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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190006	1/2	Homo sapiens	MDAMB231	DG (d)(U)C	Altei, Wanessa F.	2020	100%
Study summary Full title Inhibition of αvβ3 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles All authors Wanessa F Altei, Bianca C Pachane, Patty K Dos Santos, Lígia N M Ribeiro, Bong Hwan Sung, Alissa M Weaver, Heloisa S Selistre-de-Araújo Journal Cell Commun Signal Abstract Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from (show more...)Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. Methods: To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. Results: We find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. Conclusion: In summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs. (hide) EV-METRIC 100% (99th 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 Cell culture supernatant Sample origin immortalized Focus vesicles exosome 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 Density gradient (Differential) (ultra)centrifugation Protein markers EV: Flotillin1/ CD63/ Alix/ integrin-alpha5/ integrin-alpha2/ integrin-alphaV/ integrin-beta1/ integrin-beta3/ FN1/ COL1 non-EV: Calnexin Proteomics no EV density (g/ml) 1.11 Show all info Study aim Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Cell viability (%) 95 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) 4 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Bottom-up Rotor type Type 40 Speed (g) 100000 Duration (min) 18 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: duration (min) 180 Pelleting: rotor type TLA-100.3 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 3 Pelleting-wash: duration (min) 180 Pelleting-wash: speed (g) TLA-100.3 Density cushion Density medium EV-subtype Distinction between multiple subtypes Used subtypes Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Flotillin1/ CD63/ Alix/ integrin-alpha5/ integrin-alpha2/ integrin-alphaV/ integrin-beta1/ integrin-beta3/ FN1/ COL1 Not detected EV-associated proteins Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 107 EV concentration Yes Particle yield Yes, as number of particles per million cells 5.50E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm)

	EV190006	2/2	Homo sapiens	MDAMB231	(d)(U)C	Altei, Wanessa F.	2020	78%
Study summary Full title Inhibition of αvβ3 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles All authors Wanessa F Altei, Bianca C Pachane, Patty K Dos Santos, Lígia N M Ribeiro, Bong Hwan Sung, Alissa M Weaver, Heloisa S Selistre-de-Araújo Journal Cell Commun Signal Abstract Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from (show more...)Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. Methods: To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. Results: We find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. Conclusion: In summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs. (hide) EV-METRIC 78% (97th 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 Cell culture supernatant Sample origin immortalized Focus vesicles (shedding) microvesicle 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 (Differential) (ultra)centrifugation Protein markers EV: Flotillin1/ CD63/ Alix/ integrin-alpha5/ integrin-alpha2/ integrin-alphaV/ integrin-beta1/ integrin-beta3/ FN1/ COL1 non-EV: Calnexin Proteomics no EV density (g/ml) Show all info Study aim Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Cell viability (%) 95 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 Pelleting performed Yes Pelleting: time(min) 4 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 10000 Density gradient Only used for validation of main results Yes Type Number of initial discontinuous layers Lowest density fraction Highest density fraction Total gradient volume, incl. sample (mL) Sample volume (mL) Orientation Rotor type Speed (g) Duration (min) Fraction volume (mL) Fraction processing Pelleting: volume per fraction Pelleting: duration (min) Pelleting: rotor type Pelleting: speed (g) Pelleting-wash: volume per pellet (mL) Pelleting-wash: duration (min) Pelleting-wash: speed (g) Density cushion Density medium EV-subtype Distinction between multiple subtypes Used subtypes Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Flotillin1/ integrin-alpha5/ integrin-beta1 Not detected EV-associated proteins CD63/ Alix/ FN1/ COL1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 206 EV concentration Yes Particle yield Yes, as number of particles per million cells 9.00E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm)

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EV-TRACK ID	EV190006
species	Homo sapiens
sample type	Cell culture
cell type	MDAMB231
condition	immortalized
separation protocol	Density gradient dUC	dUC
vesicle related term	exosome	(shedding) microvesicle
Exp. nr.	1	2
EV-METRIC %	100	78