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 Athymic nude mouse Athymic nude mouse serum serum Mouse proteins + Mouse proteins Xenograft-derived proteins Human specific!
Methods - overview Nu-/ - mouse bearing a xenograft Nu-/ - mouse before inoculation Collection of serum Removal of abundant proteins (albumin, I gG, transferrin) SDS-PAGE 1D Trypsin digestion LC Separation LTQ-FT
GAPDH: 6 discriminatory human peptides 2 shared peptides HUMAN MGKVK VGVNGFGR IGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENG MOUSE ..MVK VGVNGFGR IGRLVTRAAICSGKVEIVAINDPFIDLNYMVYMFQYDSTHGKFNGTVKAENG HUMAN KLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPM MOUSE KLVINGKPITIFQERDPTNIKWGEAGAEYVVESTGVFTTMEKAGAHLKGGAKRVIISAPSADAPM HUMAN FVMGVNHEKYDNSLK IISNASCTTNCLAPLAK VIHDNFGIVEGLMTTVHAITATQKTVDGPSGKL MOUSE FVMGVNHEKYDNSLK IVSNASCTTNCLAPLAK VIHDNFGIVEGLMTTVHAITATQKTVDGPSGKL HUMAN WRDGR GALQNIIPASTGAAK AVGK VIPELDGK LTGMAFR VPTANVSVVDLTCR LEKPAKYDDIKK MOUSE WRDGR GAAQNIIPASTGAAK AVGK VIPELNGK LTGMAFR VPTPNVSVVDLTCR LEKPAKYDDIKK HUMAN VVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVK LISWYDNEFGYSNR VV MOUSE VVKQASEGPLKGILGYTEDQVVSCDFNSNSHSSTFDAGAGIALNDNFVK LISWYDNEYGYSNR VV HUMAN DLMAHMASKE MOUSE DLMAYMASKE Van den Bemd et al ., Mol Cell Proteomics 2006; 1830-1839
Results – proteins identified in HUPO Glycolysis related proteins Proteasome subunits Other proteins Proteasome subunit alpha type 4 Cathepsin Z Alpha-enolase Proteasome subunit alpha type 7 Coactosin Glutathione peroxidase 3 Proteasome subunit alpha type 6 Cofilin Nucleoside diphosphate kinase A Proteasome subunit beta type 2 Inter alpha inhibitor H3 Nucleoside diphosphate kinase B Proteasome subunit beta type 8 Lumican Fructose-bisphosphate aldolase A Proteasome subunit alpha type 1 Peroxiredoxin-2 Glyceraldehyde-3-phosphate Proteasome subunit alpha type 2 Thrombospondin-1 dehydrogenase Proteasome subunit beta type 1 Complement factor B Lactate dehydrogenase A Proteasome subunit beta type 3 14-3-3-tau Lactate dehydrogenase B Proteasome subunit beta type 4 Complement factor B Maltase-glucoamylase, intestinal Proteasome subunit beta type 5 Junction plakoglobin Triosephosphate isomerase 1 Proteasome subunit beta type 6 Prothrombin Van den Bemd et al ., Mol Cell Proteomics 2006; 1830-1839 Jansen et al ., Mol Cell Proteomics 2009; 1192-1205
Human xenograft-derived proteins So how do these cytoplasmic and nuclear proteins end up in serum? - Apoptosis / necrosis - Specific secretion HUPO meeting 2006: Irmgard Schwarte-Waldhoff Department of Internal Medicine, IMBL, Ruhr-Universität Bochum, Germany
Exosomes and Microvesicles shedding budding endocytosis PSA exocytosis merocrine apocrine Consecutive secretory pathway Théry C et al. , Nat Rev Immunol. 2009 Aug;9(8):581-93
Shao et al., Nature Medicine 18, 1835–1840 (2012)
Types of Extracellular Vesicles Main protein Vesicle Size (nm) Synthesis pathway Function markers CD9, CD63, CD81, Antigen presentation, Exosomes 50-150 CD82, Annexins, RAB Merocrine immune regulatory, proteins metastatic activity CD13,CD46, CD55, Immunosuppressive, Merocrine and Prostasomes 50-500 CD59, Annexins, RAB sperm cell motility apocrine proteins improving Oncosomes 50-500 (DIAPH3) Apocrine ND Integrins, selectins, Procoagulation and Microvesicles 100-1000 Apocrine CD40 ligand anticoagulation CR1, proteolytic Procoagulation and Ectosomes 50-1000 Apocrine enzymes anticoagulation Apoptotic vesicle 50-5000 DNA Apocrine Left over from apoptosis "Biologists would rather share a toothbrush than share a gene name" Duijvesz et al., Eur Urol. 2011;59(5):823-31.
Exosomes and Microvesicles PSA Endosomal Sorting Complexes Required for Transport (ESCRT) Ludwig & Giebel. IJB&CB 2012; 44: 11-15
THE ROLE OF EXOSOMES CD9 Characteristics Contents – 30-150 nm – proteins (novel biomarkers) – secreted by living cells – RNA (miRNAs, mRNAs) – Organ-specific transmembrane proteins FUNCTI ONAL MARKER THERAPY Duijvesz et al., Eur Urol. 2011;59(5):823-31.
