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

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 tec...

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Bibliographic Details
Published in:Veterinary & comparative oncology 2023-03, Vol.21 (1), p.36-44
Main Authors: Moccia, Valentina, Sammarco, Alessandro, Ferro, Silvia, Cavicchioli, Laura, Zappulli, Valentina
Format: Article
Language:English
Subjects:
Animals
Cell Line, Tumor
Chromatography, Gel - veterinary
CIPp
dog
Dog Diseases - metabolism
Dogs
Extracellular Vesicles
Mammary Neoplasms, Animal - metabolism
mammary tumour cell line
size exclusion chromatography
ultracentrifugation
Ultracentrifugation - methods
Ultracentrifugation - veterinary
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Summary: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.
ISSN:1476-5810
1476-5829
DOI:10.1111/vco.12858