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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210127	1/9	Homo sapiens	SK-MEL-147	(d)(U)C	García-Silva, Susana	2021	67%
Study summary Full title Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through a NGFR-dependent mechanism All authors Susana García-Silva, Alberto Benito-Martín, Laura Nogués, Alberto Hernández-Barranco, Marina S. Mazariegos, Vanesa Santos, Marta Hergueta-Redondo, Pilar Ximénez-Embún, Raghu P. Kataru, Ana Amor Lopez, Cristina Merino, Sara Sánchez-Redondo, Osvaldo Graña-Castro, Irina Matei, José Ángel Nicolás-Avila, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Lola Martínez, Manuel Pérez-Martínez, Gadea Mata, Anna Szumera-Ciećkiewicz, Iwona Kalinowska, Annalisa Saltari, Julia M. Martínez-Gómez, Sabrina A. Hogan, H. Uri Saragovi, Sagrario Ortega, Carmen Garcia-Martin, Jasminka Boskovic, Mitchell P. Levesque, Piotr Rutkowski, Andrés Hidalgo, Javier Muñoz, Diego Megías, Babak J. Mehrara, David Lyden and Héctor Peinado. Journal Nat. Cancer Abstract Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metast (show more...)Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we have analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK, NF-kB activation and ICAM-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR/p75NTR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to matched primary tumors and frequency of NGFR+ metastatic melanoma cells in lymph node correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs reinforcing lymph node pre-metastatic niche formation and metastasis. (hide) EV-METRIC 67% (94th 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 Other / small extracellular vesicles 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 Protein markers EV: Alix/ RAB27a/ NGFR/ GAPDH non-EV: Calnexin/ GM130 Proteomics yes EV density (g/ml) Not specified Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS 70 min at 100,000g;Other preparation Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 7.7 Sample volume (mL) 0.2 Orientation Top-down Rotor type Type 70 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0.75 Fraction processing Ultracentrifugation Pelleting: volume per fraction 3.25 Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins NGFR/ GAPDH/ Alix/ CD81/ CD63 Not detected EV-associated proteins RAB27a Not detected contaminants Calnexin/ GM130 Flow cytometry Type of Flow cytometry FACS Canto Calibration bead size 0,2 Detected EV-associated proteins NGFR Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 130 EV concentration Yes Particle yield Yes, as number of particles per million cells 5,70E+09

	EV210127	5/9	Mus musculus	B16-F1	(d)(U)C	García-Silva, Susana	2021	67%
Study summary Full title Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through a NGFR-dependent mechanism All authors Susana García-Silva, Alberto Benito-Martín, Laura Nogués, Alberto Hernández-Barranco, Marina S. Mazariegos, Vanesa Santos, Marta Hergueta-Redondo, Pilar Ximénez-Embún, Raghu P. Kataru, Ana Amor Lopez, Cristina Merino, Sara Sánchez-Redondo, Osvaldo Graña-Castro, Irina Matei, José Ángel Nicolás-Avila, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Lola Martínez, Manuel Pérez-Martínez, Gadea Mata, Anna Szumera-Ciećkiewicz, Iwona Kalinowska, Annalisa Saltari, Julia M. Martínez-Gómez, Sabrina A. Hogan, H. Uri Saragovi, Sagrario Ortega, Carmen Garcia-Martin, Jasminka Boskovic, Mitchell P. Levesque, Piotr Rutkowski, Andrés Hidalgo, Javier Muñoz, Diego Megías, Babak J. Mehrara, David Lyden and Héctor Peinado. Journal Nat. Cancer Abstract Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metast (show more...)Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we have analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK, NF-kB activation and ICAM-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR/p75NTR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to matched primary tumors and frequency of NGFR+ metastatic melanoma cells in lymph node correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs reinforcing lymph node pre-metastatic niche formation and metastasis. (hide) EV-METRIC 67% (94th 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 Other / small extracellular vesicles 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 Protein markers EV: Alix/ NGFR non-EV: Calnexin/ GM130 Proteomics yes EV density (g/ml) Not specified Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16-F1 EV-harvesting Medium EV-depleted medium Preparation of EDS Other preparation;70 min at 100,000g Cell viability (%) 92 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 7.7 Sample volume (mL) 0.2 Orientation Top-down Rotor type Type 70 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0.75 Fraction processing Ultracentrifugation Pelleting: volume per fraction 3.25 Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins NGFR/ Alix/ CD81/ CD9 Not detected contaminants Calnexin/ GM130 Flow cytometry Type of Flow cytometry FACS Canto Calibration bead size 0,2 Detected EV-associated proteins NGFR Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 145 EV concentration Yes Particle yield Yes, as number of particles per million cells 6,00E+09

