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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210035	2/2	Homo sapiens	A-431	(d)(U)C DG Filtration	van Dommelen, Susan M	2016	78%
Study summary Full title Cetuximab treatment alters the content of extracellular vesicles released from tumor cells All authors Susan M van Dommelen, Roy van der Meel, Wouter W van Solinge, Maria Coimbra, Pieter Vader, Raymond M Schiffelers Journal Nanomedicine Abstract Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their co (show more...)Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their content reflects the parental cell status. This study aimed to examine whether tumor cell derived EVs mirrored the cellular changes caused by treatment with cetuximab, a therapeutic antibody that blocks activation of EGF receptor (EGFR). Materials & methods: A-431 cells were treated with cetuximab for 48 h. EVs were isolated using differential centrifugation and protein content was analyzed using western blotting. Results: EV levels of EGFR and phospho-EGFR were reduced after cetuximab treatment, reflecting similar changes in the parental cells. In addition, cetuximab was found associated with EVs. Conclusion: EVs could serve as biomarkers to monitor cetuximab treatment. Association of cetuximab with EVs might influence its behavior. Keywords: EGFR; biomarker; cancer therapy; cetuximab; diagnostics; exosomes; extracellular vesicles. (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 Cetuximab 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 Density gradient Filtration Protein markers EV: TSG101/ Alix/ CD9/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ Cetuximab non-EV: Lamin-A/ Lamin-C/ ATP5-A/ Tom20 Proteomics no EV density (g/ml) 1.10-1.21 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A-431 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type JA-30.50 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type JA-30.50 Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0.4 Highest density fraction 2.5 Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Top-down Rotor type SW 40 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 4 Pelleting: duration (min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ Cetuximab/ TSG101/ Alix Not detected contaminants Lamin-A/ Lamin-C/ ATP5-A/ Tom20 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV210035	1/2	Homo sapiens	A-431	(d)(U)C Filtration	van Dommelen, Susan M	2016	56%
Study summary Full title Cetuximab treatment alters the content of extracellular vesicles released from tumor cells All authors Susan M van Dommelen, Roy van der Meel, Wouter W van Solinge, Maria Coimbra, Pieter Vader, Raymond M Schiffelers Journal Nanomedicine Abstract Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their co (show more...)Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their content reflects the parental cell status. This study aimed to examine whether tumor cell derived EVs mirrored the cellular changes caused by treatment with cetuximab, a therapeutic antibody that blocks activation of EGF receptor (EGFR). Materials & methods: A-431 cells were treated with cetuximab for 48 h. EVs were isolated using differential centrifugation and protein content was analyzed using western blotting. Results: EV levels of EGFR and phospho-EGFR were reduced after cetuximab treatment, reflecting similar changes in the parental cells. In addition, cetuximab was found associated with EVs. Conclusion: EVs could serve as biomarkers to monitor cetuximab treatment. Association of cetuximab with EVs might influence its behavior. Keywords: EGFR; biomarker; cancer therapy; cetuximab; diagnostics; exosomes; extracellular vesicles. (hide) EV-METRIC 56% (90th 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 Filtration Protein markers EV: TSG101/ Alix/ Cetuximab/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ CD9 non-EV: Lamin-A/ Lamin-C/ ATP5-A/ Tom20 Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A-431 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type JA-30.50 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type JA-30.50 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ TSG101/ Alix Not detected EV-associated proteins Cetuximab Not detected contaminants Lamin-A/ Lamin-C/ ATP5-A/ Tom20 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

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EV-TRACK ID	EV210035
species	Homo sapiens
sample type	Cell culture
cell type	A-431
condition	Cetuximab	Control condition
separation protocol	dUC Density gradient Filtration	dUC Filtration
Exp. nr.	2	1
EV-METRIC %	78	56