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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180041	1/1	Mus musculus	4T1	DG (d)(U)C	Keklikoglou I	2018	100%
Study summary Full title Chemotherapy elicits pro-metastatic extracellular vesicles in breast cancer models. All authors Keklikoglou I, Cianciaruso C, Güç E, Squadrito ML, Spring LM, Tazzyman S, Lambein L, Poissonnier A, Ferraro GB, Baer C, Cassará A, Guichard A, Iruela-Arispe ML, Lewis CE, Coussens LM, Bardia A, Jain RK, Pollard JW, De Palma M Journal Nat Cell Biol Abstract Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental s (show more...)Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca²⁺-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C⁺CCR2⁺ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy. (hide) EV-METRIC 100% (99th 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 Adj. k-factor 191 (pelleting) / 191 (washing) Protein markers EV: CD81/ CD9/ Syntenin-1 non-EV: Gp96 Proteomics yes Show all info Study aim Function, Biomarker, Biogenesis/cargo sorting, Mechanism of uptake/transfer, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 4T1 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 134000 Pelleting: adjusted k-factor 191.0 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 134000 Wash: adjusted k-factor 191.0 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 10 Highest density fraction 50 Sample volume (mL) 0.1 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 70 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 70 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 251.4 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 7.3 Western Blot Detected EV-associated proteins CD9, CD81, Syntenin-1 Not detected contaminants Gp96 ELISA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 140 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV180041
species	Mus musculus
sample type	Cell culture
cell type	4T1
condition	Control condition
separation protocol	DG (d)(U)C
Exp. nr.	1
EV-METRIC %	100