	EV210127	9/9	Mus musculus	B16-F10	(d)(U)C	García-Silva, Susana	2021	67%
Study summary Full title Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through a NGFR-dependent mechanism All authors Susana García-Silva, Alberto Benito-Martín, Laura Nogués, Alberto Hernández-Barranco, Marina S. Mazariegos, Vanesa Santos, Marta Hergueta-Redondo, Pilar Ximénez-Embún, Raghu P. Kataru, Ana Amor Lopez, Cristina Merino, Sara Sánchez-Redondo, Osvaldo Graña-Castro, Irina Matei, José Ángel Nicolás-Avila, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Lola Martínez, Manuel Pérez-Martínez, Gadea Mata, Anna Szumera-Ciećkiewicz, Iwona Kalinowska, Annalisa Saltari, Julia M. Martínez-Gómez, Sabrina A. Hogan, H. Uri Saragovi, Sagrario Ortega, Carmen Garcia-Martin, Jasminka Boskovic, Mitchell P. Levesque, Piotr Rutkowski, Andrés Hidalgo, Javier Muñoz, Diego Megías, Babak J. Mehrara, David Lyden and Héctor Peinado. Journal Nat. Cancer Abstract Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metast (show more...)Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we have analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK, NF-kB activation and ICAM-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR/p75NTR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to matched primary tumors and frequency of NGFR+ metastatic melanoma cells in lymph node correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs reinforcing lymph node pre-metastatic niche formation and metastasis. (hide) EV-METRIC 67% (94th 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 Other / small extracellular vesicles 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 Protein markers EV: Alix/ NGFR non-EV: Calnexin/ GM130 Proteomics yes EV density (g/ml) Not specified Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16-F10 EV-harvesting Medium EV-depleted medium Preparation of EDS Other preparation;70 min at 100,000g Cell viability (%) 94 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 7.7 Sample volume (mL) 0.2 Orientation Top-down Rotor type Type 70 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0.75 Fraction processing Ultracentrifugation Pelleting: volume per fraction 3.25 Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ NGFR/ CD81/ CD9 Not detected contaminants Calnexin/ GM130 Flow cytometry Type of Flow cytometry FACS Canto Calibration bead size 0,2 Detected EV-associated proteins NGFR Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 149 EV concentration Yes Particle yield Yes, as number of particles per million cells 3000000000

