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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140017	1/1	Homo sapiens	Serum	(d)(U)C DG	Di Noto G	2014	56%
Study summary Full title Immunoglobulin Free Light Chains and GAGs Mediate Multiple Myeloma Extracellular Vesicles Uptake and Secondary NfkappaB Nuclear Translocation All authors Di Noto G, Chiarini M, Paolini L, Mazzoldi EL, Giustini V, Radeghieri A, Caimi L, Ricotta D Journal Front Immunol Abstract Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistenc (show more...)Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. Monoclonal plasma cells often secrete high amounts of immunoglobulin free light chains (FLCs) that could induce tissue damage. Recently, we showed that FLCs are internalized in endothelial and myocardial cell lines and secreted in extracellular vesicles (EVs). MM serum derived EVs presented phenotypic differences if compared with monoclonal gammopathy of undetermined significance (MGUS) serum derived EVs suggesting their involvement in MM pathogenesis or progression. To investigate the effect of circulating EVs on endothelial and myocardial cells, we purified MM and MGUS serum derived EVs with differential ultracentrifugation protocols and tested their biological activity. We found that MM and MGUS EVs induced different proliferation and internalization rates in endothelial and myocardial cells, thus we tried to find specific targets in MM EVs docking and processing. Pre-treatment of EVs with anti-FLCs antibodies or heparin blocked the MM EVs uptake, highlighting that FLCs and glycosaminoglycans are involved. Indeed, only MM EVs exposure induced a strong nuclear factor kappa B nuclear translocation that was completely abolished after anti-FLCs antibodies and heparin pre-treatment. The protein tyrosine kinase c-src is present on MM circulating EVs and redistributes to the cell plasma membrane after MM EVs exposure. The anti-FLCs antibodies and heparin pre-treatments were able to block the intracellular re-distribution of the c-src kinase and the subsequent c-src kinase containing EVs production. Our results open new insights in EVs cellular biology and in MM therapeutic and diagnostic approaches. (hide) EV-METRIC 56% (92nd 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 Serum Sample origin NAY Focus vesicles 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 DG Protein markers EV: TSG101/ CD63/ Annexin5/ HSP70 non-EV: Proteomics no EV density (g/ml) 1.11-1.22 Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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 Density gradient Lowest density fraction 15 Highest density fraction 60 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ HSP70/ TSG101/ Annexin5 ELISA Detected EV-associated proteins Annexin5 Flow cytometry specific beads Selected surface protein(s) Yes Characterization: Particle analysis EM EM-type scanning EM/ atomic force EM Image type Close-up, Wide-field Report size (nm) Not reported

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EV-TRACK ID	EV140017
species	Homo sapiens
sample type	Serum
condition	NAY
separation protocol	(d)(U)C DG
Exp. nr.	1
EV-METRIC %	56