The Isolation of Morphologically Intact and Biologically Active Extracellular Vesicles From the Secretome of Cancer-Associated Adipose Tissue

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

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.

MeSH terms

  • Adipose Tissue / pathology*
  • Breast Neoplasms / metabolism*
  • Breast Neoplasms / pathology*
  • Extracellular Vesicles / metabolism*
  • Extracellular Vesicles / ultrastructure
  • Female
  • Humans
  • MCF-7 Cells
  • Proteome / metabolism*
  • Ultracentrifugation

Substances

  • Proteome