exosomes and microvesicles slides … · cd59, annexins, rab proteins merocrine and apocrine...
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Exosomes and Microvesicles
Prof. Dr. Guido Jenster Experimental Urological Oncology Erasmus MC Rotterdam [email protected]
Cancer-derived proteins in serum
Athymic nude mouse
serum
Mouse proteins
Xenograft Human prostate cancer: PC346 or PC339
Athymic nude mouse
serum
Xenograft-derived proteins Mouse proteins +
Human specific!
Methods - overview
Removal of abundant proteins (albumin, IgG, transferrin)
Nu-/- mouse before inoculation
Collection of serum
LC Separation
LTQ-FT
Trypsin digestion
SDS-PAGE 1D
Nu-/- mouse bearing a xenograft
GAPDH: 6 discriminatory human peptides 2 shared peptides
HUMAN MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENG MOUSE ..MVKVGVNGFGRIGRLVTRAAICSGKVEIVAINDPFIDLNYMVYMFQYDSTHGKFNGTVKAENG HUMAN KLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPM MOUSE KLVINGKPITIFQERDPTNIKWGEAGAEYVVESTGVFTTMEKAGAHLKGGAKRVIISAPSADAPM HUMAN FVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKL MOUSE FVMGVNHEKYDNSLKIVSNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKL HUMAN WRDGRGALQNIIPASTGAAKAVGKVIPELDGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKK MOUSE WRDGRGAAQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTPNVSVVDLTCRLEKPAKYDDIKK HUMAN VVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVV MOUSE VVKQASEGPLKGILGYTEDQVVSCDFNSNSHSSTFDAGAGIALNDNFVKLISWYDNEYGYSNRVV HUMAN DLMAHMASKE MOUSE DLMAYMASKE
Van den Bemd et al., Mol Cell Proteomics 2006; 1830-1839
Results – proteins identified in HUPO
Glycolysis related proteins
Alpha-enolase
Glutathione peroxidase 3
Nucleoside diphosphate kinase A
Nucleoside diphosphate kinase B
Fructose-bisphosphate aldolase A
Glyceraldehyde-3-phosphate dehydrogenase
Lactate dehydrogenase A
Lactate dehydrogenase B
Maltase-glucoamylase, intestinal
Triosephosphate isomerase 1
Proteasome subunits
Proteasome subunit alpha type 4
Proteasome subunit alpha type 7
Proteasome subunit alpha type 6
Proteasome subunit beta type 2
Proteasome subunit beta type 8
Proteasome subunit alpha type 1
Proteasome subunit alpha type 2
Proteasome subunit beta type 1
Proteasome subunit beta type 3
Proteasome subunit beta type 4
Proteasome subunit beta type 5
Proteasome subunit beta type 6
Other proteins
Cathepsin Z
Coactosin
Cofilin
Inter alpha inhibitor H3
Lumican
Peroxiredoxin-2
Thrombospondin-1
Complement factor B
14-3-3-tau
Complement factor B
Junction plakoglobin
Prothrombin
Van den Bemd et al., Mol Cell Proteomics 2006; 1830-1839 Jansen et al., Mol Cell Proteomics 2009; 1192-1205
HUPO meeting 2006: Irmgard Schwarte-Waldhoff Department of Internal Medicine, IMBL, Ruhr-Universität Bochum, Germany
So how do these cytoplasmic and nuclear proteins end up in serum?
- Apoptosis / necrosis
- Specific secretion
Human xenograft-derived proteins
Théry C et al., Nat Rev Immunol. 2009 Aug;9(8):581-93
PSA
Consecutive secretory pathway
Exosomes and Microvesicles
shedding budding
endocytosis
exocytosis
merocrine
apocrine
Shao et al., Nature Medicine 18, 1835–1840 (2012)
Duijvesz et al., Eur Urol. 2011;59(5):823-31.
Types of Extracellular Vesicles
"Biologists would rather share a toothbrush than share a gene name"
Vesicle Size (nm) Main protein markers Synthesis pathway Function
Exosomes 50-150CD9, CD63, CD81, CD82, Annexins, RAB proteins
MerocrineAntigen presentation, immune regulatory, metastatic activity
Prostasomes 50-500CD13,CD46, CD55, CD59, Annexins, RAB proteins
Merocrine and apocrine
Immunosuppressive, sperm cell motil ity improving
Oncosomes 50-500 (DIAPH3) Apocrine ND
Microvesicles 100-1000 Integrins, selectins, CD40 ligand
Apocrine Procoagulation and anticoagulation
Ectosomes 50-1000 CR1, proteolytic enzymes Apocrine
Procoagulation and anticoagulation
Apoptotic vesicle 50-5000 DNA Apocrine Left over from apoptosis
PSA
Exosomes and Microvesicles
Ludwig & Giebel. IJB&CB 2012; 44: 11-15
Endosomal Sorting Complexes Required for Transport (ESCRT)
Contents – proteins (novel biomarkers) – RNA (miRNAs, mRNAs) – Organ-specific transmembrane
proteins
Characteristics – 30-150 nm – secreted by living cells
CD9
THE ROLE OF EXOSOMES
Duijvesz et al., Eur Urol. 2011;59(5):823-31.
FUNCTIONAL MARKER THERAPY
- Looking for the needle in the haystack and finding the farmer’s daughter - Exosomes and microvesicles - function - how to visualize, count and track them? - how taken up by other cells? - what is inside these vesicles? - how can we use them? - Conclusions
Exosomes and Microvesicles
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
EV Research: benefits for EV uptake
What is the benefit of EVs to target cells?
