Search > Results

You searched for: EV210488 (EV-TRACK ID)

Showing 1 - 11 of 11

Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK code Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV210488 1/11 Homo sapiens Blood plasma (d)(U)C
SEC (non-commercial)
DG
van Eijndhoven MA 2016 50%

Study summary

Full title
All authors
van Eijndhoven MA, Zijlstra JM, Groenewegen NJ, Drees EE, van Niele S, Baglio SR, Koppers-Lalic D, van der Voorn H, Libregts SF, Wauben MH, de Menezes RX, van Weering JR, Nieuwland R, Visser L, van den Berg A, de Jong D, Pegtel DM
Journal
JCI insight
Abstract
Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive bioma (show more...)Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID). (hide)
EV-METRIC
50% (85th 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
Blood plasma
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
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Density gradient
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
15
Lowest density fraction
0.4M
Highest density fraction
2M
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
840-1200
Fraction volume (mL)
1
Fraction processing
None
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
1.5
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR/ RNA sequencing/ Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
50-500
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
Influx (Becton Dickinson)
Hardware adjustment
BD Influx cell sorter Influx cell sorter , which was equipped by Cytopeia and BD with: 488nm laser (200 mW)/ 561-nm laser (100 mW)/ 635-nm CUBE laser (30 mW) / SPD, containing a Mitutoyo Plan Apo 20 lens (NA 0.42), a 0.7-mm pinhole and a polarization unit with two FSC PMTs/ Optical filters/ amplifier board with 2 'fast' preamplifiers (0.12 s) and 6 'slow' preamplifiers (1.2 s)/ and Spigot software, version 6.1.
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV210488 5/11 Homo sapiens Cell culture supernatant (d)(U)C van Eijndhoven MA 2016 14%

Study summary

Full title
All authors
van Eijndhoven MA, Zijlstra JM, Groenewegen NJ, Drees EE, van Niele S, Baglio SR, Koppers-Lalic D, van der Voorn H, Libregts SF, Wauben MH, de Menezes RX, van Weering JR, Nieuwland R, Visser L, van den Berg A, de Jong D, Pegtel DM
Journal
JCI insight
Abstract
Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive bioma (show more...)Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID). (hide)
EV-METRIC
14% (38th 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
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
L1236
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Other/ 16h at 70,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
70000
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
70000
Density gradient
Only used for validation of main results
Yes
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV210488 6/11 Homo sapiens Cell culture supernatant (d)(U)C van Eijndhoven MA 2016 14%

Study summary

Full title
All authors
van Eijndhoven MA, Zijlstra JM, Groenewegen NJ, Drees EE, van Niele S, Baglio SR, Koppers-Lalic D, van der Voorn H, Libregts SF, Wauben MH, de Menezes RX, van Weering JR, Nieuwland R, Visser L, van den Berg A, de Jong D, Pegtel DM
Journal
JCI insight
Abstract
Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive bioma (show more...)Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID). (hide)
EV-METRIC
14% (38th 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
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
KMH2
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Other/ 16h at 70,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
70000
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
70000
Density gradient
Only used for validation of main results
Yes
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV210488 7/11 Homo sapiens Cell culture supernatant (d)(U)C van Eijndhoven MA 2016 14%

Study summary

Full title
All authors
van Eijndhoven MA, Zijlstra JM, Groenewegen NJ, Drees EE, van Niele S, Baglio SR, Koppers-Lalic D, van der Voorn H, Libregts SF, Wauben MH, de Menezes RX, van Weering JR, Nieuwland R, Visser L, van den Berg A, de Jong D, Pegtel DM
Journal
JCI insight
Abstract
Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive bioma (show more...)Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID). (hide)
EV-METRIC
14% (38th 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
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Ly3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Other/ 16h at 70,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
70000
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
70000
Density gradient
Only used for validation of main results
Yes
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV210488 8/11 Homo sapiens Cell culture supernatant (d)(U)C van Eijndhoven MA 2016 14%

Study summary

Full title
All authors
van Eijndhoven MA, Zijlstra JM, Groenewegen NJ, Drees EE, van Niele S, Baglio SR, Koppers-Lalic D, van der Voorn H, Libregts SF, Wauben MH, de Menezes RX, van Weering JR, Nieuwland R, Visser L, van den Berg A, de Jong D, Pegtel DM
Journal
JCI insight
Abstract
Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive bioma (show more...)Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID). (hide)
EV-METRIC
14% (38th 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
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Ly10
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Other/ 16h at 70,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
70000
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
70000
Density gradient
Only used for validation of main results
Yes
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV210488 9/11 Homo sapiens Cell culture supernatant (d)(U)C van Eijndhoven MA 2016 14%