	EV210127	2/9	Homo sapiens	SK-MEL-147	(d)(U)C	García-Silva, Susana	2021	55%
Study summary Full title Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through a NGFR-dependent mechanism All authors Susana García-Silva, Alberto Benito-Martín, Laura Nogués, Alberto Hernández-Barranco, Marina S. Mazariegos, Vanesa Santos, Marta Hergueta-Redondo, Pilar Ximénez-Embún, Raghu P. Kataru, Ana Amor Lopez, Cristina Merino, Sara Sánchez-Redondo, Osvaldo Graña-Castro, Irina Matei, José Ángel Nicolás-Avila, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Lola Martínez, Manuel Pérez-Martínez, Gadea Mata, Anna Szumera-Ciećkiewicz, Iwona Kalinowska, Annalisa Saltari, Julia M. Martínez-Gómez, Sabrina A. Hogan, H. Uri Saragovi, Sagrario Ortega, Carmen Garcia-Martin, Jasminka Boskovic, Mitchell P. Levesque, Piotr Rutkowski, Andrés Hidalgo, Javier Muñoz, Diego Megías, Babak J. Mehrara, David Lyden and Héctor Peinado. Journal Nat. Cancer Abstract Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metast (show more...)Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we have analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK, NF-kB activation and ICAM-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR/p75NTR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to matched primary tumors and frequency of NGFR+ metastatic melanoma cells in lymph node correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs reinforcing lymph node pre-metastatic niche formation and metastasis. (hide) EV-METRIC 55% (88th 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 shControl Focus vesicles Other / small extracellular vesicles 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 Protein markers EV: Alix/ B-ACTIN/ NGFR non-EV: Calnexin/ GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS Other preparation;70 min at 100,000g Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins B-ACTIN/ NGFR/ Alix/ CD81/ CD63 Not detected contaminants Calnexin/ GM130 Flow cytometry Type of Flow cytometry FACS Canto Calibration bead size 0,2 Detected EV-associated proteins NGFR Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported Particle yield NA NA

	EV210127	3/9	Homo sapiens	SK-MEL-147	(d)(U)C	García-Silva, Susana	2021	55%
Study summary Full title Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through a NGFR-dependent mechanism All authors Susana García-Silva, Alberto Benito-Martín, Laura Nogués, Alberto Hernández-Barranco, Marina S. Mazariegos, Vanesa Santos, Marta Hergueta-Redondo, Pilar Ximénez-Embún, Raghu P. Kataru, Ana Amor Lopez, Cristina Merino, Sara Sánchez-Redondo, Osvaldo Graña-Castro, Irina Matei, José Ángel Nicolás-Avila, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Lola Martínez, Manuel Pérez-Martínez, Gadea Mata, Anna Szumera-Ciećkiewicz, Iwona Kalinowska, Annalisa Saltari, Julia M. Martínez-Gómez, Sabrina A. Hogan, H. Uri Saragovi, Sagrario Ortega, Carmen Garcia-Martin, Jasminka Boskovic, Mitchell P. Levesque, Piotr Rutkowski, Andrés Hidalgo, Javier Muñoz, Diego Megías, Babak J. Mehrara, David Lyden and Héctor Peinado. Journal Nat. Cancer Abstract Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metast (show more...)Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we have analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK, NF-kB activation and ICAM-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR/p75NTR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to matched primary tumors and frequency of NGFR+ metastatic melanoma cells in lymph node correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs reinforcing lymph node pre-metastatic niche formation and metastasis. (hide) EV-METRIC 55% (88th 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 shNGFR Focus vesicles Other / small extracellular vesicles 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 Protein markers EV: Alix/ B-ACTIN/ NGFR non-EV: Calnexin/ GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS Other preparation;70 min at 100,000g Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ B-ACTIN/ NGFR/ CD81/ CD63 Not detected contaminants Calnexin/ GM130 Flow cytometry Type of Flow cytometry FACS Canto Calibration bead size 0,2 Detected EV-associated proteins NGFR Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported Particle yield NA NA

