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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130151	4/4	Homo sapiens	NAY	(d)(U)C Filtration SEC UF	Redzic JS	2013	38%
Study summary Full title Extracellular vesicles secreted from cancer cell lines stimulate secretion of MMP-9, IL-6, TGF-beta1 and EMMPRIN All authors Redzic JS, Kendrick AA, Bahmed K, Dahl KD, Pearson CG, Robinson WA, Robinson SE, Graner MW, Eisenmesser EZ Journal PLoS One Abstract Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell (show more...)Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-?1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles. (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 NAY Focus vesicles extracellular vesicles 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 (d)(U)C Filtration SEC UF Protein markers EV: EMMPRIN non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins EMMPRIN ELISA Antibody details provided? No Detected EV-associated proteins EMMPRIN Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field Report size (nm) Not reported

	EV130151	3/4	Homo sapiens	Serum	(d)(U)C Filtration IAF	Redzic JS	2013	13%
Study summary Full title Extracellular vesicles secreted from cancer cell lines stimulate secretion of MMP-9, IL-6, TGF-beta1 and EMMPRIN All authors Redzic JS, Kendrick AA, Bahmed K, Dahl KD, Pearson CG, Robinson WA, Robinson SE, Graner MW, Eisenmesser EZ Journal PLoS One Abstract Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell (show more...)Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-?1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles. (hide) EV-METRIC 13% (47th 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 Serum Sample origin NAY Focus vesicles extracellular vesicles 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 (d)(U)C Filtration IAF Protein markers EV: EMMPRIN non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) EMMPRIN Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins EMMPRIN ELISA Antibody details provided? No Detected EV-associated proteins EMMPRIN Characterization: Particle analysis None

	EV130151	1/4	Homo sapiens	Ascites	(d)(U)C Filtration IAF	Redzic JS	2013	0%
Study summary Full title Extracellular vesicles secreted from cancer cell lines stimulate secretion of MMP-9, IL-6, TGF-beta1 and EMMPRIN All authors Redzic JS, Kendrick AA, Bahmed K, Dahl KD, Pearson CG, Robinson WA, Robinson SE, Graner MW, Eisenmesser EZ Journal PLoS One Abstract Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell (show more...)Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-?1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles. (hide) EV-METRIC 0% (median: 13% 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 Ascites Sample origin NAY Focus vesicles extracellular vesicles 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 (d)(U)C Filtration IAF Protein markers EV: EMMPRIN non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) EMMPRIN Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins EMMPRIN ELISA Antibody details provided? No Detected EV-associated proteins EMMPRIN Characterization: Particle analysis None

	EV130151	2/4	Homo sapiens	Blood plasma	(d)(U)C Filtration IAF	Redzic JS	2013	0%
Study summary Full title Extracellular vesicles secreted from cancer cell lines stimulate secretion of MMP-9, IL-6, TGF-beta1 and EMMPRIN All authors Redzic JS, Kendrick AA, Bahmed K, Dahl KD, Pearson CG, Robinson WA, Robinson SE, Graner MW, Eisenmesser EZ Journal PLoS One Abstract Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell (show more...)Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-?1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles. (hide) EV-METRIC 0% (median: 22% 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 Blood plasma Sample origin NAY Focus vesicles extracellular vesicles 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 (d)(U)C Filtration IAF Protein markers EV: EMMPRIN non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) EMMPRIN Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins EMMPRIN ELISA Antibody details provided? No Detected EV-associated proteins EMMPRIN Characterization: Particle analysis None

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EV-TRACK ID	EV130151
species	Homo sapiens
sample type	Cell culture	Serum	Ascites	Blood plasma
cell type	NAY	NA	NA	NA
medium	EV Depleted
condition	NAY	NAY	NAY	NAY
separation protocol	(d)(U)C Filtration SEC UF	(d)(U)C Filtration IAF	(d)(U)C Filtration IAF	(d)(U)C Filtration IAF
Exp. nr.	4	3	1	2
EV-METRIC %	38	13	0	0