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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