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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220152	3/6	Mus musculus	67NR	(d)(U)C DG	Vardaki I	2016	78%
Study summary Full title Periostin is identified as a putative metastatic marker in breast cancer-derived exosomes. All authors Vardaki I, Ceder S, Rutishauser D, Baltatzis G, Foukakis T, Panaretakis T Journal Oncotarget Abstract Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related (show more...)Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis. (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 exosome 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 Density gradient Protein markers EV: CD63/ TSG101/ Rab5/ integrin alpha2/ integrin beta1/ periostin/ N-cadherin/ LOXL3/ LOXL4/ Glypican-1/ Glypican-4/ V-ATPase/ Alix/ beta-catenin/ E-cadherin/ Syndecan-4/ Vimentin/ GADPH/ AIF non-EV: None Proteomics yes EV density (g/ml) 1.12-1.16 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 67NR EV-harvesting Medium EV-depleted medium 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: speed (g) 120000 Wash: time (min) 120 Wash: speed (g) 120000 Density gradient Only used for validation of main results Yes Lowest density fraction 0.2 M Highest density fraction 2 M Orientation Top-down Speed (g) 120000 Duration (min) 1200 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101/ Rab5/ integrin alpha2/ integrin beta1/ periostin/ N-cadherin/ LOXL3/ LOXL4/ Glypican-1/ Glypican-4/ V-ATPase/ Alix Not detected EV-associated proteins beta-catenin/ E-cadherin/ Syndecan-4/ Vimentin/ GADPH/ AIF Flow cytometry aspecific beads Antibody details provided? Yes Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD63 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 89 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220152	4/6	Mus musculus	4T1	(d)(U)C DG	Vardaki I	2016	78%
Study summary Full title Periostin is identified as a putative metastatic marker in breast cancer-derived exosomes. All authors Vardaki I, Ceder S, Rutishauser D, Baltatzis G, Foukakis T, Panaretakis T Journal Oncotarget Abstract Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related (show more...)Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis. (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 exosome 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 Density gradient Protein markers EV: CD63/ TSG101/ Rab5/ Alix/ beta-catenin/ integrin alpha2/ integrin beta1/ periostin/ E-cadherin/ LOXL4/ Glypican-1/ V-ATPase/ Alix/ Vimentin/ GAPDH/ AIF/ N-Cadherin/ LOXL3/ Syndecan-4/ Glypican-4/ LOXL3/ N-Cadherin non-EV: None Proteomics yes EV density (g/ml) 1.12-1.16 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 4T1 EV-harvesting Medium EV-depleted medium 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: speed (g) 120000 Wash: time (min) 120 Wash: speed (g) 120000 Density gradient Only used for validation of main results Yes Lowest density fraction 0.2 M Highest density fraction 2 M Orientation Top-down Speed (g) 120000 Duration (min) 1200 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101/ Rab5/ Alix/ beta-catenin/ integrin alpha2/ integrin beta1/ periostin/ E-cadherin/ LOXL4/ Glypican-1/ V-ATPase/ Alix Not detected EV-associated proteins Vimentin/ GAPDH/ AIF/ N-Cadherin/ LOXL3/ Syndecan-4/ Glypican-4/ LOXL3/ N-Cadherin Flow cytometry aspecific beads Antibody details provided? Yes Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 102 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220152	5/6	Homo sapiens	Plasma	(d)(U)C	Vardaki I	2016	44%
Study summary Full title Periostin is identified as a putative metastatic marker in breast cancer-derived exosomes. All authors Vardaki I, Ceder S, Rutishauser D, Baltatzis G, Foukakis T, Panaretakis T Journal Oncotarget Abstract Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related (show more...)Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis. (hide) EV-METRIC 44% (66th 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 Plasma Sample origin Breast cancer, localised Focus vesicles exosome 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: E-Cadherin/ N-Cadherin/ Periostin/ CD9 non-EV: None Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Plasma 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: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins E-Cadherin/ N-Cadherin/ Periostin/ CD9 Flow cytometry aspecific beads Antibody details provided? Yes Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 95 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 8.35e+9 EM EM-type Transmission-EM Image type Close-up

	EV220152	6/6	Homo sapiens	Plasma	(d)(U)C	Vardaki I	2016	44%
Study summary Full title Periostin is identified as a putative metastatic marker in breast cancer-derived exosomes. All authors Vardaki I, Ceder S, Rutishauser D, Baltatzis G, Foukakis T, Panaretakis T Journal Oncotarget Abstract Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related (show more...)Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis. (hide) EV-METRIC 44% (66th 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 Plasma Sample origin Breast cancer, lymph-node metastasis Focus vesicles exosome 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: E-Cadherin/ N-Cadherin/ Periostin/ CD9 non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Plasma 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: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins E-Cadherin/ N-Cadherin/ Periostin/ CD9 Flow cytometry aspecific beads Antibody details provided? Yes Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 125 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 1.09e+10 EM EM-type Transmission-EM Image type Close-up

	EV220152	1/6	Homo sapiens	MCF7	(d)(U)C	Vardaki I	2016	33%
Study summary Full title Periostin is identified as a putative metastatic marker in breast cancer-derived exosomes. All authors Vardaki I, Ceder S, Rutishauser D, Baltatzis G, Foukakis T, Panaretakis T Journal Oncotarget Abstract Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related (show more...)Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis. (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 exosome 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: beta-catenin/ integrin alpha2/ integrin beta1/ periostin/ E-cadherin/ N-cadherin/ CD81/ TSG101/ ERalpha/ p53/ Glypican-1 non-EV: None Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium EV-depleted medium 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: speed (g) 120000 Wash: time (min) 120 Wash: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins beta-catenin/ integrin alpha2/ integrin beta1/ periostin/ E-cadherin/ N-cadherin/ CD81/ TSG101/ ERalpha Not detected EV-associated proteins p53/ Glypican-1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 108 EV concentration Yes

	EV220152	2/6	Homo sapiens	MDAMB231	(d)(U)C	Vardaki I	2016	33%
Study summary Full title Periostin is identified as a putative metastatic marker in breast cancer-derived exosomes. All authors Vardaki I, Ceder S, Rutishauser D, Baltatzis G, Foukakis T, Panaretakis T Journal Oncotarget Abstract Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related (show more...)Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis. (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 exosome 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: beta-catenin/ integrin alpha2/ integrin beta1/ periostin/ Glypican-1/ N-cadherin/ CD81/ TSG101/ p53/ ERalpha/ E-cadherin non-EV: None Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted medium 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: speed (g) 120000 Wash: time (min) 120 Wash: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins beta-catenin/ integrin alpha2/ integrin beta1/ periostin/ Glypican-1/ N-cadherin/ CD81/ TSG101 Not detected EV-associated proteins p53/ ERalpha/ E-cadherin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 106 EV concentration Yes

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EV-TRACK ID	EV220152
species	Mus musculus	Mus musculus	Homo sapiens	Homo sapiens	Homo sapiens	Homo sapiens
sample type	Cell culture	Cell culture	Plasma	Plasma	Cell culture	Cell culture
cell type	67NR	4T1	NA	NA	MCF7	MDAMB231
medium	EV-depleted medium	EV-depleted medium	NA	NA	EV-depleted medium	EV-depleted medium
condition	Control condition	Control condition	Breast cancer localised	Breast cancer lymph-node metastasis	Control condition	Control condition
separation protocol	dUC/ Density gradient	dUC/ Density gradient	dUC	dUC	dUC	dUC
Exp. nr.	3	4	5	6	1	2
EV-METRIC %	78	78	44	44	33	33