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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200110	1/3	Homo sapiens	NCI-H1975	(d)(U)C DG	Van Hoof R	2022	89%
Study summary Full title Intravesicular Genomic DNA Enriched by Size Exclusion Chromatography Can Enhance Lung Cancer Oncogene Mutation Detection Sensitivity. All authors Van Hoof R, Deville S, Hollanders K, Berckmans P, Wagner P, Hooyberghs J, Nelissen I Journal Int J Mol Sci Abstract Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RN (show more...)Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (</ 200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor () gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (</ 35-100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the T790M mutation in the sEVs' lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows. (hide) EV-METRIC 89% (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 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 Density gradient Protein markers EV: CD81/ HSP70/ CD63/ CD9 non-EV: Calnexin/ Ribosomal protein S6 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells NCI-H1975 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 4.7 Sample volume (mL) 0.3 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 366613 Duration (min) 960 Fraction volume (mL) 0.48 Fraction processing None Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins HSP70/ CD81 Detected contaminants Ribosomal protein S6 Not detected contaminants Calnexin Flow cytometry Type of Flow cytometry BD Influx flow cytometer Hardware adaptation to ~100nm EV's EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012). Calibration bead size 0.1/ 0.2 Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)/ Other Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type Other/ RNase A/T1 mix RNAse concentration RNase A: 0.02 mg/ml - RNase T1: 50 U/ml Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 202 EV concentration Yes Particle yield as number of particles per mililiter in the final EV fraction (500 l)/ other:: 5.00E+10 Particle analysis: flow cytometry Flow cytometer type BD Influx flow cytometer Hardware adjustment EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012). Calibration bead size 1 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200110	2/3	Homo sapiens	NCI-H1975	(d)(U)C UF qEV DG	Van Hoof R	2022	88%
Study summary Full title Intravesicular Genomic DNA Enriched by Size Exclusion Chromatography Can Enhance Lung Cancer Oncogene Mutation Detection Sensitivity. All authors Van Hoof R, Deville S, Hollanders K, Berckmans P, Wagner P, Hooyberghs J, Nelissen I Journal Int J Mol Sci Abstract Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RN (show more...)Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (</ 200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor () gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (</ 35-100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the T790M mutation in the sEVs' lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows. (hide) EV-METRIC 88% (98th 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 Commercial method Density gradient Protein markers EV: CD81/ HSP70/ CD63/ CD9 non-EV: Calnexin/ Ribosomal protein S6 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells NCI-H1975 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 4.7 Sample volume (mL) 0.3 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 366613 Duration (min) 960 Fraction volume (mL) 0.48 Fraction processing None Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins HSP70/ CD81 Not detected contaminants Calnexin/ Ribosomal protein S6 Flow cytometry Type of Flow cytometry BD Influx flow cytometer Hardware adaptation to ~100nm EV's EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012). Calibration bead size 0.1/ 0.2 Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)/ Other Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type Other/ RNase A/T1 mix RNAse concentration RNase A: 0.02 mg/ml - RNase T1: 50 U/ml Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 186 EV concentration Yes Particle yield as number of particles per mililiter in the final EV fraction (500 l)/ other:: 1.70E+10 Particle analysis: flow cytometry Flow cytometer type BD Influx flow cytometer Hardware adjustment EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012). Calibration bead size 1 Report type Not Reported EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200110	3/3	Homo sapiens	NCI-H1975	(d)(U)C Other/ exoEasy Filtration DG	Van Hoof R	2022	88%
Study summary Full title Intravesicular Genomic DNA Enriched by Size Exclusion Chromatography Can Enhance Lung Cancer Oncogene Mutation Detection Sensitivity. All authors Van Hoof R, Deville S, Hollanders K, Berckmans P, Wagner P, Hooyberghs J, Nelissen I Journal Int J Mol Sci Abstract Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RN (show more...)Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (</ 200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor () gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (</ 35-100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the T790M mutation in the sEVs' lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows. (hide) EV-METRIC 88% (98th 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 Commercial method Filtration Density gradient Protein markers EV: CD81/ HSP70/ CD63/ CD9 non-EV: Calnexin/ Ribosomal protein S6 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells NCI-H1975 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 4.7 Sample volume (mL) 0.3 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 366613 Duration (min) 960 Fraction volume (mL) 0.48 Fraction processing None Filtration steps > 0.45 µm, 0.22µm or 0.2µm Commercial kit Other/ exoEasy Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins HSP70/ CD81 Not detected contaminants Calnexin/ Ribosomal protein S6 Flow cytometry Type of Flow cytometry BD Influx flow cytometer Hardware adaptation to ~100nm EV's EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012). Calibration bead size 0.1/ 0.2 Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)/ Other Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type Other/ RNase A/T1 mix RNAse concentration RNase A: 0.02 mg/ml - RNase T1: 50 U/ml Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 250 EV concentration Yes Particle yield as number of particles per mililiter in the final EV fraction (500 l)/ other:: 2.60E+11 Particle analysis: flow cytometry Flow cytometer type BD Influx flow cytometer Hardware adjustment EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012). Calibration bead size 1 Report type Not Reported EM EM-type Transmission-EM Image type Close-up, Wide-field

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