This mouse model has been build up and tested to evaluate anticancer agents in hepatocellular carcinoma. This transgenic model can also be tested for the testing of checkpoint inhibitors and other immunoactive agents.

1. C D 8
Foldinduction
C
T
12W
16W
20W
0
2
4
6
8 **
P D -L 1
Foldinducton
C
T
12W
16W
20W
0
2
4
6
8
10
*
F o x P 3
Foldinducton
C
T
12W
16W
20W
0
2
4
6
8 *
P D -1
Foldinducton
C
T
12W
16W
20W
0
2
4
6
8
1 0 **
IL -17F
Foldinducton
C
T
12W
16W
20W
0
10
20
30
40
50
*
*
**
IL-17A
Foldinducton
C
T
12W
16W
20W
0
1
2
3
4
5
*
***
iN O S
Foldinducton
C T 12 W e e ks 16 w e e ks 20 w e e ks
0
20
40
60
80
100
CT
0.214657
2.250283
1.905416
0.314276
0.315368
12 Weeks
10.630270
30.592500
3.294606
7.235568
9.254187
0.004582
0.016403
16 weeks
33.245940
18.831890
43.264330
0.000086
24.506730
44.635080
0.000090
20 weeks
17.754420
65.123490
61.185010
22.163380
21.782630
9.849880
201.565700
*
F o x P 3
Foldinducton
C
T
12W
16W
20W
0
2
4
6
8 *
IN Fg
Foldinducton
C T 12 W e e ks 16 w e e ks 20 w e e ks
0
2
4
6
CT
0.962820
1.257308
1.094550
0.466626
1.218696
12 Weeks
0.838184
1.809183
1.735484
0.801258
2.258458
1.301647
1.688027
16 weeks
2.007414
1.784275
0.787493
1.409651
1.297144
1.735484
1.670567
20 weeks
4.014828
7.312365
4.908690
3.216158
1.790470
0.939743
5.446533
F o x P 3
Foldinducton
C
T
12W
16W
20W
0
2
4
6
8 *
*
IL -10
Foldinducton
C T 12 W e e ks 16 w e e ks 20 w e e ks
0
1
2
3
4
5
CT
1.809623
0.547408
1.329320
0.230157
1.083492
12 Weeks
0.146167
0.545514
0.309002
0.303694
0.428002
0.184372
0.898561
16 weeks
0.700127
0.425045
0.065867
1.261980
0.760853
0.443095
0.312232
20 weeks
1.939505
11.881860
1.161258
1.227471
0.457134
0.829716
1.177469
F o x P 3
Foldinducton
C
T
12W
16W
20W
0
2
4
6
8 *
*
IL -1 b
Foldinducton
C T 12 W e e ks 16 w e e ks 20 w e e ks
0.0
0.5
1.0
1.5
2.0
2.5
CT
1.470186
1.095052
1.311301
0.674083
0.449377
12 Weeks
0.545632
0.557096
0.302708
0.310142
0.215536
0.423666
1.194162
16 weeks
1.352847
2.339174
0.370103
0.886381
0.572759
0.425137
1.357544
20 weeks
1.810012
4.597976
0.782411
2.323016
0.334714
1.028826
0.662503
F o x P 3
Foldinducton
C
T
12W
16W
20W
0
2
4
6
8 *
*
# 245
o Hepatocellular carcinoma (HCC)
is a complex multistep
malignancy often arising on
underlying chronic liver disease,
requiring new therapeutic
options
o HCC is the main cause of death
in patients with non-alcoholic
steatohepatitis (NASH) and can
develop even in the absence of
cirrhosis
o There is an urgent need for
robust animal models fully
recapitulating the NASH-related
HCC carcinogenesis
Introduction
To further characterize and
develop our transgenic HCC
mouse model, focusing on
immune landscape and
specific diet-induced variants
Aim of the study
Transgenic mouse model: ASV-B is a
transgenic mouse model (C57BL/6J) that
spontaneously develops a reproducible stage-
defined HCC, with hyperplasia at week(W)8,
followed by nodular stage at W12, then diffuse
carcinoma stage at W16-20. Transgene consists
in the fusion between the antithrombin promotor
and the T oncogene of SV40, on the Y
chromosome
Immune landscape assessment: RNA was
extracted using Qiacube (Qiagen, France) from
frozen livers at W20 for immune markers
analysis using qRT-PCR (LightCycler, Roche).
