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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190025	3/4	Homo sapiens	ascites	(d)(U)C Filtration	Czystowska-Kuzmicz, Malgorzata	2019	78%
Study summary Full title Small extracellular vesicles containing arginase-1 suppress T-cell responses and promote tumor growth in ovarian carcinoma All authors Malgorzata Czystowska-Kuzmicz, Anna Sosnowska, Dominika Nowis, Kavita Ramji, Marta Szajnik, Justyna Chlebowska-Tuz, Ewa Wolinska, Pawel Gaj, Magdalena Grazul, Zofia Pilch, Abdessamad Zerrouqi, Agnieszka Graczyk-Jarzynka, Karolina Soroczynska, Szczepan Cierniak, Robert Koktysz, Esther Elishaev, Slawomir Gruca, Artur Stefanowicz, Roman Blaszczyk, Bartlomiej Borek, Anna Gzik, Theresa Whiteside, and Jakub Golab Journal Nat Commun Abstract Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (show more...)Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule – ARG1, mitigating anti-tumor immune responses. (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 ascites Sample origin ovarian cancer 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 Protein markers EV: TSG101/ CD63/ CD81/ ARG1/ EpCAM/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 8 Wash: time (min) 60 Wash: Rotor Type Type 70 Ti Wash: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TSG101/ ARG1 Not detected contaminants Calnexin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins EpCAM/ CD9/ CD81/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 125 EV concentration Yes TRPS Report type Mean Reported size (nm) 116 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190025	4/4	Homo sapiens	ovarian cyst fluid	(d)(U)C Filtration	Czystowska-Kuzmicz, Malgorzata	2019	78%
Study summary Full title Small extracellular vesicles containing arginase-1 suppress T-cell responses and promote tumor growth in ovarian carcinoma All authors Malgorzata Czystowska-Kuzmicz, Anna Sosnowska, Dominika Nowis, Kavita Ramji, Marta Szajnik, Justyna Chlebowska-Tuz, Ewa Wolinska, Pawel Gaj, Magdalena Grazul, Zofia Pilch, Abdessamad Zerrouqi, Agnieszka Graczyk-Jarzynka, Karolina Soroczynska, Szczepan Cierniak, Robert Koktysz, Esther Elishaev, Slawomir Gruca, Artur Stefanowicz, Roman Blaszczyk, Bartlomiej Borek, Anna Gzik, Theresa Whiteside, and Jakub Golab Journal Nat Commun Abstract Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (show more...)Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule – ARG1, mitigating anti-tumor immune responses. (hide) EV-METRIC 78% (50th 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 ovarian cyst fluid 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 Protein markers EV: TSG101/ ARG1/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type ovarian cyst fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 8 Wash: time (min) 60 Wash: Rotor Type Type 70 Ti Wash: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins ARG1/ CD63/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 124 EV concentration Yes TRPS Report type Mean Reported size (nm) 120 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190025	1/4	Homo sapiens	Blood plasma	(d)(U)C Filtration qEV	Czystowska-Kuzmicz, Malgorzata	2019	50%
Study summary Full title Small extracellular vesicles containing arginase-1 suppress T-cell responses and promote tumor growth in ovarian carcinoma All authors Malgorzata Czystowska-Kuzmicz, Anna Sosnowska, Dominika Nowis, Kavita Ramji, Marta Szajnik, Justyna Chlebowska-Tuz, Ewa Wolinska, Pawel Gaj, Magdalena Grazul, Zofia Pilch, Abdessamad Zerrouqi, Agnieszka Graczyk-Jarzynka, Karolina Soroczynska, Szczepan Cierniak, Robert Koktysz, Esther Elishaev, Slawomir Gruca, Artur Stefanowicz, Roman Blaszczyk, Bartlomiej Borek, Anna Gzik, Theresa Whiteside, and Jakub Golab Journal Nat Commun Abstract Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (show more...)Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule – ARG1, mitigating anti-tumor immune responses. (hide) EV-METRIC 50% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma 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 qEV Protein markers EV: TSG101/ ARG1/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ ARG1/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 128 EV concentration Yes TRPS Report type Mean Reported size (nm) 65 EV concentration Yes

	EV190025	2/4	Homo sapiens	Blood plasma	(d)(U)C Filtration qEV	Czystowska-Kuzmicz, Malgorzata	2019	50%
Study summary Full title Small extracellular vesicles containing arginase-1 suppress T-cell responses and promote tumor growth in ovarian carcinoma All authors Malgorzata Czystowska-Kuzmicz, Anna Sosnowska, Dominika Nowis, Kavita Ramji, Marta Szajnik, Justyna Chlebowska-Tuz, Ewa Wolinska, Pawel Gaj, Magdalena Grazul, Zofia Pilch, Abdessamad Zerrouqi, Agnieszka Graczyk-Jarzynka, Karolina Soroczynska, Szczepan Cierniak, Robert Koktysz, Esther Elishaev, Slawomir Gruca, Artur Stefanowicz, Roman Blaszczyk, Bartlomiej Borek, Anna Gzik, Theresa Whiteside, and Jakub Golab Journal Nat Commun Abstract Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (show more...)Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule – ARG1, mitigating anti-tumor immune responses. (hide) EV-METRIC 50% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin ovarian cancer 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 qEV Protein markers EV: TSG101/ ARG1/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ ARG1/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 126 EV concentration Yes TRPS Report type Mean Reported size (nm) 58 EV concentration Yes

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EV-TRACK ID	EV190025
species	Homo sapiens
sample type	ascites	ovarian cyst fluid	Blood plasma	Blood plasma
condition	ovarian cancer	Control condition	Control condition	ovarian cancer
separation protocol	(d)(U)C Filtration	(d)(U)C Filtration	(d)(U)C Filtration qEV	(d)(U)C Filtration qEV
Exp. nr.	3	4	1	2
EV-METRIC %	78	78	50	50