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

(2017) CELL ADHESION & MIGRATION. 11(2). p.196-204
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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 1.076-1.125g/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, BREAST-CANCER, EXOSOMES, CELLS, ADIPOCYTES, PHENOTYPE, MECHANISM

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Chicago
Jeurissen, Sarah, Glenn Vergauwen, Jan Van Deun, Lore Lapeire, Victoria Depoorter, Ilkka Miinalainen, Raija Sormunen, et al. 2017. “The Isolation of Morphologically Intact and Biologically Active Extracellular Vesicles from the Secretome of Cancer-associated Adipose Tissue.” Cell Adhesion & Migration 11 (2): 196–204.
APA
Jeurissen, S., Vergauwen, G., Van Deun, J., Lapeire, L., Depoorter, V., Miinalainen, I., Sormunen, R., et al. (2017). The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue. CELL ADHESION & MIGRATION, 11(2), 196–204.
Vancouver
1.
Jeurissen S, Vergauwen G, Van Deun J, Lapeire L, Depoorter V, Miinalainen I, et al. The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue. CELL ADHESION & MIGRATION. 2017;11(2):196–204.
MLA
Jeurissen, Sarah et al. “The Isolation of Morphologically Intact and Biologically Active Extracellular Vesicles from the Secretome of Cancer-associated Adipose Tissue.” CELL ADHESION & MIGRATION 11.2 (2017): 196–204. Print.
@article{8515626,
  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 1.076-1.125g/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.},
  author       = {Jeurissen, Sarah and Vergauwen, Glenn and Van Deun, Jan and Lapeire, Lore and Depoorter, Victoria and Miinalainen, Ilkka and Sormunen, Raija and Van den Broecke, Rudy and Braems, Geert and Cocquyt, Veronique and Denys, Hannelore and Hendrix, An},
  issn         = {1933-6918},
  journal      = {CELL ADHESION & MIGRATION},
  keywords     = {aggregation,breast cancer,characterization,exosomes,function,isolation,proliferation,BREAST-CANCER,EXOSOMES,CELLS,ADIPOCYTES,PHENOTYPE,MECHANISM},
  language     = {eng},
  number       = {2},
  pages        = {196--204},
  title        = {The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue},
  url          = {http://dx.doi.org/10.1080/19336918.2017.1279784},
  volume       = {11},
  year         = {2017},
}

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