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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180083	1/2	Mus musculus	5TGM1	DG (d)(U)C ExoQuick	Faict S	2018	78%
Study summary Full title Exosomes play a role in multiple myeloma bone disease and tumor development by targeting osteoclasts and osteoblasts. All authors Faict S, Muller J, De Veirman K, De Bruyne E, Maes K, Vrancken L, Heusschen R, De Raeve H, Schots R, Vanderkerken K, Caers J, Menu E. Journal blood cancer j Abstract Progression of multiple myeloma (MM) is largely dependent on the bone marrow (BM) microenvironment w (show more...)Progression of multiple myeloma (MM) is largely dependent on the bone marrow (BM) microenvironment wherein communication through different factors including extracellular vesicles takes place. This cross-talk not only leads to drug resistance but also to the development of osteolysis. Targeting vesicle secretion could therefore simultaneously ameliorate drug response and bone disease. In this paper, we examined the effects of MM exosomes on different aspects of osteolysis using the 5TGM1 murine model. We found that 5TGM1 sEVs, or 'exosomes', not only enhanced osteoclast activity, they also blocked osteoblast differentiation and functionality in vitro. Mechanistically, we could demonstrate that transfer of DKK-1 led to a reduction in Runx2, Osterix, and Collagen 1A1 in osteoblasts. In vivo, we uncovered that 5TGM1 exosomes could induce osteolysis in a similar pattern as the MM cells themselves. Blocking exosome secretion using the sphingomyelinase inhibitor GW4869 not only increased cortical bone volume, but also it sensitized the myeloma cells to bortezomib, leading to a strong anti-tumor response when GW4869 and bortezomib were combined. Altogether, our results indicate an important role for exosomes in the BM microenvironment and suggest a novel therapeutic target for anti-myeloma therapy. (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 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 DG (d)(U)C ExoQuick Protein markers EV: / TSG101/ CD63/ Syntenin/ CD81 non-EV: / Calreticulin Proteomics no EV density (g/ml) 1.08 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 5TGM1 EV-harvesting Medium Serum free 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: time(min) 180 Pelleting: rotor type SW 41 Ti Pelleting: 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) 12 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 10800 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ TSG101/ Syntenin/ CD81 Not detected EV-associated proteins Detected contaminants Not detected contaminants Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 114 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV180083	2/2	Mus musculus	5T33MMvt	DG (d)(U)C ExoQuick	Faict S	2018	43%
Study summary Full title Exosomes play a role in multiple myeloma bone disease and tumor development by targeting osteoclasts and osteoblasts. All authors Faict S, Muller J, De Veirman K, De Bruyne E, Maes K, Vrancken L, Heusschen R, De Raeve H, Schots R, Vanderkerken K, Caers J, Menu E. Journal blood cancer j Abstract Progression of multiple myeloma (MM) is largely dependent on the bone marrow (BM) microenvironment w (show more...)Progression of multiple myeloma (MM) is largely dependent on the bone marrow (BM) microenvironment wherein communication through different factors including extracellular vesicles takes place. This cross-talk not only leads to drug resistance but also to the development of osteolysis. Targeting vesicle secretion could therefore simultaneously ameliorate drug response and bone disease. In this paper, we examined the effects of MM exosomes on different aspects of osteolysis using the 5TGM1 murine model. We found that 5TGM1 sEVs, or 'exosomes', not only enhanced osteoclast activity, they also blocked osteoblast differentiation and functionality in vitro. Mechanistically, we could demonstrate that transfer of DKK-1 led to a reduction in Runx2, Osterix, and Collagen 1A1 in osteoblasts. In vivo, we uncovered that 5TGM1 exosomes could induce osteolysis in a similar pattern as the MM cells themselves. Blocking exosome secretion using the sphingomyelinase inhibitor GW4869 not only increased cortical bone volume, but also it sensitized the myeloma cells to bortezomib, leading to a strong anti-tumor response when GW4869 and bortezomib were combined. Altogether, our results indicate an important role for exosomes in the BM microenvironment and suggest a novel therapeutic target for anti-myeloma therapy. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C ExoQuick Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.08 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 5T33MMvt EV-harvesting Medium Serum free 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: time(min) 180 Pelleting: rotor type SW 41 Ti Pelleting: 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) 12 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 10800 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis None Protein Concentration Method BCA Western Blot Detected EV-associated proteins Not detected EV-associated proteins Detected contaminants Not detected contaminants Characterization: Lipid analysis No EM EM-type EV concentration

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EV-TRACK ID	EV180083
species	Mus musculus
sample type	Cell culture
cell type	5TGM1	5T33MMvt
condition	Control condition	Control condition
separation protocol	DG (d)(U)C ExoQuick	DG (d)(U)C ExoQuick
Exp. nr.	1	2
EV-METRIC %	78	43