Exosomes and Microvesicles - 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 Functional role of Immune cells extracellular vesicles Tissue Matrix Stromal cells Epithelial cells Endothelial cells Affect: - Immune response - Migration - Growth - Therapy response Create metastatic niche Integrins on exosomes affect organ homing
EV Research: benefits for EV uptake What is the benefit of EVs to target cells? 1) No benefit signaling EVs are taken up by the unspecific process of endocytosis 2) Signaling substrate EVs contain hormones, growth factors, RNA and DNA product 3) Acquiring (new) enzyme activity EVs contain enzymes that can generate valuable metabolites 4) Acquiring food (energy and metabolites) ATP, amino acids EVs contain essential metabolites, vitamins, etc. some with energy
Exosomes and Microvesicles: Functional activity Extracellular vesicles play a role in metastatic behavior Zomer A. et al., Cell. 2015 May 21;161(5):1046-57.
Exosomes and Microvesicles How would you isolate microvesicles? -Ultracentrifugation -Filtration (filters, chromatography) -Affinity purification -Precipitation
Exosome Research: EV isolation kits OptiPrep gradient ExoSpin ExoQuick Norgen Biotek ExoPrep ExoEasy ExoRNeasy www.slideshare.net/andyfhill/isev2014-ev-isolation-chris-gardiner Ultrafiltration YIELD PURITY SUBPOPULATIONS ACTIVITY qEV exoCaP
Exosome Research: EV visualization, counting and sizing Electron microscopy Nanoparticle Tracking (NanoSight) Brownian motion qNano Tunable Resistive Pulse Sensing EV Flow Cytometry Confocal microscopy Atomic Force Microscopy
EVQuant: direct labeling of EVs in biofluids Dye only Isolated EVs 100 uL urine after 3000 xg centrifugation Culture medium Urine microscope Hartjes T, van Royen M unpublished
EVQuant; EV production in cells Average production of EVs per cell per hour: ~2000
CrazyQuant 18-07-2016
Saliva Sup undiluted supernatant 4x dilution
Coffee undiluted supernatant 4x dilution
Cocos milk 16x dilution 64x dilution
Cola undiluted supernatant 4x dilution
Tap water 4.89E+09 2.40E+10 undiluted supernatant 4x dilution
Supernatant of bacterial culture 64x dilution 16x dilution
Nail polish 3.12E+08 4.94E+10 undiluted supernatant 4x dilution
Exosomes: Time Resolved-Fluorescence I mmuno Assay Y Y Eu Eu CD9 PCa-specific detection detection Y Y Y Y CD9 CD9 capture capture
Exosomes and Microvesicles - 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
Co-localization studies at a large time scale (Confocal) Thomas Hartjes, Martin van Royen Examine the different phases of uptake and processing of exosomes Clathrin, Exosomes Rab11, Exosomes Seconds Minutes Hours Days Rab4a (early endosome), Exosomes Exosomes, Lysosomes
Exosomes and Microvesicles - 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
EV-based Cancer Biomarkers What makes EVs from cancer cells different? Physical Properties: Shape, Size, Rigidity Presence and Homing CONTENT Number Proteins: Enzyme Activity -Intra-vesicular -Transmembrane -Extra-vesicular PROPERTIES Lipids: -types -modifications Credit: Xin Zou. www.mdlinx.com/oncology/article/796 DNA: RNA: Small/Long Metabolites -expression -species -mutations -mutations Sugars (Glycans) -modifications -modifications
Cancer Research: Liquid biopsy CIRCULATING TUMOR CELLS CELL-FREE RNA METABOLITES PROTEIN CELL-FREE DNA EXTRACELLULAR VESICLES www.exosome-rna.com PLATELETS VIRUSES MICRO-ORGANISMS LIPOPROTEINS & LIPID DROPLETS
Prostate cancer research: Exosomes as markers RNA miRNAs AR variants PCA3, TMPRSS2-ERG FGFR3 mutations Prostate-derived CD63 Kidney-derived TMPRSS2 PSMA AQP PSMA SLC12 Bladder-derived PROTEIN IL9R UPK2 NUMBER Transmembrane proteins TRPA1 Count (cancer)-derived vesicles Intra-vesicular proteins
Exosomes: Exosomal proteins from cell lines Cell lines from Cell lines from normal prostate prostate cancer RWPE1 PC346C 78 53 12 PNT2C2 VCaP 136 25 23 491 147 18 52 78 96 13 13 147 PNT2C2: - 637 proteins Prostate-specific transmembrane proteins: PSMA, RWPE1: - 476 proteins TMPRSS2 PC346C: - 274 proteins STEAP2/4 VCaP: - 896 proteins PPAP2A CD13 Duijvesz et al., PLoS One. 2013; 8:e82589
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