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. 2017 Mar 4;11(2):196-204.
doi: 10.1080/19336918.2017.1279784. Epub 2017 Feb 1.

The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue

Sarah Jeurissen  1   2   3 , Glenn Vergauwen  1   4   3 , Jan Van Deun  1   3 , Lore Lapeire  2   3 , Victoria Depoorter  1 , Ilkka Miinalainen  5 , Raija Sormunen  5 , Rudy Van den Broecke  4   3 , Geert Braems  4   3 , Véronique Cocquyt  2   3 , Hannelore Denys  2   3 , An Hendrix  1   3
Affiliations

The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue

Sarah Jeurissen et al. Cell Adh Migr. .

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.

Keywords: aggregation; breast cancer; characterization; exosomes; function; isolation; proliferation.

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Figures

Figure 1.
Figure 1.
Schematic overview of the dUC-ODG protocol to isolate EVs from cancer-associated adipose tissue-derived conditioned medium (CMCAAT). Approximately 21 g of CAAT was ex vivo cultivated in control medium. CMCAAT was harvested, centrifuged and used for further isolation by a combination of differential ultracentrifugation followed by Optiprep density gradient centrifugation.
Figure 2.
Figure 2.
Protein analysis of (non) EV-enriched proteins. EVs were isolated from the conditioned medium of cancer-associated adipose tissue by the dUC-ODG protocol. Western blot analysis of (A) EV-enriched proteins (flotillin-1, CD9 and HSP70) and adipocyte-specific protein FABP-4 and (B) cell organelle and apoptotic body proteins (GM130, prohibitin and calreticulin).
Figure 3.
Figure 3.
Morphological characterization and quantification of EV preparations by electron microscopy (EM) and nanoparticle tracking analysis (NTA). EVs were isolated from the conditioned medium of cancer-associated adipose tissue by the dUC-ODG protocol. (A) Left: Wide-field EM picture of EVs of fractions 8–9 fsrom the density gradient corresponding to the density of 1.086–1.103 g/ml. Scale bar: 200 nm. Right: Zoom in on CD63-positive and negative EVs. (B) The calculated size distribution of EVs analyzed by NTA depicted as a mean (black line) with standard error (red shaded area). Total particle number, mean particle size and modus are shown.
Figure 4.
Figure 4.
Stimulation of CREB transcription factor phosphorylation and sphere formation by EVs. EVs were isolated from the conditioned medium of cancer-associated adipose tissue by the dUC-ODG protocol. (A) Western blot analysis of phospho-CREB and total CREB from lysates of ZR75.1 cells under control conditions or treated by 1×108 EVs. (B) The number of aggregates formed by MCF7 single cells seeded in ultra-low attachment plates under control conditions or treated by 5×109 EVs for 24h. Each experiment was performed in quadruplicate.
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