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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230033	4/9	Homo sapiens	HT29	(d)(U)C UF SEC (non-commercial)	Beaumont JEJ	2024	75%
Study summary Full title Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation. All authors Beaumont JEJ, Barbeau LMO, Ju J, Savelkouls KG, Bouwman FG, Zonneveld MI, Bronckaers A, Kampen KR, Keulers TGH, Rouschop KMA Journal J Extracell Vesicles Abstract Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells tha (show more...)Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status. (hide) EV-METRIC 75% (96th 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 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 (Differential) (ultra)centrifugation Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ CD81/ Syntenin-1/ TSG101 non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT29 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 16 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ CD81/ Syntenin-1 Not detected EV-associated proteins TSG101 Not detected contaminants GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 138.62 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV230033	5/9	Homo sapiens	HT29	(d)(U)C UF SEC (non-commercial)	Beaumont JEJ	2024	75%
Study summary Full title Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation. All authors Beaumont JEJ, Barbeau LMO, Ju J, Savelkouls KG, Bouwman FG, Zonneveld MI, Bronckaers A, Kampen KR, Keulers TGH, Rouschop KMA Journal J Extracell Vesicles Abstract Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells tha (show more...)Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status. (hide) EV-METRIC 75% (96th 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 Hypoxia (0.2%O2) 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 (Differential) (ultra)centrifugation Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ CD81/ Syntenin-1/ TSG101 non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT29 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 16 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ CD81/ Syntenin-1 Not detected EV-associated proteins TSG101 Not detected contaminants GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 146.34 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV230033	6/9	Homo sapiens	HT29	(d)(U)C UF SEC (non-commercial)	Beaumont JEJ	2024	75%
Study summary Full title Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation. All authors Beaumont JEJ, Barbeau LMO, Ju J, Savelkouls KG, Bouwman FG, Zonneveld MI, Bronckaers A, Kampen KR, Keulers TGH, Rouschop KMA Journal J Extracell Vesicles Abstract Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells tha (show more...)Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status. (hide) EV-METRIC 75% (96th 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 Hypoxia (<0.02%O2) 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 (Differential) (ultra)centrifugation Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ CD81/ Syntenin-1/ TSG101 non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT29 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 16 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ CD81/ Syntenin-1 Not detected EV-associated proteins TSG101 Not detected contaminants GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 145.58 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV230033	1/9	Homo sapiens	MDA-MB-231	(d)(U)C UF SEC (non-commercial)	Beaumont JEJ	2024	50%
Study summary Full title Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation. All authors Beaumont JEJ, Barbeau LMO, Ju J, Savelkouls KG, Bouwman FG, Zonneveld MI, Bronckaers A, Kampen KR, Keulers TGH, Rouschop KMA Journal J Extracell Vesicles Abstract Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells tha (show more...)Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status. (hide) EV-METRIC 50% (87th 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 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 (Differential) (ultra)centrifugation Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ CD81/ TSG101/ Syntenin-1 non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-231 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 16 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ CD81 Not detected EV-associated proteins TSG101/ Syntenin-1 Not detected contaminants GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 160.84 EV concentration Yes

	EV230033	2/9	Homo sapiens	MDA-MB-231	(d)(U)C UF SEC (non-commercial)	Beaumont JEJ	2024	50%
Study summary Full title Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation. All authors Beaumont JEJ, Barbeau LMO, Ju J, Savelkouls KG, Bouwman FG, Zonneveld MI, Bronckaers A, Kampen KR, Keulers TGH, Rouschop KMA Journal J Extracell Vesicles Abstract Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells tha (show more...)Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status. (hide) EV-METRIC 50% (87th 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 Hypoxia (0.2%O2) 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 (Differential) (ultra)centrifugation Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ CD81/ TSG101/ Syntenin-1 non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-231 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 16 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ CD81 Not detected EV-associated proteins TSG101/ Syntenin-1 Not detected contaminants GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 156.08 EV concentration Yes

