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

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.

SUBMITTER: Jeurissen S

PROVIDER: S-EPMC5351718 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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

Jeurissen Sarah S Vergauwen Glenn G Van Deun Jan J Lapeire Lore L Depoorter Victoria V Miinalainen Ilkka I Sormunen Raija R Van den Broecke Rudy R Braems Geert G Cocquyt Véronique V Denys Hannelore H Hendrix An A

Cell adhesion & migration 20170201 2

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 ...[more]

PMID: 28146372

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Project description:BackgroundBrown adipose tissue (BAT) is considered as a primary location of adaptive thermogenesis and the thermogenic activities of brown adipocytes are also connected to generating heat and counteracting obesity. Recent studies revealed that BAT could secrete certain batokines-like factors especially small extracellular vesicles (sEVs), which contributed to the systemic consequences of BAT activities. As a newly emerging class of mediators, some long non-coding RNAs (lncRNAs) have exhibited metabolic regulatory effects in adipocyte development. However, besides the well-studied lncRNAs, the lncRNAs carried by sEVs derived from brown adipose tissue (sEV-BAT) have not been identified yet. RESULTS: In this study, we demonstrated that sEV-BAT could induce beige adipocyte differentiation both in ASCs and 3T3-L1 cells, while sEV-WAT had no corresponding effects. The lncRNA microarray assay on sEV-WAT and sEV-BAT revealed a total of 563 types of known lncRNAs were identified to be differentially expressed, among which 232 lncRNAs were upregulated and 331 lncRNAs were downregulated in sEV-BAT. Three novel candidates (AK029592, humanlincRNA1030 and ENSMUST00000152284) were selected for further validation. LncRNA-mRNA network analysis revealed candidate lncRNAs were largely embedded in cellular metabolic pathways. During adipogenic and thermogenic phenotype differentiation in ASCs and 3T3-L1 cells, only the expressions of AK029592 were upregulated. The three lncRNAs were all relatively enriched in brown adipose tissues and brown adipocytes. In different adipocytes, sEV and adipose tissue, the expression of AK029592 and ENSMUST00000152284 were remarkably decreased in obese mice compared to lean mice, while obesity state could not change the expression of humanlincRNA1030.ConclusionCollectively, our profiling study provided a comprehensive catalog for the study of lncRNAs specifically carried by sEV-BAT and indicated the potential regulatory role of certain sEV-BAT lncRNAs in thermogenesis.

Dataset Information

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

Publications

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

Similar Datasets

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