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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210377	4/8	Mus musculus	primary BM-MSCs	UF	Angioni R	2020	50%
Study summary Full title CD73 extracellular vesicles inhibit angiogenesis through adenosine A receptor signalling. All authors Angioni R, Liboni C, Herkenne S, Sánchez-Rodríguez R, Borile G, Marcuzzi E, Calì B, Muraca M, Viola A Journal J Extracell Vesicles Abstract Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory d (show more...)Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce EVs that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic EVs to inhibit tumour progression . Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis. (hide) EV-METRIC 50% (87th 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 Ultrafiltration Protein markers EV: CD9/ CD63/ CD39/ CD73/ TIMP1 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells primary BM-MSCs EV-harvesting Medium Serum free medium Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose 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 CD9/ CD63/ CD39/ CD73/ TIMP1 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV210377	6/8	Mus musculus	primary BM-MSCs	UF	Angioni R	2020	50%
Study summary Full title CD73 extracellular vesicles inhibit angiogenesis through adenosine A receptor signalling. All authors Angioni R, Liboni C, Herkenne S, Sánchez-Rodríguez R, Borile G, Marcuzzi E, Calì B, Muraca M, Viola A Journal J Extracell Vesicles Abstract Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory d (show more...)Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce EVs that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic EVs to inhibit tumour progression . Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis. (hide) EV-METRIC 50% (87th 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 Cytokines-treated 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 Ultrafiltration Protein markers EV: CD9/ CD63/ TIMP1/ CD39/ CD73 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells primary BM-MSCs EV-harvesting Medium Serum free medium Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose 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 CD9/ CD63/ TIMP1/ CD39/ CD73 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes EM EM-type Transmission?-EM Image type Close-up

	EV210377	3/8	Mus musculus	primary BM-MSCs	(d)(U)C	Angioni R	2020	33%
Study summary Full title CD73 extracellular vesicles inhibit angiogenesis through adenosine A receptor signalling. All authors Angioni R, Liboni C, Herkenne S, Sánchez-Rodríguez R, Borile G, Marcuzzi E, Calì B, Muraca M, Viola A Journal J Extracell Vesicles Abstract Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory d (show more...)Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce EVs that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic EVs to inhibit tumour progression . Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis. (hide) EV-METRIC 33% (75th 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 Protein markers EV: CD9/ CD63/ CD73/ TIMP1 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells primary BM-MSCs EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 1 Wash: time (min) 70 Wash: speed (g) 100000 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 CD9/ CD63/ CD73/ TIMP1 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210377	5/8	Mus musculus	primary BM-MSCs	(d)(U)C	Angioni R	2020	33%
Study summary Full title CD73 extracellular vesicles inhibit angiogenesis through adenosine A receptor signalling. All authors Angioni R, Liboni C, Herkenne S, Sánchez-Rodríguez R, Borile G, Marcuzzi E, Calì B, Muraca M, Viola A Journal J Extracell Vesicles Abstract Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory d (show more...)Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce EVs that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic EVs to inhibit tumour progression . Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis. (hide) EV-METRIC 33% (75th 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 Cytokines-treated 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 Protein markers EV: CD9/ CD63/ TIMP1/ CD73 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells primary BM-MSCs EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 1 Wash: time (min) 70 Wash: speed (g) 100,000 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 CD9/ CD63/ TIMP1/ CD73 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210377	1/8	Homo sapiens	primary BM-MSCs	UF	Angioni R	2020	25%
Study summary Full title CD73 extracellular vesicles inhibit angiogenesis through adenosine A receptor signalling. All authors Angioni R, Liboni C, Herkenne S, Sánchez-Rodríguez R, Borile G, Marcuzzi E, Calì B, Muraca M, Viola A Journal J Extracell Vesicles Abstract Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory d (show more...)Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce EVs that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic EVs to inhibit tumour progression . Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis. (hide) EV-METRIC 25% (64th 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 Ultrafiltration Protein markers EV: CD9/ CD73 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary BM-MSCs EV-harvesting Medium Serum free medium Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose 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 CD9/ CD73 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210377	2/8	Homo sapiens	primary BM-MSCs	UF	Angioni R	2020	25%
Study summary Full title CD73 extracellular vesicles inhibit angiogenesis through adenosine A receptor signalling. All authors Angioni R, Liboni C, Herkenne S, Sánchez-Rodríguez R, Borile G, Marcuzzi E, Calì B, Muraca M, Viola A Journal J Extracell Vesicles Abstract Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory d (show more...)Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce EVs that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic EVs to inhibit tumour progression . Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis. (hide) EV-METRIC 25% (64th 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 Cytokines-treated 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 Ultrafiltration Protein markers EV: CD9/ CD73 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary BM-MSCs EV-harvesting Medium Serum free medium Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose 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 CD9/ CD73 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210377	7/8	Mus musculus	MEF (Thermo Fisher A34960)	UF	Angioni R	2020	25%
Study summary Full title CD73 extracellular vesicles inhibit angiogenesis through adenosine A receptor signalling. All authors Angioni R, Liboni C, Herkenne S, Sánchez-Rodríguez R, Borile G, Marcuzzi E, Calì B, Muraca M, Viola A Journal J Extracell Vesicles Abstract Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory d (show more...)Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce EVs that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic EVs to inhibit tumour progression . Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis. (hide) EV-METRIC 25% (64th 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 Ultrafiltration Protein markers EV: CD63/ TIMP1 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells MEF (Thermo Fisher A34960) EV-harvesting Medium Serum free medium Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose 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 CD63/ TIMP1 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210377	8/8	Mus musculus	MEF (Thermo Fisher A34960)	UF	Angioni R	2020	25%
Study summary Full title CD73 extracellular vesicles inhibit angiogenesis through adenosine A receptor signalling. All authors Angioni R, Liboni C, Herkenne S, Sánchez-Rodríguez R, Borile G, Marcuzzi E, Calì B, Muraca M, Viola A Journal J Extracell Vesicles Abstract Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory d (show more...)Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce EVs that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic EVs to inhibit tumour progression . Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis. (hide) EV-METRIC 25% (64th 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 Cytokines-treated 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 Ultrafiltration Protein markers EV: CD63/ TIMP1 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells MEF (Thermo Fisher A34960) EV-harvesting Medium Serum free medium Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose 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 CD63/ TIMP1 Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV210377
species	Mus musculus	Mus musculus	Mus musculus	Mus musculus	Homo sapiens	Homo sapiens	Mus musculus	Mus musculus
sample type	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture
cell type	primary BM-MSCs	primary BM-MSCs	primary BM-MSCs	primary BM-MSCs	primary BM-MSCs	primary BM-MSCs	MEF (Thermo Fisher A34960)	MEF (Thermo Fisher A34960)
condition	Control condition	Cytokines-treated	Control condition	Cytokines-treated	Control condition	Cytokines-treated	Control condition	Cytokines-treated
separation protocol	Ultrafiltration	Ultrafiltration	dUC	dUC	Ultrafiltration	Ultrafiltration	Ultrafiltration	Ultrafiltration
Exp. nr.	4	6	3	5	1	2	7	8
EV-METRIC %	50	50	33	33	25	25	25	25

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