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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220155	1/3	Homo sapiens	MDAMB231	Exospin	Campos A	2018	38%
Study summary Full title Caveolin-1-containing extracellular vesicles transport adhesion proteins and promote malignancy in breast cancer cell lines. All authors Campos A, Salomon C, Bustos R, Díaz J, Martínez S, Silva V, Reyes C, Díaz-Valdivia N, Varas-Godoy M, Lobos-González L, Quest AF Journal Nanomedicine (Lond) Abstract Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-relate (show more...)Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths in women worldwide, whereby mortality is largely attributable to the development of distant metastasis. Caveolin-1 (CAV1) is a multifunctional membrane protein that is typically upregulated in the final stages of cancer and promotes migration and invasion of tumor cells. Elevated levels of CAV1 have been detected in extracellular vesicles (EVs) from advanced cancer patients. EVs are lipid enclosed vesicular structures that contain bioactive proteins, DNA and RNAs, which can be transferred to other cells and promote metastasis. Therefore, we hypothesized that CAV1 containing EVs released from breast cancer cells may enhance migration and invasion of recipient cells. EVs were purified from conditioned media of MDA-MB-231 wild-type (WT), MDA-MB-231 (shCAV1/ possessing the plasmid pLKO.1 encoding a 'small hairpin' directed against CAV1) and MDA-MB-231 (shC) short hairpin control cells. Nanoparticle tracking analysis revealed an average particle size of 40-350 nm for all preparations. As anticipated, CAV1 was detected in MDA-MB-231 WT and shC EVs, but not in MDA-MB-231 (shCAV1) EVs. Mass spectrometry analysis revealed the presence of specific cell adhesion-related proteins, such as Cyr61, tenascin (TNC) and S100A9 only in WT and shC, but not in shCAV1 EVs. Importantly, EVs containing CAV1 promoted migration and invasion of cells lacking CAV1. We conclude that the presence of CAV1 in EVs from metastatic breast cancer cells is associated with enhanced migration and invasiveness of recipient cells in vitro, suggesting that intercellular communication promoted by EVs containing CAV1 will likely favor metastasis in vivo. (hide) EV-METRIC 38% (79th 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 Control condition 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 Commercial method Protein markers EV: CD9/ Alix/ TSG101/ CAV1/ beta-actin non-EV: Calnexin Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Commercial kit Exospin Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins CAV1/ CD9/ Alix/ TSG101 Not detected EV-associated proteins Beta-actin Not detected contaminants Calnexin Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA EM Image type Wide-field Report size (nm) 50-100

	EV220155	2/3	Homo sapiens	MDAMB231	Exospin	Campos A	2018	38%
Study summary Full title Caveolin-1-containing extracellular vesicles transport adhesion proteins and promote malignancy in breast cancer cell lines. All authors Campos A, Salomon C, Bustos R, Díaz J, Martínez S, Silva V, Reyes C, Díaz-Valdivia N, Varas-Godoy M, Lobos-González L, Quest AF Journal Nanomedicine (Lond) Abstract Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-relate (show more...)Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths in women worldwide, whereby mortality is largely attributable to the development of distant metastasis. Caveolin-1 (CAV1) is a multifunctional membrane protein that is typically upregulated in the final stages of cancer and promotes migration and invasion of tumor cells. Elevated levels of CAV1 have been detected in extracellular vesicles (EVs) from advanced cancer patients. EVs are lipid enclosed vesicular structures that contain bioactive proteins, DNA and RNAs, which can be transferred to other cells and promote metastasis. Therefore, we hypothesized that CAV1 containing EVs released from breast cancer cells may enhance migration and invasion of recipient cells. EVs were purified from conditioned media of MDA-MB-231 wild-type (WT), MDA-MB-231 (shCAV1/ possessing the plasmid pLKO.1 encoding a 'small hairpin' directed against CAV1) and MDA-MB-231 (shC) short hairpin control cells. Nanoparticle tracking analysis revealed an average particle size of 40-350 nm for all preparations. As anticipated, CAV1 was detected in MDA-MB-231 WT and shC EVs, but not in MDA-MB-231 (shCAV1) EVs. Mass spectrometry analysis revealed the presence of specific cell adhesion-related proteins, such as Cyr61, tenascin (TNC) and S100A9 only in WT and shC, but not in shCAV1 EVs. Importantly, EVs containing CAV1 promoted migration and invasion of cells lacking CAV1. We conclude that the presence of CAV1 in EVs from metastatic breast cancer cells is associated with enhanced migration and invasiveness of recipient cells in vitro, suggesting that intercellular communication promoted by EVs containing CAV1 will likely favor metastasis in vivo. (hide) EV-METRIC 38% (79th 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 shC 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 Commercial method Protein markers EV: CD9/ Alix/ TSG101/ CAV1/ beta-actin non-EV: Calnexin Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Commercial kit Exospin Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins CAV1/ CD9/ Alix/ TSG101 Not detected EV-associated proteins Beta-actin Not detected contaminants Calnexin Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA EM Image type Wide-field Report size (nm) 50-100

