Using the Mouse as a Model
System in Cancer Research
Matthew Ramsey, PhD
Brigham and Women’s Hospital
Department of Dermatology
Why study cancer in the mouse?
•
Protein-coding regions of the mouse and human genomes are ~85% identical
•
•
•
Tumors evolve and grow in the context of non-tumor cells
Local microenvironment of tumor cells can impact tumor growth
•
•
•
Very similar physiology
Reasonably short lifespan
Genetic homogeneity reduces variation in system
Ease of genetic engineering results in immense genetic toolkit
Getting to treatments that benefit patients
• Models need to be designed to reflect human
tumor genetics
• Intervention studies need to be performed in
established tumors
• Treatment schedules need to reflect realities of
patient treatment
• Tumors need to REGRESS after treatment!
How do we make murine
cancer models?
The “Hallmarks of Cancer”
Douglas Hanahan and Robert A. Weinberg, Hallmarks of Cancer: The Next Generation, Cell, Volume 144, Issue 5, 2011, 646 - 674
Generation of Squamous Cell Carcinoma by
chemical carcinogens
Normal
Dysplasia
DMBA
DMBA
TPA
Papilloma
SCC
Genetically-defined tumor models
Tumor suppressors
Genes which contribute to tumorigenesis
when inactivated
(p53, p16INK4a)
Oncogenes
Genes which drive tumorigenesis by being
activated
(K-RasG12D)
Making
Transgenic
Mice
From: Gilbert, Scott F., Developmental Biology, Tenth Edition, Sunderland (MA): Sinauer Associates, 2013
First tumor-prone transgenic mice
Key early tumor models
•
Richard Palmiter lab:
Em-myc
B-Cell lymphoma
(Adams et al, Nature,1985)
•
Phillip Leder lab:
MMTV-c-Myc,
mammary adenocarcinoma
(Stewart et al, Cell, 1984)
•
Douglas Hanahan lab:
RIP-Tag
Pancreatic Islet tumors
(Hanahan D., Nature, 1985)
Examining tumor-suppressor genes in the
mouse using germline knock-outs
1
2
1b
p15Ink4b
1a
p16Ink4a
Cdk4/6
Cyclin D
Tumor Suppressor
Oncogene
2
p19Arf
Mdm2
p21Cip1
pRb
E2F
Modified from: Kim and Sharpless, Cell 2006.
p53
3
Map of the murine Ink4a/Arf Locus and targeting constructs
p16INK4a / p19ARF
p15Ink4b
Ex 1
B
Ex 2
B
Ex 1b
R1
Acc
R1
Ex 2
Ex 1a
1b Probe
SacI
X
R1
X
SacI
1a Probe
Xho
Xmn
BssHII
TK
Ex 3
Not
1 kb
TK
PGKNeo
PGKNeo
R1
Exon 1b targeting construct
Exon 1a targeting construct
WT 4.8 kb
WT 8.0 kb
KO 2.2 kb
KO 9.8 kb
EcoR1 Digest
= LoxP Site
Sac I digest
X
Making
germ-line
knock-out
mice
From: Gilbert, Scott F., Developmental Biology, Tenth Edition, Sunderland (MA): Sinauer Associates, 2013
Tumor Incidence In Mice Lacking
p19Arf and/or p16Ink4a
Spontaneous Tumors
Tumor Free Survival
100
Wild-type
50
p16Ink4a -/-
p16Ink4a/p19Arf -/p19Arf -/0
0
20
40
60
80
100
Weeks
Sharpless, NE et al, Oncogene, 2004
Studying Activated Oncogenes
Cre-LoxP system for conditional modification
of genomic DNA
Image from: http://cre.jax.org/introduction.html
Conditionally activated oncogenic alleles
Adenoviral CRE
Dental Floss
Lox-STOP-Lox-Rosa26-LacZ
Adeno-Cre
Adeno-Empty
Lung tumors in K-RasG12D Lkb1-/- mice show
all histologies of NSCLC
Sq
Ad
LCC
Mixed
SCC
Inducible alleles
Doxycycline-regulated alleles
Tet-Off
Tet-On
From: Mouse Models of Cancer: A Laboratory Manual, Cold Spring Harbor Protocols,
Spatial and temporal regulation of CreRecombinase using CreER alleles
Tamoxifen
CYTOPLASM
NUCLEUS
Keratin 14 promoter
Cre Recombinase Estrogen Receptor
K14-CreER
Tumor Volume (mm3)
Examining tumor maintenance using CreER
alleles in GEMM mice with chemical induction
Percent Tumor Free
100
80
60
40
p53+/+
p53+/p< 0.0001
20
200
175
150
125
100
75
50
25
0
Papilloma
SCC
0
50 100 150 200 250
Days After Start of DMBA Treatment
0
0
50
100
150
200
Days After Start of DMBA Treatment
SCC
Papilloma
Ramsey et al., J Clin Invest, 2013
Cre-mediated excision of p63 in vivo
Keratin 14 promoter
Cre Recombinase Estrogen Receptor
K14-CreER
4
5
6
7
8
p63flox
Cutaneous SCC
+ Tamoxifen
8
4
p63Brdm3
Keratin 14
p63
(p63-ablated)
5
7
6
Excision of p63 in SCC in vivo results in
rapid tumor regression
Apoptosis
p63 intact
p63 ablated
2.0
Tamoxifen
% CC3 Positive
1.8
1.4
1.2
1.0
10
8
6
4
2
0
0.6
0.4
0.2
*
Day 6
*
Day 8
Proliferation
p63 intact
p63 ablated
0.8
p63L/L; WT
p63+/L; WT
p63L/L; K14-CreER
p63+/L; K14-CreER
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Days after start of treatment
% Ki67 Positive
Relative Tumor Volume
1.6
25
20
15
10
5
0
*
Day 6
**
Day 8
Ramsey et al., J Clin Invest, 2013
CRISPER-Cas models
What is CRISPER/Cas?
What is CRISPER/Cas?
PAM =Protospacer adjacent motif
Hui Yang, Haoyi Wang & Rudolf Jaenisch, Generating genetically modified mice using CRISPR/Cas-mediated genome engineering, Nature Protocols 9, 1956–1968 (2014)
Advantages of CRISPER/Cas
• Highly efficient targeting of genomic locus in ES cells
• Can make small insertions and deletions, generate
conditional alleles, generate specific point mutations,
insert reporters, or insert tags
• Multiplexing of alleles: Can infect cells with multiple
sgRNAs at one time
• Increased speed of generating complex genotypes
Hui Yang, Haoyi Wang & Rudolf Jaenisch, Generating genetically modified mice using CRISPR/Cas-mediated genome engineering, Nature Protocols 9, 1956–1968 (2014)
Generating liver tumors using CRISPER/Cas
W Xue et al. Nature 000, 1-5 (2014) doi:10.1038/nature13589
Limitations of Crisper/Cas9 system
• Target sequence must have an adjacent PAM motif for
sgRNA to correctly interact with genomic DNA
• Multiple different mutations are induced which might not
interfere with gene function
• Works best if function of gene is known (ie, targeting of
functional domains)
• Frequency and nature of off-target effects are not known
• Low efficiency of delivery to organs (like liver)