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Blood Circulating Exosomes Contain Distinguishable Fractions of Free and Cell-Surface-Associated Vesicles

Author(s):

Svetlana Tamkovich*, Oleg Tutanov, Anastasia Efimenko, Alina Grigor’eva, Elena Ryabchikova, Natalia Kirushina, Valentin Vlassov, Vsevolod Tkachuk and Pavel Laktionov  

Abstract:


Background: Considering exosomes as intercellular transporters, thus inevitably interacting with plasma membrane and large available surface of blood cells, we wonder if a fraction of circulating exosomes is associated with the surface of blood cells.

Objective: The aim of this study was to develop an efficient protocol for isolating exosomes associated with the surface of blood cells and to further investigate the characteristics of this fraction in healthy state and during the development of breast cancer, as well as its possible implication for use in diagnostic applications.

Methods: Blood samples were collected from healthy females (HFs) and breast cancer patients (BCPs). Exosomes extracted from blood plasma and eluted from the surface of blood cells were isolated by ultrafiltration with subsequent ultracentrifugation.

Results: Transmission electron microscopy (TEM), along with immunogold labeling, demonstrated the presence of exosomes among membrane-wrapped extracellular vesicles (EVs) isolated from both plasma and blood cell eluates. TEM, nanoparticle tracking analysis, and NanoOrange protein quantitation data showed that cell-associated exosomes constituted no less than 2/3 of total blood exosome number. Exosomes, ranged 50–70 nm in size, prevailed in blood of breast cancer patients, whereas smaller exosomes (30–50 nm) were mostly observed in blood of healthy women. Analysis of specific proteins and RNAs in exosomes circulating in blood demonstrated the significant differences in the packing density of the polymers in exosomes of HFs and BCPs. Preliminary data indicated that detection of cancer-specific miRNA (miR-103, miR-191, miR-195) in exosomes associated with the fraction of red blood cells allowed to discriminate HFs and BCPs more precisely compared to cell-free exosomes circulating in plasma.

Conclusion: Our data provide the basis for using blood cell-associated exosomes for diagnostic applications.

Keywords:

Exosomes, extracellular vesicles, blood cells, cell-associated exosomes, miRNA, breast cancer.

Affiliation:

Laboratory of Molecular Medicine, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Science, Novosibirsk, Laboratory of Molecular Medicine, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Science, Novosibirsk, Institute for Regenerative Medicine, Lomonosov Moscow State University, Moscow, Laboratory of Molecular Medicine, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Science, Novosibirsk, Laboratory of Molecular Medicine, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Science, Novosibirsk, Department of Mammology, National Novosibirsk Regional Oncologic Dispensary, Novosibirsk, Laboratory of Molecular Medicine, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Science, Novosibirsk, Institute for Regenerative Medicine, Lomonosov Moscow State University, Moscow, Laboratory of Molecular Medicine, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Science, Novosibirsk



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