Study summary

Full title
All authors
van Eijndhoven MA, Zijlstra JM, Groenewegen NJ, Drees EE, van Niele S, Baglio SR, Koppers-Lalic D, van der Voorn H, Libregts SF, Wauben MH, de Menezes RX, van Weering JR, Nieuwland R, Visser L, van den Berg A, de Jong D, Pegtel DM
Journal
JCI insight
Abstract
Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive bioma (show more...)Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID). (hide)
EV-METRIC
14% (38th 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
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HL4
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Other/ 16h at 70,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
70000
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
70000
Density gradient
Only used for validation of main results
Yes
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV210488 10/11 Homo sapiens Cell culture supernatant (d)(U)C van Eijndhoven MA 2016 14%

Study summary

Full title
All authors
van Eijndhoven MA, Zijlstra JM, Groenewegen NJ, Drees EE, van Niele S, Baglio SR, Koppers-Lalic D, van der Voorn H, Libregts SF, Wauben MH, de Menezes RX, van Weering JR, Nieuwland R, Visser L, van den Berg A, de Jong D, Pegtel DM
Journal
JCI insight
Abstract
Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive bioma (show more...)Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID). (hide)
EV-METRIC
14% (38th 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
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HL5
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Other/ 16h at 70,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
70000
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
70000
Density gradient
Only used for validation of main results
Yes
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV210488 11/11 Homo sapiens Cell culture supernatant (d)(U)C van Eijndhoven MA 2016 14%

Study summary

Full title
All authors
van Eijndhoven MA, Zijlstra JM, Groenewegen NJ, Drees EE, van Niele S, Baglio SR, Koppers-Lalic D, van der Voorn H, Libregts SF, Wauben MH, de Menezes RX, van Weering JR, Nieuwland R, Visser L, van den Berg A, de Jong D, Pegtel DM
Journal
JCI insight
Abstract
Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive bioma (show more...)Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID). (hide)
EV-METRIC
14% (38th 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
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
IK140508
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Other/ 16h at 70,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
70000
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
70000
Density gradient
Only used for validation of main results
Yes
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV210488 2/11 Homo sapiens Blood plasma (d)(U)C
SEC (non-commercial)
van Eijndhoven MA 2016 0%

Study summary

Full title
All authors
van Eijndhoven MA, Zijlstra JM, Groenewegen NJ, Drees EE, van Niele S, Baglio SR, Koppers-Lalic D, van der Voorn H, Libregts SF, Wauben MH, de Menezes RX, van Weering JR, Nieuwland R, Visser L, van den Berg A, de Jong D, Pegtel DM
Journal
JCI insight
Abstract
Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive bioma (show more...)Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID). (hide)
EV-METRIC
0% (median: 17% 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
Blood plasma
Sample origin
Classical Hodgkin lymphoma
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
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
1.5
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR/ RNAsequencing/ Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
50-500
EV concentration
Yes
EV210488 3/11 Homo sapiens Blood plasma (d)(U)C
SEC (non-commercial)
van Eijndhoven MA 2016 0%

Study summary

Full title
All authors
van Eijndhoven MA, Zijlstra JM, Groenewegen NJ, Drees EE, van Niele S, Baglio SR, Koppers-Lalic D, van der Voorn H, Libregts SF, Wauben MH, de Menezes RX, van Weering JR, Nieuwland R, Visser L, van den Berg A, de Jong D, Pegtel DM
Journal
JCI insight
Abstract
Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive bioma (show more...)Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID). (hide)
EV-METRIC
0% (median: 17% 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
Blood plasma
Sample origin
Systemic lupus erythematosus
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
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
1.5
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV210488 4/11 Homo sapiens Serum (d)(U)C
SEC (non-commercial)
van Eijndhoven MA 2016 0%

Study summary

Full title
All authors
van Eijndhoven MA, Zijlstra JM, Groenewegen NJ, Drees EE, van Niele S, Baglio SR, Koppers-Lalic D, van der Voorn H, Libregts SF, Wauben MH, de Menezes RX, van Weering JR, Nieuwland R, Visser L, van den Berg A, de Jong D, Pegtel DM
Journal
JCI insight
Abstract
Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive bioma (show more...)Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID). (hide)
EV-METRIC
0% (median: 13% 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
Classical Hodgkin lymphoma
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
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
1.5
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
1 - 11 of 11
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV210488
species
Homo
sapiens
sample type
Blood
plasma
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Blood
plasma
Blood
plasma
Serum
cell type
NA
L1236
KMH2
Ly3
Ly10
HL4
HL5
IK140508
NA
NA
NA
medium
NA
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
NA
NA
NA
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Classical
Hodgkin
lymphoma
Systemic
lupus
erythematosus
Classical
Hodgkin
lymphoma
separation protocol
dUC/
Size-exclusion
chromatography
(non-commercial)/
Density
gradient
dUC
dUC
dUC
dUC
dUC
dUC
dUC
dUC/
Size-exclusion
chromatography
(non-commercial)
dUC/
Size-exclusion
chromatography
(non-commercial)
dUC/
Size-exclusion
chromatography
(non-commercial)
Exp. nr.
1
5
6
7
8
9
10
11
2
3
4
EV-METRIC %
50
14
14
14
14
14
14
14
0
0
0