experimental models of hcc - klcsg.or.krklcsg.or.kr/pdf/conference_9/01.pdf · chemical...
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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.