Pierre Busson

« Tumor microenvironment, exosomes and microRNAs in solid tumors »

CNRS UMR 8126

Gustave Roussy - Villejuif

Exploration of host-tumor relationships and search for

novel biomarkers in Nasopharyngeal Carcinomas

Summary

Introduction - NPC and exosomes carrying galectin-9 Plasma BART micro-RNAs in NPC patients Tumor exosomes containing the LMP1 oncoprotein

Nasopharyngeal carcinomas or NPC

- Human malignant epithelial tumor with a

lymphoepithelial pattern

- Major public health problem in a number of emerging

countries

- Multi-factorial disease constantly associated with the

Epstein-Barr virus

- Biological heterogeneity

BART microRNAs

Non-translated RNAs Proteins

Expression of EBV-encoded products in NPCs

IF detection of the EBNA1

protein in NPC cells

Viral and cellular oncogenesis of NPC

Electron micrography of an

Epstein-Barr virus particle

EBNA1

LMP1

LMP2

BARF1

EBERs

TR TR Ori-P

Contribution of EBV

Contribution of cellular alterations

- p53 rarely mutated - frequent silencing of CDKN2A (9p21) and RASSF1 (3p21) - amplification of CCND1 (11q13) - amplification of the LTb-R gene (12p13.31)

NPC : biological ressources :

patient-derived xenografts (PDX) :

IGR (C15-PDX, C17-PDX et C18-PDX)

CUHK Hong-Kong (X666, X99186, X1915, X2117)

NPC cell lines propagated in culture :

C666-1 (Hong-Kong)

C17-PIC (IGR)

Y-27632 Rho kinases I and II pharmacological inhibitor

Vérillaud et al. - Infectious Agents and Cancer - 2012

Galectin -9 : - protein binding b-galactosyl residues - immunomodulatory with a predominant immunosuppressive activity - non-classical secretion with contribution of exosomes

Exosomes :

Exosomes 40-100nm

Microvesicles 100 nm-1µm

MVB

Endosomes

Lysosomes

- Produced by most cell types including malignant cells - Can carry nucleic acids - Can present immunmodulatory ligands on their surface

Key points on exosomes and galectin-9

NH2 COOH

β-Galactoside binding sites

CRD 2 CRD 1

Gal β(1→4) Glc.

Gal-9 : a dual role in immunity

Controversies about the gal-9 membrane receptors : - Tim-3 has other ligands - Gal-9 has other receptors (PDI, IgE ? CD40 ?)

anti-gal-9 beads Control

6

.

anti-galectin-9

magnetic beads

NPC tumor

exosomes

Detection of galectin-9 at the surface of NPC tumor exosomes

Detection of tumor exosomes carrying galectin-9 in the plasma of NPC patients (collaboration with Joël Guigay)

Immunosuppressive effects of tumor exosomes carrying galectin-9

Klibi et al. Blood 2009 N. Delhem and O. Morales (Lille) JNCI in revision

NPC plasmas Non NPC

Galectin-9

- NPC : 16/17 positive for gal9 exosomes

- non-NPC 1/8 positive

NPC and galectin-9 : background :

100 mm

Galectin-9 in plasma samples of

patients infected by HCV

(Mengshol et al., PlosOne 2010)

Patient #8 (non

infected tissue) Patient #2 (VHC

infection)

Patient #5

(VHB infection)

Detection of galectin-9 in liver tissue sections

Abundant production of galectin-9 in NPC and chronic viral hepatitis

Galectin-9 in NPC : - extremely abundant in NPC - secreted in the extra-cellular medium at least in part bound to tumor exosomes Staining with 1G3 Mo Ab

Galectin-9 in chronic active viral hepatitis and hepatocarcinomas :

(x100)

Summary

Introduction - NPC and exosomes carrying galectin-9 Plasma BART micro-RNAs in NPC patients Tumor exosomes containing the LMP1 oncoprotein

Functions of BART microRNAs

TR TR Ori-P

TR TR

Ori-P

Bart RNAs

Bart RNAs

In malignant NPC cells:

In the lytic cycle :

At least three types of functions :

- Repress cellular pro-apoptotic genes like PUMA

- Partially repress the viral lytic cycle

- Modulate expression of viral oncoproteins

- miR-BART5

- miR-BART22

- miR-BART2

Puma

Viral DNA-

polymerase

LMP2 viral

oncoprotein

Rationale of the study

Anti-EBV

serum

antibodies

Plasma EBV

DNA

Population

screening Partially No

Risk

stratification No Partially

Assessment

of treatment

efficacy

No Partially

Long term

monitoring No Partially

Gyn Oncol 2008

2008

MicroRNAs detected in all biofluids

– Plasma

– Saliva

– Cerebrospinal fluid

– Urine

– Malignant effusion

– Capture by platelets (Nilsson et al., Blood 2011)

PLASMA C666-1

PLASMA C17

PLASMA CAPI PLASMA C15

0

0,02

0,04

0,06

0,08

0,1

0,12

2 -ΔCT

0

0,5

1

1,5

2

0

5

10

15

20

0

0,5

1

1,5

2

2,5

3

C15 C66 C17 CAPI C15 C66 C17 CAPI C15 C66 C17 CAPI C15 C66 C17 CAPI

miR-BART1-5p miR-BART5 miR-BART7 miR-BART13

Detection of EBV BART microRNAs in plasma samples from

NPC xenografted mice

NPC Carcinoma of unknown primary (non-infected by EBV)

miR-146a miR-BART 1-5p ΔRn

Gourzones et al, Virology Journal, 2010

Gourzones et al. Virology J. 2010

Detection of EBV BART microRNAs in plasma samples from NPC and control patients

Weeks after diagnosis 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Radio-chemotherapy

