Coordinación científica:

Dr. Rafael López López

Complejo Hospitalario Universitario de Santiago de

Compostela

Organizado

por: Sede:

San Francisco Hotel Monumento

Campillo de San Francisco, 3 - Santiago de

Compostela

Extracellular Vesicles in Liquid Biopsies

and Cancer

Prof. Dr. Guido Jenster

Experimental Urological Oncology

Erasmus MC Rotterdam, The Netherlands

Théry C et al., Nat Rev Immunol. 2009 Aug;9(8):581-93

PSA

Consecutive secretory pathway

Exosomes and Microvesicles: EVs

shedding budding

endocytosis

exocytosis

merocrine

EVs contain proteins and RNAs from host cell

Research: Biomarker

Functional

Therapy

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

PLATELETS

VIRUSES

MICRO-

ORGANISMS

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

Exosomes and Microvesicles: Functional activity

Zomer A. et al., Cell. 2015 May 21;161(5):1046-57.

Exosome Research: commercial EV isolation kits and visualization of EV

OptiPrep gradient

ExoQuick

ExoSpin ExoPrep

ExoEasy

ExoRNeasy

qEV

exoCaP

Norgen Biotek

Electron microscopy qNano

Tunable Resistive Pulse Sensing

Nanoparticle Tracking (NanoSight)

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Hartjes T, van Royen M unpublished

EVQuant; EV production in cells

Average production of

EVs per cell per hour:

~2000

Blood & Urine contain

1-10 109 EVs/ml

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

EVQuant: Biomarker detection (multicolor imaging)

Detect individual vesicles with different phenotypes

Membrane labeling TM-Protein1-alexa647 TM-Protein2-alexa488

Hartjes T, van Royen M, unpublished

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

EVs: Time-Resolved Fluorescence Immuno Assay

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

Nanobody BioPanning EV-Nanobodies Joke Veldhoven, Janne Leivo, Guido Jenster

Urinary EVs from men with and without PCa

Y Y PCa PCa

Y Y N N

TR-FIA:

Time-Resolved

Fluorescence

ImmunoAssay

Llama

EVs from PCa cell lines

Immunize

Nanobody

Library

Diagnosis and prognosis of urogenital diseases

Urinary RNA and DNA from:

Kidney

Bladder

Prostate

Other organs

Representing:

Infection

Cancer

Stones

Chronic disease

Congenital diseases

Transplantation

Children, women

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

ERG

www.urinome.nl

TMPRSS2

RNA Next Generation Sequencing

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