Extracellular Vesicles in cancer Exosomes and Microvesicles Prof. - - PowerPoint PPT Presentation

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Extracellular Vesicles in cancer Exosomes and Microvesicles Prof. - - PowerPoint PPT Presentation

Mar 14, 2024 460 likes •950 views

Extracellular Vesicles in cancer 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

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Extracellular Vesicles in cancer

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: EV isolation kits OptiPrep gradient ExoQuick ExoSpin ExoPrep ExoEasy ExoRNeasy qEV exoCaP Norgen Biotek

www.slideshare.net/andyfhill/isev2014-ev-isolation-chris-gardiner

Ultrafiltration

YIELD PURITY SUBPOPULATIONS ACTIVITY

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Exosome Research: EV visualization, counting and sizing

Electron microscopy qNano Tunable Resistive Pulse Sensing Nanoparticle Tracking (NanoSight) Brownian motion Confocal microscopy Atomic Force Microscopy EV Flow Cytometry

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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

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EVQuant; EV production in cells

Average production of EVs per cell per hour:

~2000

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CrazyQuant

18-07-2016

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Saliva

Sup undiluted supernatant 4x dilution

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Coffee

undiluted supernatant 4x dilution

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Cocos milk

16x dilution 64x dilution

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Cola

undiluted supernatant 4x dilution

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Tap water

4.89E+09 2.40E+10

undiluted supernatant 4x dilution

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Supernatant of bacterial culture

16x dilution 64x dilution

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Nail polish

undiluted supernatant 4x dilution

3.12E+08 4.94E+10

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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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EV-based Cancer Biomarkers

What makes EVs from cancer cells different?

Number Physical Properties: Shape, Size, Rigidity RNA: Small/Long

  • expression
  • mutations
  • modifications

DNA:

  • species
  • mutations
  • modifications

Proteins:

  • Intra-vesicular
  • Transmembrane
  • Extra-vesicular

Lipids:

  • types
  • modifications

Metabolites Enzyme Activity Sugars (Glycans) Presence and Homing

Credit: Xin Zou. www.mdlinx.com/oncology/article/796

CONTENT PROPERTIES

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CIRCULATING TUMOR CELLS CELL-FREE RNA

Cancer Research: Liquid biopsy

PROTEIN METABOLITES EXTRACELLULAR VESICLES CELL-FREE DNA PLATELETS VIRUSES MICRO-ORGANISMS LIPOPROTEINS & LIPID DROPLETS

www.exosome-rna.com

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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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Exosome Research: commercial EV isolation kits and visualization of EV

The EV biomarker Checklist:

  • Select:
  • Type of Disease and Clinical Need
  • Type of Biofluid and Biobank (type & number of patients)
  • EV Characteristic to Study (Protein, RNA, Number, etc.)
  • Test and Optimize:
  • Biobank: collection & storage SOPs
  • (remove cells, -80°C, THP, serum vs plasma)
  • Model System
  • EV Isolation Protocol
  • EV RNA/Protein/Lipid Extraction/Detection Protocol
  • Check:
  • MISEV and EV-TRACK for the EV requirements and reporting
  • TMUG Worksheet (Tumor Marker Utility Grading)
  • REMARK (Reporting Marker studies)
  • STARD (Reporting Studies of Diagnostic Accuracy)
  • Perform the EV Experiments
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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?

Biomarkers

  • Disease markers (risk, diagnosis, prognosis, predictive, monitoring)

Therapeutics

  • Drug delivery systems

– Homing vehicles – Drug carriers (small molecules, siRNAs, enzymes) (liposomes)

  • Immune modulation and vaccination
  • Disrupt tumor-environment communication

– Inhibit exosome production – Inhibit exosome uptake

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Prostate Cancer Biomarkers: AR-V7 expression in EVs

AR-V7 expression in Extracellular Vesicles from plasma

AR-V7 is a predictive marker for resistance to enzalutamide and abiraterone

Del Re M. et al. Eur Urol 2016 Aug 26. pii: S0302-2838(16)30479-1

Progression-free survival

Plasma (1-2 ml) ExoRNeasy (Qiagen) digital droplet PCR

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Prostate Cancer Biomarkers: PD-L1 expression in EVs

Del Re M. et al. Br J Cancer. 2018;118(6):820-824.

Melanoma PD-L1 EV-protein response to pembrolizumab Melanoma and NSCLC PD-L1 EV-RNA (ddPCR) response to nivolumab/pembrolizumab

Chen G et al. Nature. 2018;560(7718):382-386.

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Men without PCa Men with PCa

CD63 immunoassay after correction for urinary PSA, is higher in men with PCa

Duijvesz D. & Versluis Y. et al., Int J Cancer. 2015;137(12):2869-78

Urine biomarkers: Extracellular vesicles

100 uL urine after 3000 xg centrifugation

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Conclusions

  • Human xenograft-derived proteins can be identified in serum from

grafted mice

  • Extracellular vesicles contain proteins and RNAs, but not a random

selection

  • Extracellular vesicles are taken up via endocytosis
  • Extracellular vesicles can be captured and measured and are

markers for disease

  • Extracellular vesicles 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