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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210002	1/3	Mus musculus	dissociated tissues	(d)(U)C	Delcorte, Ophélie	2022	78%
Study summary Full title BRAF V600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment All authors Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux Journal Biomediscines Abstract Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide) EV-METRIC 78% (83rd 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 dissociated tissues 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 No extra separation steps Protein markers EV: Alix/ CD63/ Flotillin1/ CD9/ CD81 non-EV: Calnexin/ PDI Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type dissociated tissues Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 80 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 7 Wash: time (min) 90 Wash: Rotor Type Type 80 Ti Wash: speed (g) 150000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ Alix/ CD81 Not detected contaminants Calnexin/ PDI Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 0,01 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Per dissociated thyroid;Yes, other: 1,50E+09 EM EM-type Transmission-EM/ Scanning-EM Image type Close-up, Wide-field

	EV210002	2/3	Mus musculus	dissociated tissues	(d)(U)C	Delcorte, Ophélie	2022	67%
Study summary Full title BRAF V600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment All authors Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux Journal Biomediscines Abstract Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide) EV-METRIC 67% (33rd 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 dissociated tissues Sample origin BRAFV600E-induced tissue (Doxycyxline 2*24h) 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 No extra separation steps Protein markers EV: Alix/ CD63/ Flotillin1/ CD9/ CD81 non-EV: Calnexin/ PDI Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type dissociated tissues Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 80 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 7 Wash: time (min) 90 Wash: Rotor Type Type 80 Ti Wash: speed (g) 150000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ Alix/ CD81 Not detected contaminants Calnexin/ PDI Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 0,01 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Per dissociated thyroid;Yes, other: 3,30E+09 EM EM-type Transmission-EM Image type Close-up

	EV210002	3/3	Mus musculus	dissociated tissues	(d)(U)C	Delcorte, Ophélie	2022	67%
Study summary Full title BRAF V600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment All authors Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux Journal Biomediscines Abstract Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide) EV-METRIC 67% (33rd 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 dissociated tissues Sample origin BRAFV600E-induced tissue (Doxycyxline4*24h) 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 No extra separation steps Protein markers EV: Alix/ CD63/ Flotillin1/ CD9/ CD81 non-EV: Calnexin/ PDI Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type dissociated tissues Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 80 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 7 Wash: time (min) 90 Wash: Rotor Type Type 80 Ti Wash: speed (g) 150000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ Alix/ CD9/ CD63/ CD81 Not detected contaminants Calnexin/ PDI Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 0,01 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Per dissociated thyroid;Yes, other: 1,20E+10 EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV210002
species	Mus musculus
sample type	dissociated tissues
condition	Control condition	BRAFV600E-induced tissue (Doxycyxline 2*24h)	BRAFV600E-induced tissue (Doxycyxline4*24h)
separation protocol	dUC No extra separation steps	dUC No extra separation steps	dUC No extra separation steps
Exp. nr.	1	2	3
EV-METRIC %	78	67	67