1) No benefit EVs are taken up by the unspecific process of endocytosis
2) Signaling EVs contain hormones, growth factors, RNA and DNA
3) Acquiring (new) enzyme activity EVs contain enzymes that can generate valuable metabolites
4) Acquiring food (energy and metabolites) EVs contain essential metabolites, some with energy
signaling
substrate
product
ATP, amino acids vitamins, etc.
Exosomes and Microvesicles: Functional activity
Zomer A. et al., Cell. 2015 May 21;161(5):1046-57.
Extracellular vesicles play a role in metastatic behavior
Exosomes and Microvesicles
How would you isolate microvesicles?
-Ultracentrifugation
-Filtration (filters, chromatography)
-Affinity purification
-Precipitation
Exosome Research: commercial EV isolation kits
OptiPrep gradient
ExoQuick
ExoSpin
ExoPrep
ExoEasy ExoRNeasy
qEV
exoCaP
Norgen Biotek
Exosomes and Microvesicles
How would you visualize and count microvesicles?
-Electron microscopy
-Labeling (PKH26) and confocal microscopy
-ELISA
-Fluorescence flow cytometry
-Tunable Resistive Pulse Sensing (qNano)
-Light scattering / Brownian motion (NanoSight)
• Without EV purification • No washing of unbound dye • Automation in 96 wells format
EVQuant: How does it work?
Sample preparation
• Isolated EVs • Culture medium • Urine • Serum/Plasma
Martin van Royen & Thomas Hartjes
EVQuant; How does it work?
RAW image Image processing EV quantification
Image analysis using open source software (ImageJ) Calculated bead concentrations correlated linearly with the measured bead concentrations
Beads (100nm)
r2=0.99
EVQuant; How does it work?
Isolated EVs Dye only
Culture medium Urine
EVQuant; EV production in cells
CrazyQuant
18-07-2016
Exosomes: Time Resolved-Fluorescence Immuno Assay
Y
Y
Y CD9 capture
CD9 detection
Eu
Y
Y
Y CD9 capture
PCa-specific detection
Eu
- Looking for the needle in the haystack and finding the farmer’s daughter - Exosomes and microvesicles - function - how to visualize, count and track them? - how taken up by other cells? - what is inside these vesicles? - how can we use them? - Conclusions
Exosomes and Microvesicles
Co-localization studies at a large time scale (Confocal)
Examine the different phases of uptake and processing of exosomes
Seconds Hours Days Minutes
Clathrin, Exosomes
Rab4a (early endosome), Exosomes Exosomes, Lysosomes
Rab11, Exosomes
Thomas Hartjes, Martin van Royen
- Looking for the needle in the haystack and finding the farmer’s daughter - Exosomes and microvesicles - function - how to visualize, count and track them? - how taken up by other cells? - what is inside these vesicles? - how can we use them? - Conclusions
Exosomes and Microvesicles
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
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
53 12
78
18
25
491 23
52
136
78
147
96 13 13
147
PNT2C2
RWPE1 PC346C
VCaP
Exosomes: Exosomal proteins from cell lines
Cell lines from normal prostate
Cell lines from prostate cancer
PNT2C2: - 637 proteins RWPE1: - 476 proteins PC346C: - 274 proteins VCaP: - 896 proteins
Duijvesz et al., PLoS One. 2013; 8:e82589
Prostate-specific transmembrane proteins: PSMA, TMPRSS2 STEAP2/4 PPAP2A CD13
Exosomes: Marker selection and Western blotting
PNT2C2 RWPE1 PC346C VCaP
cells
exos
omes
cells
exos
omes
cells
exos
omes
cells
exos
omes
Duijvesz et al., PLoS One. 2013; 8:e82589
Vesiclepedia: Kalra et al., PLoS Biol. 2012; 10:e1001450
Exosomes: Affymetrix Exon array analyses
PCa cell line RNA
Exosomal RNA
Exosomal RNA
Exosomes: Affymetrix Exon array analyses
Differences in mRNA profile of cells and exosomes?
Differences in mRNA profile of exosomes from cancer vs normal
Cell lines Exosomes Normal PCa
Exosomal RNA RNA
PCA3 TMPRSS2-ERG snoRNA lncRNA
Diagnosis and prognosis of urogenital diseases: The Urinome Project
RNAseq of urine: TMPRSS2-ERG fusion transcript detected in a man with PCa
TMPRSS2
ERG
- Looking for the needle in the haystack and finding the farmer’s daughter - Exosomes and microvesicles - function - how to visualize, count and track them? - how taken up by other cells? - what is inside these vesicles? - how can we use them? - Conclusions
Exosomes and Microvesicles
How can we use them?
• Drug delivery systems – Homing vehicles
– Drug carriers (small molecules, siRNAs, enzymes) (liposomes)
• Immune modulation
• Disrupt tumor-environment communication
• Disease markers (risk, diagnosis, prognosis, predictive, monitoring)
• Targets for therapy? – Inhibit exosome production
– Inhibit exosome uptake
Prostate Cancer Biomarkers: AR-V7 expression in CTCs
AR-V7 expression in Extracellular Vesicles from plasma
AR-V7 is a predictive marker for resistance to enzalutamide and abiraterone
Progression-free survival
1-2 ml plasma; ExoEasy (Qiagen); digital droplet PCR
Del Re M. et al. Eur Urol 2016 Aug 26. pii: S0302-2838(16)30479-1
Exosome TR-FIA
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
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
Conclusions
• Human xenograft-derived proteins can be identified in serum from grafted mice
• Exosomes contain proteins and RNAs, but not a random selection
• Exosomes are taken up via endocytosis
• Exosomes can be captured and measured and are markers for disease
• Exosomes and their content are biomarkers, have a biological function and are potential therapeutic vehicles and targets
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