Objective: This study tried to evaluate whether 8% polyethylene glycol (PEG) 6000 precipitation combined with differential ultracentrifugation (PPDU) was an efficient and practical method for the enrichment and purification of extracellular vesicles (EVs) derived from the culture supernatant of human ovarian cancer cell line A2780 and from body fluids of patients with high-grade serous carcinoma (HGSC).
Methods: PPDU was used to enrich and purify the EVs derived from body fluids of patients with HSGC and cell culture supernatant of subclones of human ovarian cancer cell line A2780 with high/low invasive capacity (named as A-H/A-L, respectively). Transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA) were used to identificate the EVs size and distribution. Western blots (WB) were used to detect the expression of CD9, CD63, Alix and Calnexin. The high-purity EVs derived from the cell culture supernatant of A-H/A-L were detected by the protein profile. Expression of integrins (ITGs) αV, β 1 and β 3 in the EVs derived from body fluids of HGSC patients was also evaluated.
Results: The diameter of EVs was about 30– 260 nm observed under the TEM. Under the NTA identification, the peak size of EVs was ranged from 70 to 159nm. EVs derived from different specimens did not significantly differ in mean size and peak size. Presence of CD9, CD63 and Alix and absence of Calnexin were confirmed in the EVs. The protein concentrations of EVs’ sample extracted from A-H/A-L cell culture supernatant were 0.36μg/μL and 0.20μg/μL, respectively. The total amount of protein obtained from 300ul EVs was 108.02ug and 61.44ug, respectively. Totally, 2397 peptides and 952 proteins were identified by isobaric tags for relative and absolute quantitation (ITRAQ). The expression of ITGαV, β 1, and β 3 in the EVs from plasma and ascites of HGSC patients was significantly higher than the control group (plasma: all P< 0.0001; ascites: P=0.036, 0.001 and 0.004, respectively). The expression level of ITGαV and β 1 in EVs of HGSC’s ascites was significantly higher than that in plasma (P= 0.004, 0.001, respectively). The expression of ITGβ 3 was also slightly elevated in EVs-derived HGSC patients’ ascites (P=0.492).
Conclusion: PPDU was an efficient and practical method to enrich EVs from body fluids and cell culture supernatant. The characteristic expression of ITGαV, β 1 and β 3 in ascites and plasma EVs of patients with HGSC provided useful information on the development of EVs in HGSC.
Keywords: EVs, PEG6000 precipitation, HGSC, body fluids, cell culture supernatant