The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue. Issue 2 (4th March 2017)

• Record Type: Journal Article
• Authors: Jeurissen, Sarah; Vergauwen, Glenn; Van Deun, Jan; Lapeire, Lore; Depoorter, Victoria; Miinalainen, Ilkka; Sormunen, Raija; Van den Broecke, Rudy; Braems, Geert; Cocquyt, Véronique; Denys, Hannelore; Hendrix, An
• Is Part Of: Cell adhesion & migration. Volume 11:Issue 2(2017)
• Page Start: 196
• Page End: 204
• Publication Date: 2017-03-04
• Subjects: aggregation -- breast cancer -- characterization -- exosomes -- function -- isolation -- proliferation; Cell adhesion -- Periodicals; Cell migration -- Periodicals; Cell interaction -- Periodicals; 574.876
• DOI: 10.1080/19336918.2017.1279784
• Languages: English
• ISSNs: 1933-6918

Abstract

ABSTRACT: Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to favor invasive growth and metastasis. Extracellular vesicles (EVs) are nanometer-sized vesicles secreted by different cell types that shuttle proteins and nucleic acids to establish cell-cell communication. To study the role of EVs released by cancer-associated adipose tissue in breast cancer progression and metastasis a standardized EV isolation protocol that obtains pure EVs and maintains their functional characteristics is required. We implemented differential ultracentrifugation as a pre-enrichment step followed by OptiPrep density gradient centrifugation (dUC-ODG) to isolate EVs from the conditioned medium of cancer-associated adipose tissue. A combination of immune-electron microscopy, nanoparticle tracking analysis (NTA) and Western blot analysis identified EVs that are enriched in flotillin-1, CD9 and CD63, and sized between 20 and 200 nm with a density of 1.076–1.125 g/ml. The lack of protein aggregates and cell organelle proteins confirmed the purity of the EV preparations. Next, we evaluated whether dUC-ODG isolated EVs are functionally active. ZR75.1 breast cancer cells treated with cancer-associated adipose tissue-secreted EVs from breast cancer patients showed an increased phosphorylation of CREB. MCF-7 breast cancer cells treated with adipose tissue-derived EVs exhibited a stronger propensity to form cellular aggregates. In conclusion, dUC-ODG purifies EVs from conditioned medium of cancer-associated adipose tissue, and these EVs are morphologically intact and biologically active.