Characterization and function of extracellular vesicles in a canine mammary tumour cell line: Ultracentrifugation versus size exclusion chromatography

Abstract

Extracellular vesicles (EVs) are cell‐derived membrane‐bound vesicles involved in many biological processes such as tumour progression. For years, ultracentrifugation (UC) has been considered the gold standard for EV isolation but limited purity and integrity allowed the diffusion of alternative techniques. In this study, EVs were isolated from a canine mammary tumour cell line using UC and size exclusion chromatography (SEC) and analysed for size and concentration by nanoparticle tracking analysis (NTA) and for protein expression by western blot (WB). EV autocrine effect on cell proliferation, migration and invasiveness was then evaluated in vitro. In all samples, particles were in the EV size range (50–1000 nm), with a higher concentration in UC than in SEC samples (1011 and 1010 particles/ml respectively), and expressed EV markers (Alix, CD9). Functional assays did not show statistically significant difference among conditions, but EV treatment slightly increased cell proliferation and invasiveness and treatment with SEC‐isolated EVs slightly enhanced cell migration compared to UC‐isolated EVs. In conclusion, the main differences between the two isolation techniques are the quantity of the final EV‐product and slight differences on EV functionality, which should be further explored to better highlight the real autocrine effect of tumoral EVs.

Isolation and Characterization of Cetacean Cell-Derived Extracellular Vesicles

“…As such, the absence of expression of TSG101 in our SEC-derived EVs might be explained by a lower quantity of TSG101 compared to UC-EVs, which might be related to the isolation of different EV subtypes with lower TSG101 expression. In relation to our previous study on canine mammary tumor cell-derived EVs, while integrin-beta and TSG101 were not tested as EV markers, CD9 was present in both UC and SEC EVs [23]. Therefore, we demonstrated that all the tested EV markers are also conserved and expressed in cetacean-derived EVs.…”

“…The aim of this study was to isolate and characterize EVs from two cetaceans' cell lines using two different isolation techniques. Similar studies have already been performed on other in vitro models [23]. However, considering the novelty of using cetacean cell lines, the fact that EVs released in vitro from cetaceans have never been described, and that EV size, concentration, and marker expression can vary according to the EV source, here we report our detailed protocol and findings.…”

“…Despite the fact that the particle concentration of UC EVs at NTA was similar, if slightly higher (7 × 10 11 particles/mL), the SEC EV concentration from canine mammary tumor cells was higher (10 10 particles/mL) than the concentration SEC EVs from cetaceans (ref). This might be explained by the fact that cancer cells have been demonstrated to generally shed more EVs [23].…”

“…EVs were isolated from two plates with semi-confluent cells by UC or SEC, as already described [23]. Briefly, the medium from each plate was centrifuged at 300× g and at 2000× g for 10 min at 4 • C, to remove any cell/cell debris.…”