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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210056	1/2	Mus musculus	WEHI3B	DG (d)(U)C	Gu, Xiaoyu	2015	25%
Study summary Full title Improved vaccine efficacy of tumor exosome compared to tumor lysate loaded dendritic cells in mice All authors Xiaoyu Gu, Ulrike Erb, Markus W Büchler, Margot Zöller Journal Int J Cancer Abstract Leukemia immunotherapy frequently does not meet expectation, one of the handicaps being tumor exosom (show more...)Leukemia immunotherapy frequently does not meet expectation, one of the handicaps being tumor exosome (TEX)-promoted immunosuppression. We here asked, using the mouse myeloid leukemia WEHI3B and the renal cell carcinoma line RENCA, whether dendritic cell (DC) vaccination suffices to counterregulate TEX-induced immunosuppression and whether TEX could serve as tumor antigen for DC-loading. DC-vaccination significantly prolonged the survival time of WEHI3B-bearing mice, TEX-loaded DC (DC-TEX) being superior to lysate-loaded DC (DC-lys), even an excess of TEX not interfering with immune response induction. The superior response to DC-TEX was accompanied by an increase in WEHI3B-specific CD4+ T cells, evaluated by trogocytosis and proliferation. Similar findings accounted for DC loaded with RENCA TEX. TEX was efficiently taken-up by DC and TEX uptake supported CD11c, MHCII and IL12 upregulation in DC. Importantly, TEX was partly recruited into the MHCII-loading compartment such that TEX presentation time and recovery in T cells significantly exceeded that of tumor-lysate. Thus, TEX did not drive DC into a suppressive phenotype and were a superior antigen due to higher efficacy of TEX-presentation that is supported by prolonged persistence, preferential processing in the MHCII-loading compartment and pronounced trogocytosis by T helper cells. TEX is present in tumor patients' sera. TEX, recovered and enriched from patients' sera, might well provide an optimized, individual-specific antigen source for DC-loading and vaccination. (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 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 DG (d)(U)C Protein markers EV: CD24/ CD34/ CD90/ CD117/ SCA1/ CD16/32/ CD31/ CD44/ CD9/ CD63/ CD81/ MFGE8/ HSP70/ IL3/ IL6/ IL7/ CCR9/ CXCR3/ CXCR4 non-EV: None Proteomics no EV density (g/ml) Not specified Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells WEHI3B EV-harvesting Medium Serum free medium 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) 90 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 90 Wash: Rotor Type Not specified Wash: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 0.25M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Bottom-up Rotor type Not specified Speed (g) 150000 Duration (min) 900 Fraction volume (mL) Not specified Fraction processing Not specified Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD24/ CD34/ CD90/ CD117/ SCA1/ CD16/32/ CD31/ CD44/ CD9/ CD63/ CD81/ MFGE8/ HSP70/ IL3/ IL6/ IL7/ CCR9/ CXCR3/ CXCR4 Characterization: Lipid analysis No

	EV210056	2/2	Mus musculus	RENCA	DG (d)(U)C	Gu, Xiaoyu	2015	14%
Study summary Full title Improved vaccine efficacy of tumor exosome compared to tumor lysate loaded dendritic cells in mice All authors Xiaoyu Gu, Ulrike Erb, Markus W Büchler, Margot Zöller Journal Int J Cancer Abstract Leukemia immunotherapy frequently does not meet expectation, one of the handicaps being tumor exosom (show more...)Leukemia immunotherapy frequently does not meet expectation, one of the handicaps being tumor exosome (TEX)-promoted immunosuppression. We here asked, using the mouse myeloid leukemia WEHI3B and the renal cell carcinoma line RENCA, whether dendritic cell (DC) vaccination suffices to counterregulate TEX-induced immunosuppression and whether TEX could serve as tumor antigen for DC-loading. DC-vaccination significantly prolonged the survival time of WEHI3B-bearing mice, TEX-loaded DC (DC-TEX) being superior to lysate-loaded DC (DC-lys), even an excess of TEX not interfering with immune response induction. The superior response to DC-TEX was accompanied by an increase in WEHI3B-specific CD4+ T cells, evaluated by trogocytosis and proliferation. Similar findings accounted for DC loaded with RENCA TEX. TEX was efficiently taken-up by DC and TEX uptake supported CD11c, MHCII and IL12 upregulation in DC. Importantly, TEX was partly recruited into the MHCII-loading compartment such that TEX presentation time and recovery in T cells significantly exceeded that of tumor-lysate. Thus, TEX did not drive DC into a suppressive phenotype and were a superior antigen due to higher efficacy of TEX-presentation that is supported by prolonged persistence, preferential processing in the MHCII-loading compartment and pronounced trogocytosis by T helper cells. TEX is present in tumor patients' sera. TEX, recovered and enriched from patients' sera, might well provide an optimized, individual-specific antigen source for DC-loading and vaccination. (hide) EV-METRIC 14% (44th 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 DG (d)(U)C Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) Not specified Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells RENCA EV-harvesting Medium Serum free medium 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) 90 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 90 Wash: Rotor Type Not specified Wash: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 0.25M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Bottom-up Rotor type Not specified Speed (g) 150000 Duration (min) 900 Fraction volume (mL) Not specified Fraction processing Not specified Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No

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EV-TRACK ID	EV210056
species	Mus musculus
sample type	Cell culture
cell type	WEHI3B	RENCA
condition	Control condition	Control condition
separation protocol	DG (d)(U)C	DG (d)(U)C
Exp. nr.	1	2
EV-METRIC %	25	14