	EV220155	3/3	Homo sapiens	MDAMB231	Exospin	Campos A	2018	38%
Study summary Full title Caveolin-1-containing extracellular vesicles transport adhesion proteins and promote malignancy in breast cancer cell lines. All authors Campos A, Salomon C, Bustos R, Díaz J, Martínez S, Silva V, Reyes C, Díaz-Valdivia N, Varas-Godoy M, Lobos-González L, Quest AF Journal Nanomedicine (Lond) Abstract Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-relate (show more...)Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths in women worldwide, whereby mortality is largely attributable to the development of distant metastasis. Caveolin-1 (CAV1) is a multifunctional membrane protein that is typically upregulated in the final stages of cancer and promotes migration and invasion of tumor cells. Elevated levels of CAV1 have been detected in extracellular vesicles (EVs) from advanced cancer patients. EVs are lipid enclosed vesicular structures that contain bioactive proteins, DNA and RNAs, which can be transferred to other cells and promote metastasis. Therefore, we hypothesized that CAV1 containing EVs released from breast cancer cells may enhance migration and invasion of recipient cells. EVs were purified from conditioned media of MDA-MB-231 wild-type (WT), MDA-MB-231 (shCAV1/ possessing the plasmid pLKO.1 encoding a 'small hairpin' directed against CAV1) and MDA-MB-231 (shC) short hairpin control cells. Nanoparticle tracking analysis revealed an average particle size of 40-350 nm for all preparations. As anticipated, CAV1 was detected in MDA-MB-231 WT and shC EVs, but not in MDA-MB-231 (shCAV1) EVs. Mass spectrometry analysis revealed the presence of specific cell adhesion-related proteins, such as Cyr61, tenascin (TNC) and S100A9 only in WT and shC, but not in shCAV1 EVs. Importantly, EVs containing CAV1 promoted migration and invasion of cells lacking CAV1. We conclude that the presence of CAV1 in EVs from metastatic breast cancer cells is associated with enhanced migration and invasiveness of recipient cells in vitro, suggesting that intercellular communication promoted by EVs containing CAV1 will likely favor metastasis in vivo. (hide) EV-METRIC 38% (79th 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 shCAV1 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 Commercial method Protein markers EV: CD9/ Alix/ TSG101/ CAV1/ beta-actin non-EV: Calnexin Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Commercial kit Exospin Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins CD9/ Alix/ TSG101 Not detected EV-associated proteins Beta-actin/ CAV1 Not detected contaminants Calnexin Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA EM Image type Wide-field Report size (nm) 50-100

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EV-TRACK ID	EV220155
species	Homo sapiens
sample type	Cell culture
cell type	MDAMB231
condition	Control condition	shC	shCAV1
separation protocol	Exospin	Exospin	Exospin
Exp. nr.	1	2	3
EV-METRIC %	38	38	38