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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV180041 1/1 Mus musculus Cell culture supernatant DG
dUC
Keklikoglou I 2018 100%

Study summary

Full title
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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
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
Sample Condition
Control condition
EV-producing cells
4T1
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
40
Isolation Method
Differential ultracentrifugation
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: 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
Density medium
Sucrose
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 Concentration
7.3
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, Syntenin-1
Not detected contaminants
Gp96
ELISA
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Proteomics database
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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