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
;
2020-01-01
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
