Introduction: Extracellular vesicles (EVs) are membrane-bound vesicles produced by cells that play a key role in intercellular communication. This study aimed to evaluate the autocrine effect of canine and feline mammary tumour-derived EVs, comparing two different EV isolation methods. Materials and Methods: EVs were isolated from canine (CIPp) and feline (FMCp) mammary tumour cell lines by ultracentrifugation and by size exclusion chromatography (SEC). A wound healing assay, a transwell migration/invasion assay and a proliferation assay were performed on CIPp and FMCp treated with CIPp- and FMCp-derived EVs, respectively. EVs isolated by ultracentrifugation and by SEC were compared for purity using the COlorimetric NANoplasmonic (CONAN) assay and the BCA protein assay. Results: CIPp treated with CIPp EVs and FMCp treated with FMCp EVs showed a higher migration (P <0.0001) in the wound healing assay with no differences between ultracentrifugation and SEC. EVs purified by SEC induced a higher proliferation rate (P <0.05) in both cell lines when compared with EVs isolated by ultracentrifugation. CONAN assay confirmed a higher EV purity of SEC fractions. BCA assay showed a higher protein content in the EV pellet obtained by ultracentrifugation when compared with EVs purified by SEC. Discussion: An autocrine effect of canine and feline mammary cancer-derived EVs on cell migration and proliferation was demonstrated. The effect of EVs isolated by ultracentrifugation might be enhanced by the co-presence of soluble proteins, while the functionality of EVs purified by SEC might be increased by their better preservation, which needs to be further investigated.

Canine and Feline Mammary Tumours: In-vitro Study of the Autocrine Effect of Extracellular Vesicles Isolated by Ultracentrifugation and Size Exclusion Chromatography

Romano, M.
Investigation
;
Radeghieri, A.
Conceptualization
;
Bergese, P.
Conceptualization
;
2020

Abstract

Introduction: Extracellular vesicles (EVs) are membrane-bound vesicles produced by cells that play a key role in intercellular communication. This study aimed to evaluate the autocrine effect of canine and feline mammary tumour-derived EVs, comparing two different EV isolation methods. Materials and Methods: EVs were isolated from canine (CIPp) and feline (FMCp) mammary tumour cell lines by ultracentrifugation and by size exclusion chromatography (SEC). A wound healing assay, a transwell migration/invasion assay and a proliferation assay were performed on CIPp and FMCp treated with CIPp- and FMCp-derived EVs, respectively. EVs isolated by ultracentrifugation and by SEC were compared for purity using the COlorimetric NANoplasmonic (CONAN) assay and the BCA protein assay. Results: CIPp treated with CIPp EVs and FMCp treated with FMCp EVs showed a higher migration (P <0.0001) in the wound healing assay with no differences between ultracentrifugation and SEC. EVs purified by SEC induced a higher proliferation rate (P <0.05) in both cell lines when compared with EVs isolated by ultracentrifugation. CONAN assay confirmed a higher EV purity of SEC fractions. BCA assay showed a higher protein content in the EV pellet obtained by ultracentrifugation when compared with EVs purified by SEC. Discussion: An autocrine effect of canine and feline mammary cancer-derived EVs on cell migration and proliferation was demonstrated. The effect of EVs isolated by ultracentrifugation might be enhanced by the co-presence of soluble proteins, while the functionality of EVs purified by SEC might be increased by their better preservation, which needs to be further investigated.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11379/527233
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