	EV230033	3/9	Homo sapiens	MDA-MB-231	(d)(U)C UF SEC (non-commercial)	Beaumont JEJ	2024	50%
Study summary Full title Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation. All authors Beaumont JEJ, Barbeau LMO, Ju J, Savelkouls KG, Bouwman FG, Zonneveld MI, Bronckaers A, Kampen KR, Keulers TGH, Rouschop KMA Journal J Extracell Vesicles Abstract Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells tha (show more...)Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status. (hide) EV-METRIC 50% (87th 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 Hypoxia (<0.02%O2) 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 (Differential) (ultra)centrifugation Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ CD81/ TSG101/ Syntenin-1 non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-231 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 16 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ CD81 Not detected EV-associated proteins TSG101/ Syntenin-1 Not detected contaminants GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 157.16 EV concentration Yes

	EV230033	7/9	Homo sapiens	U87	(d)(U)C UF SEC (non-commercial)	Beaumont JEJ	2024	50%
Study summary Full title Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation. All authors Beaumont JEJ, Barbeau LMO, Ju J, Savelkouls KG, Bouwman FG, Zonneveld MI, Bronckaers A, Kampen KR, Keulers TGH, Rouschop KMA Journal J Extracell Vesicles Abstract Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells tha (show more...)Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status. (hide) EV-METRIC 50% (87th 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 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 (Differential) (ultra)centrifugation Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ CD81/ Syntenin-1/ TSG101 non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U87 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 16 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ CD81/ Syntenin-1 Not detected EV-associated proteins TSG101 Not detected contaminants GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 160.56 EV concentration Yes

	EV230033	8/9	Homo sapiens	U87	(d)(U)C UF SEC (non-commercial)	Beaumont JEJ	2024	50%
Study summary Full title Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation. All authors Beaumont JEJ, Barbeau LMO, Ju J, Savelkouls KG, Bouwman FG, Zonneveld MI, Bronckaers A, Kampen KR, Keulers TGH, Rouschop KMA Journal J Extracell Vesicles Abstract Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells tha (show more...)Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status. (hide) EV-METRIC 50% (87th 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 Hypoxia (0.2%O2) 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 (Differential) (ultra)centrifugation Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ CD81/ Syntenin-1/ TSG101 non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U87 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 16 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ CD81/ Syntenin-1 Not detected EV-associated proteins TSG101 Not detected contaminants GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 167.54 EV concentration Yes

	EV230033	9/9	Homo sapiens	U87	(d)(U)C UF SEC (non-commercial)	Beaumont JEJ	2024	50%
Study summary Full title Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation. All authors Beaumont JEJ, Barbeau LMO, Ju J, Savelkouls KG, Bouwman FG, Zonneveld MI, Bronckaers A, Kampen KR, Keulers TGH, Rouschop KMA Journal J Extracell Vesicles Abstract Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells tha (show more...)Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status. (hide) EV-METRIC 50% (87th 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 Hypoxia (<0.02%O2) 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 (Differential) (ultra)centrifugation Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ CD81/ Syntenin-1/ TSG101 non-EV: GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U87 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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 No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 16 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ CD81/ Syntenin-1 Not detected EV-associated proteins TSG101 Not detected contaminants GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 164 EV concentration Yes

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EV-TRACK ID	EV230033
species	Homo sapiens
sample type	Cell culture
cell type	HT29	HT29	HT29	MDA-MB-231	MDA-MB-231	MDA-MB-231	U87	U87	U87
condition	Control condition	Hypoxia (0.2%O2)	Hypoxia (<0.02%O2)	Control condition	Hypoxia (0.2%O2)	Hypoxia (<0.02%O2)	Control condition	Hypoxia (0.2%O2)	Hypoxia (<0.02%O2)
separation protocol	dUC/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)
Exp. nr.	4	5	6	1	2	3	7	8	9
EV-METRIC %	75	75	75	50	50	50	50	50	50

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