Subtypes of tumour cell‐derived small extracellular vesicles having differently externalized phosphatidylserine

Abstract

Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS + -enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy.

Characterization and internalization of small extracellular vesicles released by human primary macrophages derived from circulating monocytes

“…The raw blotting images from gradient assays are shown in S6 Fig. Our data showed that CD63 colocalized with CD81 in fractions F2 to F4 (densities between 1,117 to 1,181 g/mL), a buoyant property reported for sEVs from endosomal origin [59,60].…”

“…This effect could be a consequence of high-speed centrifugation of culture medium in combination with lipophilic dyes such as PKH, which induce the formation of artifacts with different sizes and morphologies that can be detected by fluorescence microscopy [58,87,88]. The specialized literature has also reported a multitude of contaminants in vesicles separated by multi-step methods [60,89,90]. Moreover, we believe that staining EVs with diameter smaller (<200 nm) than the diffraction limit of light of the confocal microscopy may also have potentially favored the visualization of other large fluorescent dots in our preparations, probably corresponding to clusters of positive PKH26-labeled sEV without the possibility to discriminate one vesicle from another [91,92].…”

Cell Stress Induced Stressome Release Including Damaged Membrane Vesicles and Extracellular HSP90 by Prostate Cancer Cells

“…CD9 has been known as a marker of exosomes, small vesicles secreted from cells via exocytosis. However, recent studies pointed out the heterogeneity of EVs and exosomes [29,[102][103][104][105]. Therefore, for example, some EVs contained many CD9, while the other EVs might not contain CD9.…”

Extracellular Vesicles as an Efficient and Versatile System for Drug Delivery

“…The main lipid contributor of EVs is their lipid bilayer membrane, whose structure resembles the plasma membrane of cells and is quite different from other single-layered lipids in the circulation such as cholesterol in blood lipoproteins [ 72 , 73 ]. With regard to molecular composition, EV lipids are enriched in phosphatidylserine (PS) which may influence the uptake of EVs [ 74 , 75 ]. EVs from different parental cells may also have different lipid profiles.…”