Transplantation of autologous extracellular vesicles for ...
Extracellular Vesicles in Liquid Biopsies and...
Transcript of Extracellular Vesicles in Liquid Biopsies and...
Coordinación científica:
Dr. Rafael López López
Complejo Hospitalario Universitario de Santiago de
Compostela
Organizado
por: Sede:
San Francisco Hotel Monumento
Campillo de San Francisco, 3 - Santiago de
Compostela
Extracellular Vesicles in Liquid Biopsies
and Cancer
Prof. Dr. Guido Jenster
Experimental Urological Oncology
Erasmus MC Rotterdam, The Netherlands
Théry C et al., Nat Rev Immunol. 2009 Aug;9(8):581-93
PSA
Consecutive secretory pathway
Exosomes and Microvesicles: EVs
shedding budding
endocytosis
exocytosis
merocrine
EVs contain proteins and RNAs from host cell
Research: Biomarker
Functional
Therapy
CIRCULATING TUMOR
CELLS
Urine
Serum
CELL-FREE
RNA
Prostate Cancer Research: Liquid biopsy
PROTEIN
METABOLITES
EXTRACELLULAR
VESICLES (EV-RNA; EV-
PROTEINS; EV-DNA)
CELL-FREE
DNA
PLATELETS
VIRUSES
MICRO-
ORGANISMS
Immune cells
Tissue Matrix
Stromal cells
Epithelial cells
Endothelial cells
Affect:
- Immune response
- Migration
- Growth
- Therapy response
Create metastatic niche
Exosomes and Microvesicles
Integrins on exosomes
affect organ homing
Functional role of
extracellular vesicles
Exosomes and Microvesicles: Functional activity
Zomer A. et al., Cell. 2015 May 21;161(5):1046-57.
Exosome Research: commercial EV isolation kits and visualization of EV
OptiPrep gradient
ExoQuick
ExoSpin ExoPrep
ExoEasy
ExoRNeasy
qEV
exoCaP
Norgen Biotek
Electron microscopy qNano
Tunable Resistive Pulse Sensing
Nanoparticle Tracking (NanoSight)
EVQuant: direct labeling of EVs in biofluids
Isolated EVs Dye only
Culture medium Urine
microscope
100 uL urine after
3000 xg centrifugation
Hartjes T, van Royen M unpublished
EVQuant; EV production in cells
Average production of
EVs per cell per hour:
~2000
Blood & Urine contain
1-10 109 EVs/ml
NUMBER
RNA
TMPRSS2
CD63
PSMA
Prostate cancer research: Exosomes as markers
PROTEIN
PSMA
UPK2
TRPA1
IL9R
SLC12
AQP
Prostate-derived
Bladder-derived
Kidney-derived
miRNAs
AR variants
PCA3, TMPRSS2-ERG
FGFR3 mutations
Transmembrane proteins
Intra-vesicular proteins Count (cancer)-derived vesicles
EVQuant: Biomarker detection (multicolor imaging)
Detect individual vesicles with different phenotypes
Membrane labeling TM-Protein1-alexa647 TM-Protein2-alexa488
Hartjes T, van Royen M, unpublished
100 uL urine
after
3000 xg centrifugation
N=16 N=16 N=12 N=37 N=41
DRE
PCa+
DRE
PCa-
noDRE
PCa+
Rad
Pros
Women
EVs: Time-Resolved Fluorescence Immuno Assay
Exosome TR-FIA: normalize for amount prostate fluid
Men without PCa Men with PCa
CD63 TR-FIA after correction for urinary PSA, is higher in men with PCa
Duijvesz et al., Int J Cancer. 2015 Dec 15;137(12):2869-78
Nanobody BioPanning EV-Nanobodies Joke Veldhoven, Janne Leivo, Guido Jenster
Urinary EVs from men with and without PCa
Y Y PCa PCa
Y Y N N
TR-FIA:
Time-Resolved
Fluorescence
ImmunoAssay
Llama
EVs from PCa cell lines
Immunize
Nanobody
Library
Diagnosis and prognosis of urogenital diseases
Urinary RNA and DNA from:
Kidney
Bladder
Prostate
Other organs
Representing:
Infection
Cancer
Stones
Chronic disease
Congenital diseases
Transplantation
Children, women
and men RNAseq of urine: TMPRSS2-ERG fusion transcript detected in a man with PCa
ERG
www.urinome.nl
TMPRSS2
RNA Next Generation Sequencing
Prostate Cancer Research: The future
Next Generation
Sequencing
RT-PCR
ELISA / TR-FIA
EVQuant
New Assays
CTCs
Exosomes
cfDNA
Thanks to:
Ron van Schaik
Clinical Chemistry, EMC
Flip Jansen
Diederick Duijvesz
Natasja Dits
Mirella van den Berg
Matthijs Bekkers
David Nieuwenhuikse
Joke Zweistra
Wytske van Weerden
Chris Bangma
Urology, EMC
Ewout Hoorn
Mahdi Salih
Nefrology, EMC
Marzia Del Re
Stefania Crucitta
Romano Danesi
Clin & Exp Medicine, Pisa, Italy
Martin van Royen
Thomas Hartjes
Adriaan Houtsmuller
Ellen Zwarthoff
Pathology, EMC
Kim Pettersson
Janne Leivo
Turku University, Finland
All volunteers and patients for
their samples and cooperation
Roy van der Meel
Raymond Schiffelers
University Utrecht
John Hayes
Bacteriology, EMC
Youri Hoogstrate
Andrew Stubbs
Bioinformatics, EMC
Harmen van de Werken
Job van Riet
CCBC, EMC