 4-7 — CRC patients with metabolic disorders, 8-10 — colon polyps with metabolic disorders (3a, 3b). Lanes 1, 2 — CRC patients without metabolic disorders, 3-5 — CRC patients with metabolic disorders, 6-7 — colon polyps with metabolic disorders (3c). Western blotting. * — differences are significant compared to the group of CRC patients without metabolic disorders, p < 0.05, n = 20.

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