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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190012	1/3	Mus musculus	4T1	DC (d)(U)C Filtration qEV UF	Vu LT	2019	38%
Study summary Full title Tumor-secreted extracellular vesicles promote the activation of cancer-associated fibroblasts via the transfer of microRNA-125b. All authors Vu LT, Peng B, Zhang DX, Ma V, Mathey-Andrews CA, Lam CK, Kiomourtzis T, Jin J, McReynolds L, Huang L, Grimson A, Cho WC, Lieberman J, Le MT. Journal J Extracell Vesicles Abstract Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions (show more...)Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions with other cells in the tumour microenvironment. To identify host cells that naturally take up EVs from tumour cells, we created breast cancer cell lines secreting fluorescent EVs. These fluorescent EVs are taken up most robustly by fibroblasts within the tumour microenvironment. RNA sequencing indicated that miR-125b is one of the most abundant microRNAs secreted by mouse triple-negative breast cancer 4T1 and 4TO7 cells. Treatment with 4T1 EVs leads to an increase in fibroblast activation in isogenic 4TO7 tumours, which is reversed by blocking miR-125b in 4T1 EVs; hence, miR-125b delivery by EVs is responsible for fibroblast activation in mouse tumour models. miR-125b is also secreted by human breast cancer cells and the uptake of EVs from these cells significantly increases cellular levels of miR-125b and expression of multiple cancer-associated fibroblast markers in resident fibroblasts. Overexpression of miR-125b in both mouse and human fibroblasts leads to an activated phenotype similar to the knockdown of established miR-125b target mRNAs. These data indicate that miR-125b is transferred through EVs from breast cancer cells to normal fibroblasts within the tumour microenvironment and contributes to their development into cancer-associated fibroblasts. (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 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 DC (d)(U)C Filtration qEV UF Protein markers EV: TSG101/ Alix/ CD63 non-EV: Beta-Actin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 4T1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 90 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 No Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) Not spec Membrane type Regenerated cellulose Commercial kit qEV Density cushion Density medium Sucrose Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins TSG101/ Alix Not detected contaminants Beta-Actin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD63 Detected EV-associated proteins CD63 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR; RNA sequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 50 U 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 Wide-field

	EV190012	3/3	Homo sapiens	4T07	(d)(U)C Filtration	Vu LT	2019	17%
Study summary Full title Tumor-secreted extracellular vesicles promote the activation of cancer-associated fibroblasts via the transfer of microRNA-125b. All authors Vu LT, Peng B, Zhang DX, Ma V, Mathey-Andrews CA, Lam CK, Kiomourtzis T, Jin J, McReynolds L, Huang L, Grimson A, Cho WC, Lieberman J, Le MT. Journal J Extracell Vesicles Abstract Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions (show more...)Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions with other cells in the tumour microenvironment. To identify host cells that naturally take up EVs from tumour cells, we created breast cancer cell lines secreting fluorescent EVs. These fluorescent EVs are taken up most robustly by fibroblasts within the tumour microenvironment. RNA sequencing indicated that miR-125b is one of the most abundant microRNAs secreted by mouse triple-negative breast cancer 4T1 and 4TO7 cells. Treatment with 4T1 EVs leads to an increase in fibroblast activation in isogenic 4TO7 tumours, which is reversed by blocking miR-125b in 4T1 EVs; hence, miR-125b delivery by EVs is responsible for fibroblast activation in mouse tumour models. miR-125b is also secreted by human breast cancer cells and the uptake of EVs from these cells significantly increases cellular levels of miR-125b and expression of multiple cancer-associated fibroblast markers in resident fibroblasts. Overexpression of miR-125b in both mouse and human fibroblasts leads to an activated phenotype similar to the knockdown of established miR-125b target mRNAs. These data indicate that miR-125b is transferred through EVs from breast cancer cells to normal fibroblasts within the tumour microenvironment and contributes to their development into cancer-associated fibroblasts. (hide) EV-METRIC 17% (54th 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 Filtration Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 4T07 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 90 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) 90 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 25 Wash: time (min) 90 Wash: Rotor Type SW 32 Ti Wash: speed (g) 110000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis None Protein Concentration Method BCA Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA-sequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 50 U Characterization: Lipid analysis No

	EV190012	2/3	Homo sapiens	CA1a	DC (d)(U)C Filtration qEV UF	Vu LT	2019	0%
Study summary Full title Tumor-secreted extracellular vesicles promote the activation of cancer-associated fibroblasts via the transfer of microRNA-125b. All authors Vu LT, Peng B, Zhang DX, Ma V, Mathey-Andrews CA, Lam CK, Kiomourtzis T, Jin J, McReynolds L, Huang L, Grimson A, Cho WC, Lieberman J, Le MT. Journal J Extracell Vesicles Abstract Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions (show more...)Tumour cells release large quantities of extracellular vesicles (EVs) to mediate their interactions with other cells in the tumour microenvironment. To identify host cells that naturally take up EVs from tumour cells, we created breast cancer cell lines secreting fluorescent EVs. These fluorescent EVs are taken up most robustly by fibroblasts within the tumour microenvironment. RNA sequencing indicated that miR-125b is one of the most abundant microRNAs secreted by mouse triple-negative breast cancer 4T1 and 4TO7 cells. Treatment with 4T1 EVs leads to an increase in fibroblast activation in isogenic 4TO7 tumours, which is reversed by blocking miR-125b in 4T1 EVs; hence, miR-125b delivery by EVs is responsible for fibroblast activation in mouse tumour models. miR-125b is also secreted by human breast cancer cells and the uptake of EVs from these cells significantly increases cellular levels of miR-125b and expression of multiple cancer-associated fibroblast markers in resident fibroblasts. Overexpression of miR-125b in both mouse and human fibroblasts leads to an activated phenotype similar to the knockdown of established miR-125b target mRNAs. These data indicate that miR-125b is transferred through EVs from breast cancer cells to normal fibroblasts within the tumour microenvironment and contributes to their development into cancer-associated fibroblasts. (hide) EV-METRIC 0% (median: 14% 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 DC (d)(U)C Filtration qEV UF Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CA1a EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 90 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 No Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) Not spec Membrane type Regenerated cellulose Commercial kit qEV Density cushion Density medium Sucrose Other Name other separation method qEV Characterization: Protein analysis None Protein Concentration Method BCA Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 50 U Characterization: Lipid analysis No

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EV-TRACK ID	EV190012
species	Mus musculus	Homo sapiens	Homo sapiens
sample type	Cell culture	Cell culture	Cell culture
cell type	4T1	4T07	CA1a
condition	Control condition	Control condition	Control condition
separation protocol	DC (d)(U)C Filtration qEV UF	(d)(U)C Filtration	DC (d)(U)C Filtration qEV UF
Exp. nr.	1	3	2
EV-METRIC %	38	17	0