Exosomes and Microvesicles

Prof. Dr. Guido Jenster Experimental Urological Oncology Erasmus MC Rotterdam g.jenster@erasmusmc.nl

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