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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210078	1/5	Homo sapiens	KHOS	(d)(U)C Filtration ExoQuick	Macklin, Rebecca	2016	38%
Study summary Full title Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones All authors Rebecca Macklin, Haolu Wang, Dorothy Loo, Sally Martin, Andrew Cumming, Na Cai, Rebecca Lane, Natalia Saenz Ponce, Eleni Topkas, Kerry Inder, Nicholas A Saunders, Liliana Endo-Munoz Journal Oncotarget Abstract Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pu (show more...)Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pulmonary metastasis. Unfortunately, the mechanistic basis for metastasis remains unclear. Tumor-derived extracellular vesicles (EVs) have been shown to play critical roles in cell-to-cell communication and metastatic progression in other cancers, but their role in OS has not been explored. We show that EVs secreted by cells derived from a highly metastatic clonal variant of the KHOS cell line can be internalized by a poorly metastatic clonal variant of the same cell line and induce a migratory and invasive phenotype. This horizontal phenotypic transfer is unidirectional and provides evidence that metastatic potential may arise via interclonal co-operation. Proteomic analysis of the EVs secreted by highly metastatic OS clonal variants results in the identification of a number of proteins and G-protein coupled receptor signaling events as potential drivers of OS metastasis and novel therapeutic targets. Finally, multiphoton microscopy with fluorescence lifetime imaging in vivo, demonstrated a preferential seeding of lung tissue by EVs derived from highly metastatic OS clonal variants. Thus, we show that EVs derived from highly metastatic clonal variants of OS may drive metastatic behaviour via interclonal co-operation and preferential colonization of the lungs. (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 HiMet-C6 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 Commercial method Protein markers EV: CD81/ HSP70/ CD63 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells KHOS EV-harvesting Medium EV-depleted medium Preparation of EDS Overnight (16h) at >=100,000g + 0.2 µm filtration Cell count Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70 Not detected EV-associated proteins CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Modus Reported size (nm) 85 EM EM-type Transmission-EM Image type Close-up

	EV210078	3/5	Homo sapiens	KHOS	(d)(U)C Filtration ExoQuick	Macklin, Rebecca	2016	25%
Study summary Full title Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones All authors Rebecca Macklin, Haolu Wang, Dorothy Loo, Sally Martin, Andrew Cumming, Na Cai, Rebecca Lane, Natalia Saenz Ponce, Eleni Topkas, Kerry Inder, Nicholas A Saunders, Liliana Endo-Munoz Journal Oncotarget Abstract Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pu (show more...)Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pulmonary metastasis. Unfortunately, the mechanistic basis for metastasis remains unclear. Tumor-derived extracellular vesicles (EVs) have been shown to play critical roles in cell-to-cell communication and metastatic progression in other cancers, but their role in OS has not been explored. We show that EVs secreted by cells derived from a highly metastatic clonal variant of the KHOS cell line can be internalized by a poorly metastatic clonal variant of the same cell line and induce a migratory and invasive phenotype. This horizontal phenotypic transfer is unidirectional and provides evidence that metastatic potential may arise via interclonal co-operation. Proteomic analysis of the EVs secreted by highly metastatic OS clonal variants results in the identification of a number of proteins and G-protein coupled receptor signaling events as potential drivers of OS metastasis and novel therapeutic targets. Finally, multiphoton microscopy with fluorescence lifetime imaging in vivo, demonstrated a preferential seeding of lung tissue by EVs derived from highly metastatic OS clonal variants. Thus, we show that EVs derived from highly metastatic clonal variants of OS may drive metastatic behaviour via interclonal co-operation and preferential colonization of the lungs. (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 (d)(U)C Filtration Commercial method Protein markers EV: CD81/ HSP70/ CD63 non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells KHOS EV-harvesting Medium EV-depleted medium Preparation of EDS Overnight (16h) at >=100,000g + 0.2 µm filtration Cell count Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ CD81 Characterization: Lipid analysis No

	EV210078	4/5	Homo sapiens	KHOS	(d)(U)C Filtration ExoQuick	Macklin, Rebecca	2016	25%
Study summary Full title Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones All authors Rebecca Macklin, Haolu Wang, Dorothy Loo, Sally Martin, Andrew Cumming, Na Cai, Rebecca Lane, Natalia Saenz Ponce, Eleni Topkas, Kerry Inder, Nicholas A Saunders, Liliana Endo-Munoz Journal Oncotarget Abstract Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pu (show more...)Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pulmonary metastasis. Unfortunately, the mechanistic basis for metastasis remains unclear. Tumor-derived extracellular vesicles (EVs) have been shown to play critical roles in cell-to-cell communication and metastatic progression in other cancers, but their role in OS has not been explored. We show that EVs secreted by cells derived from a highly metastatic clonal variant of the KHOS cell line can be internalized by a poorly metastatic clonal variant of the same cell line and induce a migratory and invasive phenotype. This horizontal phenotypic transfer is unidirectional and provides evidence that metastatic potential may arise via interclonal co-operation. Proteomic analysis of the EVs secreted by highly metastatic OS clonal variants results in the identification of a number of proteins and G-protein coupled receptor signaling events as potential drivers of OS metastasis and novel therapeutic targets. Finally, multiphoton microscopy with fluorescence lifetime imaging in vivo, demonstrated a preferential seeding of lung tissue by EVs derived from highly metastatic OS clonal variants. Thus, we show that EVs derived from highly metastatic clonal variants of OS may drive metastatic behaviour via interclonal co-operation and preferential colonization of the lungs. (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 LoMet-C4 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 Commercial method Protein markers EV: CD81/ HSP70/ CD63 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells KHOS EV-harvesting Medium EV-depleted medium Preparation of EDS Overnight (16h) at >=100,000g + 0.2 µm filtration Cell count Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Modus Reported size (nm) 99

