<p>A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles from Culture Supernatant of Human Ovarian Cancer Cell Line A2780 and Body Fluids of High-Grade Serous Carcinoma Patients</p>

Abstract

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, β1 and β3 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, β1, and β3 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 β1 in EVs of HGSC's ascites was significantly higher than that in plasma (P= 0.004, 0.001, respectively). The expression of ITGβ3 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, β1 and β3 in ascites and plasma EVs of patients with HGSC provided useful information on the development of EVs in HGSC.

MicroRNA 421 induces the formation of high-invasive cell subsets of ovarian cancer from low-invasive cell subsets mediated by exosomes by activating the PI3K/AKT pathway

“…The morphology and size of exosomes were observed by TEM using negative staining with copper mesh, as described previously 12,27 . Exosomes precipitates were resuspended using 50-100 µL 2% PFA, loaded onto a formvar-carbon-coated grid, and incubated for 20 min.…”

“…Exosomes are related to the formation of a tumor heterogeneous microenvironment 11 . One of our previous studies revealed that exosomes derived from OC cells contain speci c proteins related to OC genesis and metastasis 12 .…”

The potential therapeutic role of camel milk exosomes – A review

“…First, a low speed centrifugation (600-1000× g) is operated to defat the milk and precipitate the cells, then 10×10 3 -16×10 3 g is used to precipitate milk proteins, and finally, one or numerous multi-ultracentrifugations at 100×10 3 -200×10 3 g are utilized to separate the EXOs (Yassin et al, 2016;El-Kattawy et al, 2021). Commercially, several supply materials are used, including volume-excluding polymers for isolation of milk EXOs such as ExoQuick (System Biosciences, Palo Alto, CA, USA), containing PEG 8000 kDa; Total Exosome Isolation (Thermo Fisher Scientific, Waltham, MA, USA), comprising polyvinyl, polyethylene glycol, and dextrans with a molecular weight above 1000 kDa, and their mixtures with other approaches (Jiao et al, 2020). Additionally, the immunomagnetic approach is applied to separate EXOs through a semipermeable membrane (Bunggulawa et al, 2018).…”

Immunomagnetic Separation Method Integrated with the Strep-Tag II System for Rapid Enrichment and Mild Release of Exosomes

“…However, it not only requires expensive equipment and long operation time but also has relatively low recovery rates (5–25%) . Other conventional approaches, including polymer-based precipitation, , size exclusion chromatography, (SEC), and ultrafiltration, usually suffer from various drawbacks as they usually result in low purity, are time-consuming, and may cause mechanical damage to exosomes. Physical characteristics of exosomes have allowed many microfluidic technologies, such as viscoelastic, acoustics, − dielectrophoretic, and nanoscale deterministic lateral displacement, , to be applied in exosome separation.…”