	EV210127	8/9	Mus musculus	B16-F1R2	(d)(U)C	García-Silva, Susana	2021	55%
Study summary Full title Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through a NGFR-dependent mechanism All authors Susana García-Silva, Alberto Benito-Martín, Laura Nogués, Alberto Hernández-Barranco, Marina S. Mazariegos, Vanesa Santos, Marta Hergueta-Redondo, Pilar Ximénez-Embún, Raghu P. Kataru, Ana Amor Lopez, Cristina Merino, Sara Sánchez-Redondo, Osvaldo Graña-Castro, Irina Matei, José Ángel Nicolás-Avila, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Lola Martínez, Manuel Pérez-Martínez, Gadea Mata, Anna Szumera-Ciećkiewicz, Iwona Kalinowska, Annalisa Saltari, Julia M. Martínez-Gómez, Sabrina A. Hogan, H. Uri Saragovi, Sagrario Ortega, Carmen Garcia-Martin, Jasminka Boskovic, Mitchell P. Levesque, Piotr Rutkowski, Andrés Hidalgo, Javier Muñoz, Diego Megías, Babak J. Mehrara, David Lyden and Héctor Peinado. Journal Nat. Cancer Abstract Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metast (show more...)Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we have analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK, NF-kB activation and ICAM-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR/p75NTR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to matched primary tumors and frequency of NGFR+ metastatic melanoma cells in lymph node correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs reinforcing lymph node pre-metastatic niche formation and metastasis. (hide) EV-METRIC 55% (88th 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 Other / small extracellular vesicles 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 Protein markers EV: Alix/ B-ACTIN/ NGFR non-EV: Calnexin/ GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16-F1R2 EV-harvesting Medium EV-depleted medium Preparation of EDS 70 min at 100,000g;Other preparation Cell viability (%) 92 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ NGFR/ B-ACTIN/ CD81/ CD9 Not detected contaminants Calnexin/ GM130 Flow cytometry Type of Flow cytometry FACS Canto Calibration bead size 0,2 Detected EV-associated proteins NGFR Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148 EV concentration Yes Particle yield Yes, as number of particles per million cells 2500000000

	EV210127	6/9	Mus musculus	B16-F1R2	(d)(U)C	García-Silva, Susana	2021	44%
Study summary Full title Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through a NGFR-dependent mechanism All authors Susana García-Silva, Alberto Benito-Martín, Laura Nogués, Alberto Hernández-Barranco, Marina S. Mazariegos, Vanesa Santos, Marta Hergueta-Redondo, Pilar Ximénez-Embún, Raghu P. Kataru, Ana Amor Lopez, Cristina Merino, Sara Sánchez-Redondo, Osvaldo Graña-Castro, Irina Matei, José Ángel Nicolás-Avila, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Lola Martínez, Manuel Pérez-Martínez, Gadea Mata, Anna Szumera-Ciećkiewicz, Iwona Kalinowska, Annalisa Saltari, Julia M. Martínez-Gómez, Sabrina A. Hogan, H. Uri Saragovi, Sagrario Ortega, Carmen Garcia-Martin, Jasminka Boskovic, Mitchell P. Levesque, Piotr Rutkowski, Andrés Hidalgo, Javier Muñoz, Diego Megías, Babak J. Mehrara, David Lyden and Héctor Peinado. Journal Nat. Cancer Abstract Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metast (show more...)Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we have analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK, NF-kB activation and ICAM-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR/p75NTR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to matched primary tumors and frequency of NGFR+ metastatic melanoma cells in lymph node correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs reinforcing lymph node pre-metastatic niche formation and metastasis. (hide) EV-METRIC 44% (84th 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 CRISPR/Cas9 control Focus vesicles Other / small extracellular vesicles 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 Protein markers EV: CD81/ B-ACTIN/ NGFR non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16-F1R2 EV-harvesting Medium EV-depleted medium Preparation of EDS Other preparation;70 min at 100,000g Cell viability (%) 93 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins NGFR/ B-ACTIN/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported Particle yield NA NA

