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Experimental Models of HCC

2015.02.14

Hepatocellular carcinoma

HCC, a complex disease

• Wide range of risk factors

• Multistep carcinogenesis

• Genetically and clinically heterogeneous

Fibrosis, Cirrhosis and HCC

• Fibrosis/Cirrhosis is the primary risk factor

• present in 80 to 90% of patients with HCC

• Most animal models for HCC do not feature

liver fibrosis

Mouse models for HCC

1. Chemically induced models

2. Transplantation models

3. GEM models

4. TG models: Non-germline

Chemically induced models

• Mutagenic 혹은 hepatotoxic chemicals 처리를 통해 정상적인 마우스 혹은 랫 에서의 암을 유발

• Natural course of disease progression 을 가장 잘 보여줄 수 있음

• Fibrosis/Cirrhosis 동반할 수 있음

• 암생성이 마우스마다 편차가 심하고 암발생 시점도 매우 길어 예측하기 어려움

Chemical Carcinogenesis

Chemical Time to deveop tumor

% of mice with HCC

Metastasis Remarks

DEN 45-104 weeks

80-100% in male

/ Poorly reproducible

Aflatoxine High grade HCC 92-

110 weeks

interstrain differences

Yes Suitable model for AFB-induced HCC

in humans

CCl4 104 weeks 50-94% Yes

Thioacetamide 50-80 weeks

70-100% /

Peroxisome proliferators

50-100 weeks

Depends on strain

Yes PP tumorigenicity in humans not

known

Choline deficient diet

50-52 weeks

100% / Steatohepatitis

Transplantation models

Xenograft models

• 인간의 간암 세포주나 간암 조직을 면역 결핍된 쥐

(예, nude, SCID 마우스)에 이식

• Tumor Immunology, micro environment 연구에

적합하지 않음

• Abnormal vascularization

Transplantation models

Syngeneic models

• Spontaneous, chemically induced tumor 를 정상적

인 면역 기능을 갖는syngeneic mouse나 rat 에 이식

• Tumor-immune system interaction,

Immunotherapy, cancer vaccine 등의 연구에 이용

Rat transplantation models

• Nude Rat models

- Rowett nude rats (rnu) and New Zealand nude

rats (nznu)

• Syngeneic rat models

- McA-RH7777 rat hepatoma cells to Buffalo rats

(intrahepatic metastasis)

- N1-S1 rat HCC cells to Sprague-Dawley rats

Rabbit transplantation model

• The VX2 carcinoma cells derived from a virus-

induced tumors in a rabbit

• Papilloma origin

Transplantation models

Ectopic models

– (간)암세포를 피하에 주입하여 암발생

– (간)암이 잘 생기고 성장을 쉽게 관찰함

– 이질적인 환경에서 성장

Transplantation models

• Orthotopic models

– 간암 조직을 직접 간에 이식하거나 간암세포주를 주

입하여 간에서 성장하게 함

– 미세환경이나 전이능력의 관점에서 ectopic model

보다 우수함

– 수술 과정이 어렵고 간암 생성 효율이 떨어짐

– 간암 성장 관찰이 어려움

GEM models for Cancer

• 정상적인 면역 기능을 갖는 마우스에서 자생적으로 간

암이 발생되도록 하는 모델

• 다양한 암유전자에 의해 발생하는 간암모델 제작

• 암유전자의 발현 시기, 장소를 조절

• 제작하는데 많은 비용과 시간이 소요됨

• Fibrosis 의 부재

Ubi. pro. oncogene

ubiquitous

alb pro. oncogene

Liver specific

Tissue specificity

tetO Pro.

TSP rTta tetO Pro. oncogene

Oncogene

Dox +

Tet-On

Inducible (Tet-on system)

Genetic manipulation in TG

P53

Exon3

Exon2 Exon3 loxP

Cre

Exon2

Exon2

Cre system

loxP Exon1

P53 genetic locus

X Cre Tg mouse

Cre

Exon3 Exon1

Exon1 No functional proteins

Genes Incidence rate

(%) Latency (weeks)

AAT 100 52-90 APC-/- 67 38 c-myc 55 65-90 E2F-1 33 52 EGF 100 24-36 HBx 84 52-104

HCV core 32 80-105 NEMO-/- 100 10-12 P53-/- 10.9 14

PTEN-/- 66 44 SV40 T-antigen 100 20

TAK1-/- 80 39 TGF- α 50 >52

c-myc + E2F1 100 26-39 c-myc + EGF 100 12-18

c-myc + TGF-α 17.5 16 β-catenin* +

H-rasG12V 100 8

K-rasG12D + HBx 62.5 34 P53-/- + c-myc 75 21

PTEN-/- + GRP94-/- 80 25

HCC models generated by traditional GEM methodology

TG models: Non-germline

1. 기존의 유전자 마우스 모델 제작에 소요되는 시

간과 비용을 줄임

2. Better temporal and spatial control

Hydrodynamic transfection of transposons

Hydrodynamic Injection

Sleeping Beauty Transposon System

Adapted from Hackett et al. (2005) Adv. Genet. 54:189

HT + SB transposons

CAG HrasG12V IR/DR IR/DR

LTR shp53

PGK SB Transposase

A

+ CAG cMyc

IR/DR IR/DR

combination of oncogenes IR/DR IR/DR

…, and etc

Co-expression of oncogenes induced HCC in the liver

cMyc+shP53

cMyc+HrasG12V HrasG12V+shP53

EGFP

~ 1 month ~ 2 month

~ 7 month

Ju et al PLoS One 2013;8(3):e59869

Liver cancer models via HT

HCC model developed via HT of mAkt

12 weeks

28 weeks

ICC model developed via HT of

mAkt and NICD and

Preclinical application

Control Akt inhibitor VEGF inhibitor Combination

Kim et al., 2015 (unpublished data)

Preclinical application

*P<0.001

Lee et al., 2015 (unpublished data)

Regular Diet Low Carbohydrate Diet

Generation of HCC model with fibrosis

Hydrodynamic Injection

CCl4 injection (twice a weeks)

2 weeks

* Carbon tetrachloride (CCl4), 1mg/kg,

HCC with fibrosis

cMYC cMYC Plus CCl4 CCl4

cMYC + CCl4 Histology

Summary

• Transgenic liver cancer models expressing various kinds of oncogenes were easily generated using the HI plus SB methods

• The mouse models for HCC can be efficiently applied to various pre-clinical studies.

• HCC models with fibrosis can be easily generated by combining HT method and CCl4 treatment.