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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170004	1/1	Homo sapiens	Adipose tissue	DG (d)(U)C Filtration	Jeurissen S	2017	88%
Study summary Full title The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue. All authors Jeurissen S, Vergauwen G, Van Deun J, Lapeire L, Depoorter V, Miinalainen I, Sormunen R, Van den Broecke R, Braems G, Cocquyt V, Denys H, Hendrix A Journal Cell Adh Migr Abstract Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to (show more...)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. (hide) EV-METRIC 88% (91st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Adipose tissue Sample origin breast cancer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Filtration Adj. k-factor 138.6 (pelleting) / 138.6 (washing) Protein markers EV: CD63/ HSP70/ FABP4/ Flotillin-1/ CD9 non-EV: Prohibitin/ GM130/ Calreticulin Proteomics no Show all info Study aim Function, Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Adipose tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 138.6 Wash: time (min) 120 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 138.6 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 1 Orientation Top-down (sample migrates downwards) Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 297.9 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins CD9, Flotillin-1, HSP70, FABP4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 116 EV concentration Yes Particle yield 2.2 10E09 EM EM-type Immune-EM EM protein CD63 Image type Close-up, Wide-field Report size (nm) 20-200

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EV-TRACK ID	EV170004
species	Homo sapiens
sample type	Adipose tissue
condition	breast cancer
separation protocol	DG (d)(U)C Filtration
Exp. nr.	1
EV-METRIC %	88