Exosomes and Microvesicles Prof. Dr. Guido Jenster Experimental - - PowerPoint PPT Presentation

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Exosomes and Microvesicles Prof. Dr. Guido Jenster Experimental - - PowerPoint PPT Presentation

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Exosomes and Microvesicles Prof. Dr. Guido Jenster Experimental Urological Oncology Erasmus MC Rotterdam g.jenster@erasmusmc.nl Cancer-derived proteins in serum Xenograft Human prostate cancer: PC346 or PC339 Athymic nude mouse Athymic nude

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Exosomes and Microvesicles

  • Prof. Dr. Guido Jenster

Experimental Urological Oncology Erasmus MC Rotterdam g.jenster@erasmusmc.nl

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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!

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Methods - overview

Removal of abundant proteins (albumin, I gG, transferrin) Nu-/ - mouse before inoculation Collection of serum LC Separation LTQ-FT Trypsin digestion SDS-PAGE 1D Nu-/ - mouse bearing a xenograft

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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

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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

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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

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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

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Shao et al., Nature Medicine 18, 1835–1840 (2012)

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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 50-150

CD9, CD63, CD81, CD82, Annexins, RAB proteins Merocrine Antigen presentation, immune regulatory, metastatic activity

Prostasomes 50-500

CD13,CD46, CD55, CD59, Annexins, RAB proteins Merocrine and apocrine Immunosuppressive, sperm cell motility 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

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PSA

Exosomes and Microvesicles

Ludwig & Giebel. IJB&CB 2012; 44: 11-15

Endosomal Sorting Complexes Required for Transport (ESCRT)

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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.

FUNCTI ONAL MARKER THERAPY

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  • 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

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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

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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.

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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

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Exosomes and Microvesicles

How would you isolate microvesicles?

  • Ultracentrifugation
  • Filtration (filters, chromatography)
  • Affinity purification
  • Precipitation
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Exosome Research: commercial EV isolation kits OptiPrep gradient ExoQuick ExoSpin ExoPrep ExoEasy ExoRNeasy qEV exoCaP Norgen Biotek

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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)
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  • 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

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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

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EVQuant; How does it work?

Isolated EVs Dye only Culture medium Urine

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EVQuant; EV production in cells

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CrazyQuant

18-07-2016

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Exosomes: Time Resolved-Fluorescence I mmuno Assay

Y Y Y

CD9 capture CD9 detection Eu

Y Y Y

CD9 capture PCa-specific detection Eu

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  • 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

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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

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  • 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

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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

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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

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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

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Exosomes: Marker selection and Western blotting

PNT2C2 RWPE1 PC346C VCaP

cells exosomes cells exosomes cells exosomes cells exosomes

Duijvesz et al., PLoS One. 2013; 8:e82589 Vesiclepedia: Kalra et al., PLoS Biol. 2012; 10:e1001450

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Exosomes: Affymetrix Exon array analyses

PCa cell line RNA Exosomal RNA Exosomal RNA

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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

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Diagnosis and prognosis of urogenital diseases: The Urinome Project

RNAseq of urine: TMPRSS2-ERG fusion transcript detected in a man with PCa

TMPRSS2 ERG

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  • 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

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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

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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

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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

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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

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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

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Prostate Cancer Research: The future Next Generation Sequencing RT-PCR ELISA / TR-FIA EVQuant New Assays CTCs Exosomes cfDNA

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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