The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue.
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.
a Laboratory of Experimental Cancer Research , Department of Radiation Oncology and Experimental Cancer Research, Ghent University , Ghent , Belgium.|b Department of Medical Oncology and Department of Gynaecology , Ghent University Hospital , Ghent , Belgium.|d Cancer Research Institute Ghent (CRIG) , Ghent , Belgium.
Sarah
S
Jeurissen
a Laboratory of Experimental Cancer Research , Department of Radiation Oncology and Experimental Cancer Research, Ghent University , Ghent , Belgium.|c Department of Gynaecology , Ghent University Hospital , Ghent , Belgium.|d Cancer Research Institute Ghent (CRIG) , Ghent , Belgium.
Glenn
G
Vergauwen
a Laboratory of Experimental Cancer Research , Department of Radiation Oncology and Experimental Cancer Research, Ghent University , Ghent , Belgium.|d Cancer Research Institute Ghent (CRIG) , Ghent , Belgium.
Jan
J
Van Deun
b Department of Medical Oncology and Department of Gynaecology , Ghent University Hospital , Ghent , Belgium.|d Cancer Research Institute Ghent (CRIG) , Ghent , Belgium.
Lore
L
Lapeire
a Laboratory of Experimental Cancer Research , Department of Radiation Oncology and Experimental Cancer Research, Ghent University , Ghent , Belgium.
Victoria
V
Depoorter
e Biocenter Oulu and Departments of Pathology , University of Oulu and Oulu University Hospital , Oulu , Finland.
Ilkka
I
Miinalainen
e Biocenter Oulu and Departments of Pathology , University of Oulu and Oulu University Hospital , Oulu , Finland.
Raija
R
Sormunen
c Department of Gynaecology , Ghent University Hospital , Ghent , Belgium.|d Cancer Research Institute Ghent (CRIG) , Ghent , Belgium.
Rudy
R
Van den Broecke
c Department of Gynaecology , Ghent University Hospital , Ghent , Belgium.|d Cancer Research Institute Ghent (CRIG) , Ghent , Belgium.
Geert
G
Braems
b Department of Medical Oncology and Department of Gynaecology , Ghent University Hospital , Ghent , Belgium.|d Cancer Research Institute Ghent (CRIG) , Ghent , Belgium.
Véronique
V
Cocquyt
b Department of Medical Oncology and Department of Gynaecology , Ghent University Hospital , Ghent , Belgium.|d Cancer Research Institute Ghent (CRIG) , Ghent , Belgium.
Hannelore
H
Denys
a Laboratory of Experimental Cancer Research , Department of Radiation Oncology and Experimental Cancer Research, Ghent University , Ghent , Belgium.|d Cancer Research Institute Ghent (CRIG) , Ghent , Belgium.
An
A
Hendrix
D000273:Adipose Tissue / Q000473:pathology*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D000067128:Extracellular Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D020543:Proteome / Q000378:metabolism*; D014461:Ultracentrifugation
D020543:Proteome
aggregation; breast cancer; characterization; exosomes; function; isolation; proliferation