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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200098	1/3	Homo sapiens	PNT1A	(d)(U)C qEV UF	Millan, Christopher	2021	78%
Study summary Full title Extracellular Vesicles from 3D Engineered Microtissues Harbor Disease-Related Cargo Absent in EVs from 2D Cultures All authors Christopher Millan, Lukas Prause, Queralt Vallmajo-Martin, Natalie Hensky, Daniel Eberli Journal Advanced Healthcare Materials Abstract Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce cl (show more...)Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce clinically relevant cancer phenotypes in vitro. However, their effect on molecular cargo of secreted extracellular vesicles (EVs) has not yet been investigated. Here, the impact of hydrogel-based 3D engineered microtissues on EVs secreted by benign and malignant prostate cells is assessed. Compared to 2D cultures, yield of EVs per cell is significantly increased for cancer cells cultured in 3D. Whole transcriptome sequencing and proteomics of 2D-EV and 3D-EV samples reveal stark contrasts in molecular cargo. For one cell type in particular, LNCaP, enrichment is observed exclusively in 3D-EVs of GDF15, FASN, and TOP1, known drivers of prostate cancer progression. Using imaging flow cytometry in a novel approach to validate a putative EV biomarker, colocalization in single EVs of GDF15 with CD9, a universal EV marker, is demonstrated. Finally, in functional assays it is observed that only 3D-EVs, unlike 2D-EVs, confer increased invasiveness and chemoresistance to cells in 2D. Collectively, this study highlights the value of engineered 3D microtissue cultures for the study of bona fide EV cargoes and their potential to identify biomarkers that are not detectable in EVs secreted by cells cultured in standard 2D conditions. (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 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 (d)(U)C Commercial method UF Protein markers EV: TSG101/ Alix/ CD9 non-EV: Calnexin/ Argonaute2 Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PNT1A EV-harvesting Medium EV-depleted medium Preparation of EDS Ultrafiltration 100kDa cutoff Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 12 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 110,000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV 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 CD9/ TSG101/ Alix Not detected contaminants Calnexin/ Argonaute2 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type RNA sequencing;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 120

	EV200098	2/3	Homo sapiens	LNCaP	(d)(U)C qEV UF	Millan, Christopher	2021	78%
Study summary Full title Extracellular Vesicles from 3D Engineered Microtissues Harbor Disease-Related Cargo Absent in EVs from 2D Cultures All authors Christopher Millan, Lukas Prause, Queralt Vallmajo-Martin, Natalie Hensky, Daniel Eberli Journal Advanced Healthcare Materials Abstract Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce cl (show more...)Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce clinically relevant cancer phenotypes in vitro. However, their effect on molecular cargo of secreted extracellular vesicles (EVs) has not yet been investigated. Here, the impact of hydrogel-based 3D engineered microtissues on EVs secreted by benign and malignant prostate cells is assessed. Compared to 2D cultures, yield of EVs per cell is significantly increased for cancer cells cultured in 3D. Whole transcriptome sequencing and proteomics of 2D-EV and 3D-EV samples reveal stark contrasts in molecular cargo. For one cell type in particular, LNCaP, enrichment is observed exclusively in 3D-EVs of GDF15, FASN, and TOP1, known drivers of prostate cancer progression. Using imaging flow cytometry in a novel approach to validate a putative EV biomarker, colocalization in single EVs of GDF15 with CD9, a universal EV marker, is demonstrated. Finally, in functional assays it is observed that only 3D-EVs, unlike 2D-EVs, confer increased invasiveness and chemoresistance to cells in 2D. Collectively, this study highlights the value of engineered 3D microtissue cultures for the study of bona fide EV cargoes and their potential to identify biomarkers that are not detectable in EVs secreted by cells cultured in standard 2D conditions. (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 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 (d)(U)C Commercial method UF Protein markers EV: TSG101/ Alix/ GDF15/ CD9 non-EV: Calnexin/ Argonaute2 Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCaP EV-harvesting Medium EV-depleted medium Preparation of EDS Ultrafiltration 100kDa cutoff Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 12 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 110,000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV 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 CD9/ TSG101/ Alix Not detected contaminants Calnexin/ Argonaute2 Flow cytometry Antibody details provided? No Detected EV-associated proteins CD9/ GDF15 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type RNAsequencing;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Amnis ImageStreamX MkII Hardware adjustment 60x objective, 7um core diameter, low flow rate. Particles with high aspect ratio, negligible SSC, and high fluorescent were considered positively stained EVs Calibration bead size 1000 Report type Not Reported EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 120

	EV200098	3/3	Homo sapiens	PC3	(d)(U)C qEV UF	Millan, Christopher	2021	78%
Study summary Full title Extracellular Vesicles from 3D Engineered Microtissues Harbor Disease-Related Cargo Absent in EVs from 2D Cultures All authors Christopher Millan, Lukas Prause, Queralt Vallmajo-Martin, Natalie Hensky, Daniel Eberli Journal Advanced Healthcare Materials Abstract Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce cl (show more...)Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce clinically relevant cancer phenotypes in vitro. However, their effect on molecular cargo of secreted extracellular vesicles (EVs) has not yet been investigated. Here, the impact of hydrogel-based 3D engineered microtissues on EVs secreted by benign and malignant prostate cells is assessed. Compared to 2D cultures, yield of EVs per cell is significantly increased for cancer cells cultured in 3D. Whole transcriptome sequencing and proteomics of 2D-EV and 3D-EV samples reveal stark contrasts in molecular cargo. For one cell type in particular, LNCaP, enrichment is observed exclusively in 3D-EVs of GDF15, FASN, and TOP1, known drivers of prostate cancer progression. Using imaging flow cytometry in a novel approach to validate a putative EV biomarker, colocalization in single EVs of GDF15 with CD9, a universal EV marker, is demonstrated. Finally, in functional assays it is observed that only 3D-EVs, unlike 2D-EVs, confer increased invasiveness and chemoresistance to cells in 2D. Collectively, this study highlights the value of engineered 3D microtissue cultures for the study of bona fide EV cargoes and their potential to identify biomarkers that are not detectable in EVs secreted by cells cultured in standard 2D conditions. (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 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 (d)(U)C Commercial method UF Protein markers EV: Alix/ TSG101/ CD9 non-EV: Calnexin/ Argonaute2 Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium EV-depleted medium Preparation of EDS Ultrafiltration 100kDa cutoff Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 12 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 110,000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV 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 Alix/ CD9/ TSG101 Not detected contaminants Calnexin/ Argonaute2 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type RNAsequencing;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 120

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EV-TRACK ID	EV200098
species	Homo sapiens
sample type	Cell culture
cell type	PNT1A	LNCaP	PC3
condition	Control condition	Control condition	Control condition
separation protocol	(d)(U)C qEV UF	(d)(U)C qEV UF	(d)(U)C qEV UF
Exp. nr.	1	2	3
EV-METRIC %	78	78	78