Immune populations were also assessed using
automated immunohistochemistry (Bond Max,
Leica)
NASH model: ASV-B transgenic mice were
exposed to 5 different diet regimen: classic,
high-fat, containing 22% of vegetal oil and 0.2%
cholesterol, enriched in saturated fatty
acid+1.25% cholesterol, and containing 21%
milkfat + 1.25% cholesterol. All mice fed with
special diets also received 30% fructose in drink
water
Statistic: All data are mean ± standard
deviation (SD). Statistical significance was
calculated by two-sided unpaired Student's t test
(Prism)
Material and Methods
Results
Contact:
araballand@afr-oncology.com
Conclusions
o Our model pertains to the few genetic engineered mouse model for HCC, easy to use with a stage-defined tumor progression
o ASV-B recapitulates immune modifications observed in inflamed human HCC
o Using specific regimen in the ASV-B model, we have been able to develop a phenotype mimicking human NASH pattern, to be confirmed by
additional date analysis
o ASV-B model is a useful tool to test new treatments in HCC
Immune characterization and diet-variants of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B)
Stage-defined HCC mouse model
Liver volume (Ultrasound) and angiogenesis (Doppler) at each
step of carcinogenesis showed a significant increase in
transgenic mice compared to controls
ASV-B model: Pattern of angiogenesis
ASV-B model:
liver volume and angiogenesis assessment
LamininArteriography Desmin α-SMA
W16:
Normal liver
W16:
HCC liver
Arterialization Capillarization
W8 W12 W16
0
2
4
6
8
Livervolume(mm3)
WT
ASV-B
***
***
***
W8 W12 W16
0
10
20
30
40
BloodFlowinhepaticartery(m/s)
WT
ASV-B
**
***
***
Ultrasound Doppler
Hyperplasia at W8, nodular stage at W12, and diffuse
carcinoma stage at W16-20. Along with tumor development,
angiogenesis increased in HCC livers
ASV-B model displays marked arterialization with intense arterial
flow in liver tumor. Sinusoids in tumor nodules are tortuous and
dilated, surrounded by activated hepatic stellate cells (SMA
high/desmin low)
Annemilaï Tijeras-Raballand1, Benoit Rousseau2,3*, Christian Hobeika4,5*, Patricia Hainaud6, Philippe Bonnin3,7, Aurélie Rodrigues3, Fouad Ladfil3, Marc Pocard4,5, Valérie Paradis8, Mohamed Bouattour9, Armand de Gramont1, Eric Raymond10, Evelyne Dupuy6, Clarisse Eveno4,5, Sandrine Faivre9
1AFR Oncology, Boulogne-Billancourt, France ; 2Medical and Pharmacology Department, Henri Mondor University Hospital, Créteil, France; 3Inserm U955, Innovation and Drug Development Unit, Créteil, France; 4 Inserm U965, Paris, France; 5 Digestive and Cancer Surgery Department, Lariboisière University Hospital, Paris;6Institut des Vaisseaux et du Sang, Paris, France; 7Clinical Physiology-Functionnal Investigations Department , Lariboisière
University Hospital, Paris; 8Pathology Department, Beaujon University Hospital, Clichy, France; 9Medical Oncology Department, Beaujon University Hospital, France; 10Medical Oncology Department, Saint-Joseph Hospital, Paris, France
30th EORTC/AACR/NCI symposium
Dublin (13-16 November)
ASV-B mouse model characterization A new NASH model derived form ASV-B mice
Randomisation at 6W
High-fat diet plus 30% fructose in drink water (blue)
Classic diet plus 22% vegetal oil, 0.2% cholesterol and
30% fructose in drink water (orange)
n=50
Classic diet (yellow)
Classic diet with saturated fatty acids, 1.25% cholesterol
and 30% fructose in drink water (green)
Classic diet plus 21% milkfat, 1.25% cholesterol
and 30% fructose in drink water (red)
Study design
ASV-B transgenic mice were exposed to 5 different diet regimen. Liver
volume, angiogenesis and blood sugar were assessed every 4 weeks.
Sacrifice were performed at W12 (4/group) and W20 (6/group) for
blood and liver sampling
NASH pattern in ASV-B mice fed with specific diet-variants
Specific diet-variant induced different liver phenotype. The orange
regimen will be further investigated to confirm the features mimicking
the human NASH underlying disease
Fusion between the antithrombin promotor and the T oncogene of SV40
W12: NodularW8: Hyperplasia
* x20
*
x20x20
HE staining
W16: Diffuse
x20x20 x20
CD31 staining
HE
CD31
W16:
Normal
W16:
Tumor
Immune profile of ASV-B mice
As frequently observed in human inflammatory HCC, CD8,
Foxp3, INOS, CD11b, PD-1, PD-L1, IL1β, IFN-γ, TNF-α, IL17A
and IL17F mRNA expression increased at W20 as compared to
controls
N W12 W16 W20 N W12 W16 W20 N W12 W16 W20 N W12 W16 W20 N W12 W16 W20
N W12 W16 W20 N W12 W16 W20 N W12 W16 W20 N W12 W16 W20 N W12 W16 W20
CD8 FoxP3 iNOS PD-1 PD-L1
IL-10 IL-1b IL-17a 1L-17fINGg