	EV210127	7/9	Mus musculus	B16-F1R2	(d)(U)C	García-Silva, Susana	2021	44%
Study summary Full title Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through a NGFR-dependent mechanism All authors Susana García-Silva, Alberto Benito-Martín, Laura Nogués, Alberto Hernández-Barranco, Marina S. Mazariegos, Vanesa Santos, Marta Hergueta-Redondo, Pilar Ximénez-Embún, Raghu P. Kataru, Ana Amor Lopez, Cristina Merino, Sara Sánchez-Redondo, Osvaldo Graña-Castro, Irina Matei, José Ángel Nicolás-Avila, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Lola Martínez, Manuel Pérez-Martínez, Gadea Mata, Anna Szumera-Ciećkiewicz, Iwona Kalinowska, Annalisa Saltari, Julia M. Martínez-Gómez, Sabrina A. Hogan, H. Uri Saragovi, Sagrario Ortega, Carmen Garcia-Martin, Jasminka Boskovic, Mitchell P. Levesque, Piotr Rutkowski, Andrés Hidalgo, Javier Muñoz, Diego Megías, Babak J. Mehrara, David Lyden and Héctor Peinado. Journal Nat. Cancer Abstract Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metast (show more...)Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we have analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK, NF-kB activation and ICAM-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR/p75NTR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to matched primary tumors and frequency of NGFR+ metastatic melanoma cells in lymph node correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs reinforcing lymph node pre-metastatic niche formation and metastasis. (hide) EV-METRIC 44% (84th 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 CRISPR/Cas9 Ngfr Focus vesicles Other / small extracellular vesicles 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 Protein markers EV: CD81/ B-ACTIN/ NGFR non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16-F1R2 EV-harvesting Medium EV-depleted medium Preparation of EDS Other preparation;70 min at 100,000g Cell viability (%) 94 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins NGFR/ B-ACTIN/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported Particle yield NA NA

	EV210127	4/9	Homo sapiens	Primary melanocytes	(d)(U)C	García-Silva, Susana	2021	33%
Study summary Full title Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through a NGFR-dependent mechanism All authors Susana García-Silva, Alberto Benito-Martín, Laura Nogués, Alberto Hernández-Barranco, Marina S. Mazariegos, Vanesa Santos, Marta Hergueta-Redondo, Pilar Ximénez-Embún, Raghu P. Kataru, Ana Amor Lopez, Cristina Merino, Sara Sánchez-Redondo, Osvaldo Graña-Castro, Irina Matei, José Ángel Nicolás-Avila, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Lola Martínez, Manuel Pérez-Martínez, Gadea Mata, Anna Szumera-Ciećkiewicz, Iwona Kalinowska, Annalisa Saltari, Julia M. Martínez-Gómez, Sabrina A. Hogan, H. Uri Saragovi, Sagrario Ortega, Carmen Garcia-Martin, Jasminka Boskovic, Mitchell P. Levesque, Piotr Rutkowski, Andrés Hidalgo, Javier Muñoz, Diego Megías, Babak J. Mehrara, David Lyden and Héctor Peinado. Journal Nat. Cancer Abstract Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metast (show more...)Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we have analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK, NF-kB activation and ICAM-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR/p75NTR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to matched primary tumors and frequency of NGFR+ metastatic melanoma cells in lymph node correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs reinforcing lymph node pre-metastatic niche formation and metastasis. (hide) EV-METRIC 33% (74th 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 Other / small extracellular vesicles 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 Protein markers EV: Alix/ NGFR non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary melanocytes EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ NGFR Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported Particle yield NA NA

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EV-TRACK ID	EV210127
species	Homo sapiens	Mus musculus	Mus musculus	Homo sapiens	Homo sapiens	Mus musculus	Mus musculus	Mus musculus	Homo sapiens
sample type	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture
cell type	SK-MEL-147	B16-F1	B16-F10	SK-MEL-147	SK-MEL-147	B16-F1R2	B16-F1R2	B16-F1R2	Primary melanocytes
medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	Serum free medium
condition	Control condition	Control condition	Control condition	shControl	shNGFR	Control condition	CRISPR/Cas9 control	CRISPR/Cas9 Ngfr	Control condition
separation protocol	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C
Exp. nr.	1	5	9	2	3	8	6	7	4
EV-METRIC %	67	67	67	55	55	55	44	44	33

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