0

50

100

150

200

2 -∆Ct(BART7-Cel39)X10

0

50

100

150

200

0

50

100

150

200

Initial work-up : no metastatic lesions

Detection of ebv-miR-BART 7 by Q-RT-PCR in plasma samples

1st plasma sample 2nd sample

miR-BART7 in the plasma of an NPC patient : one observation suggesting a parallel evolution

3nd sample

No metastatic lesions Bone and liver metastases

Detection of plasma miR-BART-17 in a series of NPC patients and non-NPC controls

• Prospective series

•Absolute copy number

quantification

•Spike-in controls (cel-miR-52)

Next question : How to increase

sensitivity ?

Non-vesicular carriers

- lipoproteins (<50 nm) carry microRNAs

(Vickers et al., Nat Cell Biol. 2011)

- Ago2-containing extra-cellular ribonucleoparticles

(Arroyo et al., PNAS 2011)

? ?

?

?

Vesicular carriers

• microvesicles result from burgeoning of the plasma

membrane

• exosomes : nanovesicles derived from

« multivesicular bodies » (MVB)

• despite distinct biogenesis, similarities in protein

composition

• carriers of various species of RNAs

100nm

HDL lipoprotein

Exosomes 40-100nm (r = 1.1)

Microvesicles (or shedding vesicles)

100 nm-1µm

MVB

Endosomes

Lysosomes

Fractionation of plasma samples on a KBr gradient (Collaboration with Maryse Guérin, Pitié-Salpetrière)

0

10

20

30

40

50

0 10 20 30 40

0

20

40

60

80

100

120

0 5 10 15 20 25 30

VLDL LDL

HDL

Cholestérol

Hsa-miR-16/cel-miR-39

Ebv-miR-BART7/cel-miR-39

Ebv-miR-BART17/cel-miR-39

Cholestérol

N°fraction

8 12 17 19 20 22 25 28 Fraction #

CD63

CD63 western blot

=>only miR 16 co-purify with plasma exosomes

=>no co-purification of these microRNAs with

HDL

exosomes

Distribution of miR-BART7 and 17 and miR-16

in plasma fractions

N

fraction

VLDL

LDL

HDL

miRNA

anti-human

Ago2

antibodies

anti-mouse

Ago2

antibodies

2) Immunoprecipitation

and wash

3) RNA

purification

4) RT-PCR for ebv-miR-

BART7

Ago2 Immunoprecipitation and miR-BART7 detection

Human

Ago2 1) Mix plasma with beads

- miR-BART7 is immunoprecipitated from xenografted mouse plasma using anti-human

Ago2, but not anti-mouse Ago2

- so far, miR-BART7 immunoprecipitation not successful from human plasma

0

100

200

300

400

500

600

IP human Ago2 IP mouse Ago2

plasma from C15 mice

copies ebv-miR-BART7/µl

Plasma from an NPC

patient

No significant difference

with human and mouse

anti-Ago2

Plasma from xenografted mice

Anti-human

Ago2

Anti-mouse

Ago2

Ago2 Immunoprecipitation and miR-BART7 detection

Plasma BART microRNAs in NPC :

conclusions and perspectives

-Sensitivity and specificity to be increased - Quantification problems :

- absolute quantification ? - for references use of cellular miRs not sensitive to hemolysis (better than spike-in)

- Use of total blood (taking in account circulating tumor cells, platelets lipoproteins– no need for carrier RNA)

- Detection of spliced viral messenger RNAs

- Interesting perspectives of digital PCR

Summary

Introduction - NPC and exosomes carrying galectin-9 Plasma BART micro-RNAs in NPC patients Tumor exosomes containing the LMP1 oncoprotein

300g 10’

1900 g 15’

12 000 g 35’

110 000g pellet

Flottation (D2O/sucrose cushion – r = 1.2)

Purified exosomes (r = 1.10 – 1.19)

Cells

Dead cells/Cell debris/most apoptotic bodies (r = 1.24 – 1.28)

Microvesicles (r = 1.16)

Conditionned medium or diluted plasma sample

Pellet of dense material

Sequential purification of microvesicles, exosomes and « Lex » from conditionned culture medium of tumor cells

- Lamparski et al. J. Immunol. Methods 2002

- Klibi et al. Blood 2009

Acknowledgments

- Claire Lhuillier (anti-gal 9 N-Ab) - Benjamin Vérillaud - Eugénie Mussard - Nikiforos Kapetanakis - Natalie Oker - Aurore Gelin - Anne-Sophie Jimenez - Clément Barjon

CUHK – Hong-Kong : Kwok-Wai Lo Gal-Pharma – Takamatsu – Japon : Toshiro Niki

Inserm U757 Orsay: Olivier Dellis Curie Institute and Genosplice Damarys Loew (protéomique) Florent Dingli Thomas Rio Frio (séquençage) Frédéric Lemoine (bioinformatique) Pierre de la Grange (bioinformatique) UMR 8161 – Institut de Biologie de Lille : Nadira Delhem – Olivier Morales