	EV210078	5/5	Homo sapiens	HOS	(d)(U)C Filtration ExoQuick	Macklin, Rebecca	2016	25%
Study summary Full title Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones All authors Rebecca Macklin, Haolu Wang, Dorothy Loo, Sally Martin, Andrew Cumming, Na Cai, Rebecca Lane, Natalia Saenz Ponce, Eleni Topkas, Kerry Inder, Nicholas A Saunders, Liliana Endo-Munoz Journal Oncotarget Abstract Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pu (show more...)Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pulmonary metastasis. Unfortunately, the mechanistic basis for metastasis remains unclear. Tumor-derived extracellular vesicles (EVs) have been shown to play critical roles in cell-to-cell communication and metastatic progression in other cancers, but their role in OS has not been explored. We show that EVs secreted by cells derived from a highly metastatic clonal variant of the KHOS cell line can be internalized by a poorly metastatic clonal variant of the same cell line and induce a migratory and invasive phenotype. This horizontal phenotypic transfer is unidirectional and provides evidence that metastatic potential may arise via interclonal co-operation. Proteomic analysis of the EVs secreted by highly metastatic OS clonal variants results in the identification of a number of proteins and G-protein coupled receptor signaling events as potential drivers of OS metastasis and novel therapeutic targets. Finally, multiphoton microscopy with fluorescence lifetime imaging in vivo, demonstrated a preferential seeding of lung tissue by EVs derived from highly metastatic OS clonal variants. Thus, we show that EVs derived from highly metastatic clonal variants of OS may drive metastatic behaviour via interclonal co-operation and preferential colonization of the lungs. (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 (d)(U)C Filtration Commercial method Protein markers EV: CD81/ HSP70/ CD63 non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HOS EV-harvesting Medium EV-depleted medium Preparation of EDS Overnight (16h) at >=100,000g + 0.2 µm filtration Cell count Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Not detected EV-associated proteins HSP70/ Characterization: Lipid analysis No

	EV210078	2/5	Homo sapiens	KHOS	(d)(U)C Filtration UF	Macklin, Rebecca	2016	0%
Study summary Full title Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones All authors Rebecca Macklin, Haolu Wang, Dorothy Loo, Sally Martin, Andrew Cumming, Na Cai, Rebecca Lane, Natalia Saenz Ponce, Eleni Topkas, Kerry Inder, Nicholas A Saunders, Liliana Endo-Munoz Journal Oncotarget Abstract Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pu (show more...)Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pulmonary metastasis. Unfortunately, the mechanistic basis for metastasis remains unclear. Tumor-derived extracellular vesicles (EVs) have been shown to play critical roles in cell-to-cell communication and metastatic progression in other cancers, but their role in OS has not been explored. We show that EVs secreted by cells derived from a highly metastatic clonal variant of the KHOS cell line can be internalized by a poorly metastatic clonal variant of the same cell line and induce a migratory and invasive phenotype. This horizontal phenotypic transfer is unidirectional and provides evidence that metastatic potential may arise via interclonal co-operation. Proteomic analysis of the EVs secreted by highly metastatic OS clonal variants results in the identification of a number of proteins and G-protein coupled receptor signaling events as potential drivers of OS metastasis and novel therapeutic targets. Finally, multiphoton microscopy with fluorescence lifetime imaging in vivo, demonstrated a preferential seeding of lung tissue by EVs derived from highly metastatic OS clonal variants. Thus, we show that EVs derived from highly metastatic clonal variants of OS may drive metastatic behaviour via interclonal co-operation and preferential colonization of the lungs. (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 HiMet-C6 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 UF Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells KHOS EV-harvesting Medium EV-depleted medium Preparation of EDS Overnight (16h) at >=100,000g + 0.2 µm filtration Cell count Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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: time(min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100 000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100 000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Not specified Protein Concentration Method Bradford Characterization: Lipid analysis No

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EV-TRACK ID	EV210078
species	Homo sapiens
sample type	Cell culture
cell type	KHOS	KHOS	KHOS	HOS	KHOS
condition	HiMet-C6	Control condition	LoMet-C4	Control condition	HiMet-C6
separation protocol	(d)(U)C Filtration ExoQuick	(d)(U)C Filtration ExoQuick	(d)(U)C Filtration ExoQuick	(d)(U)C Filtration ExoQuick	(d)(U)C Filtration UF
Exp. nr.	1	3	4	5	2
EV-METRIC %	38	25	25	25	0