detail about cancer

1. How To Choose Your Animal Model
Peter D. Aplan MD
Senior Investigator
Genetics Branch

2. Outline of today’s talk
• Framework for cancer (leukemia) research.
• Approaches to model cancer in mice.
• Considerations in choosing a model.

3. Framework for cancer research
• “Cancer is a genetic disease”
– Acquired/inherited
– GCR/Single nuc changes
• “Cancer is a developmental disease”
– Drosophila developmental mutants (Wnt, Runt, Trithorax, Notch)
• Cancer as an infectious disease
– Invasion of normal tissues by rogue cells
– Treated with small, cytotoxic molecules (often in combination)
– Relapse if not completely eradicated
– Important role for the immune system in eradicating rogue cells
“Progress in science depends on new techniques, new discoveries and new
ideas, probably in that order.” Sydney Brenner, circa 1980

4. Koch’s postulates
• The microorganism must be found in abundance in all
organisms suffering from the disease.
• The microorganism must be isolated from a diseased
organism and grown in pure culture.
• The cultured microorganism should cause disease when
introduced into a healthy organism.
• The microorganism must be re-isolated from the inoculated,
diseased experimental host and identified as being identical
to the original specific causative agent.
Identify the lesion, isolate the lesion, recapitulate the lesion.

5. Koch’s postulates
• The microorganism must be found in abundance in all organisms
suffering from the disease.
• The microorganism must be isolated from a diseased organism and
grown in pure culture.
• The cultured microorganism should cause disease when introduced into
a healthy organism.
• The microorganism must be re-isolated from the inoculated, diseased
experimental host and identified as being identical to the original
specific causative agent.
Koch’s postulates (adapted for cancer)
• The (cancer gene) must be found in abundance in all organisms
suffering from the (particular subtype of cancer).
• The (cancer gene) must be isolated from a diseased organism.
• The (cancer gene) should cause disease when introduced into
a healthy organism.
• The (cancer gene) must be re-isolated from (expressed in) the
inoculated, diseased experimental host and identified as being
identical to the original specific causative agent.

6. Koch postulates 1 and 2 –identify and isolate the
lesion—Gene Discovery
• 1960s-1990s--- Karyotype
• New tools in the 1990s
– Expression arrays
– SNP, aCGH arrays
– Gene/genome re-sequencing
“Progress in science depends on new techniques, new discoveries and new
ideas, probably in that order.” Sydney Brenner, circa 1980
Comment— a corollary of this postulate might suggest that key insights
come from studies that are not particularly “hypothesis driven”.
New/revised ideas on clonal evolution and pre-malignant lesions.

7. Koch Postulate 3 and 4—Recapitulate the disease--Utility of
animal models for human malignancies
• In vivo verification of putative disease genes.
• Understand disease biology.
• Pre-clinical evaluation of therapeutic approaches (impetus for
development of palbociclib –trade name Ibrance-- stemmed from mouse breast
cancer caused by Cdk4/CyclinD).
• Understand natural history of disease process—clinical
presentation of cancer is often a very late stage of disease
evolution. Allows for study of pre-malignant lesions.
• Murine hematopoiesis similar—not identical—to human
hematopoiesis.

8. How To Choose Your Animal Model
Animals used as biomedical research models
“all models are approximations”
Species Advantage Disadvantage
Flies Small, short generation time Not vertebrates
Fish Short generation time, clear embryos Not mammals
Mice Mammals, relatively small, many
genetic “tools” available
Not primate; last common
ancestor 100 M yrs ago
Rats Same as mice. Bigger than mice. Fewer genetic “tools”.
Bigger than mice.
Dogs Outbred species, useful for BMT Costly, pets, ethical issues.
Non-human
primates
Similar to humans. Similar to humans.
“2015 Mouse 101 Course”

9. Classification of murine cancer models
• Spontaneous
• Chemically induced
• Viral induced
• Genetically engineered
• Xenotransplant
• Tissue transplant (mostly hematopoietic models)

10. Spontaneous
• Most spontaneous cancers rare in outbred mice.
• Can be age-related (hepatoma, lung adenoma).
• Investigators noted that certain strains of inbred mice were
pre-disposed to develop cancer (skin, breast, leukemia).
• Why?—answers led to seminal advances in understanding
cancer biology.
– Vertical transmission and viral etiology.
– Concept and cloning of modifier loci (ex. Mom1)

11. Chemically induced
• Random mutagenesis:
– Use low dose mutagen – ie, ethylnitrosourea (ENU)
– Can obtain germline (mutagenize sperm) or acquired mutants
– Study phenotype of homozygotes, heterozygotes
– Large scale projects. Lots of mouse cages, genotyping
– Cloning involved loci is laborious, many backcrosses, need genetic
maps.
– Demonstrated utility:
• L1210, P388 leukemias (induced by 3-methylcholantrene) used for drug screening
and modeling in vitro
• Min (multiple intestinal neoplasia) mutant caused by Apc truncation; led to cloning
of Apc gene and critical insights into colon cancer and Familial Adenomatous
Polyposis
• Skin cancers and coal/cigarette tar

12. Viral induced cancer
• MMTV (mouse mammary tumor virus)
• MULV (murine leukemia virus)
• Vertically transmitted disease (mother’s milk)
• Retroviral Insertional Mutagenesis

13. Retrovirus integration into the genome can
transform cells by either oncogene activation or
tumor suppressor gene inactivation
Proto-oncogene
Oncogene
Virus
Tumor suppressor
Tumor suppressor
Gene activation
Viral promoter insertion and
enhancer activation
Gene inactivation
Virus
Retroviral Insertional Mutagenesis

14. Retroviral Insertional Mutagenesis
• Retroviral insertion is random at first
approximation
• Transformation leads to a growth advantage and
clonal expansion which results in tumor
formation
• Proviral Integration serves as a tag by which
nearby genes are identified through ligation-
mediated PCR.
• Multiple viral integrations occur in each mouse
• Common integration sites (CIS) identify nearby
candidate genes

15. Retroviral Insertional Mutagenesis
• Utility:
– Discovery of Hoxa9, Meis1, Evi1, Etv6 role in
leukemia
– Discovery of Wnt-1 (β-catenin pathway),
Fgf3/4/8, Notch-4 role in breast cancer
– Limited by cell type tropism
– Extension to most cell types through Sleeping
Beauty Transposon

16. Transplantation of modified tissue
• Primarily used to study hematopoietic malignancy
• Transduce (WT or modified) HSC with viral vector
carrying gene of interest
• (Select transduced cells)
• Transplant into lethally irradiated recipients
• Utility:
– Relatively quick experiments
– Can test several gene variants (mutants) simultaneously
– Can test combinations of genes
– Irradiation of recipients, integration sites, and in vitro
selection can be confounding variables

17. Xeno (foreign)-grafts
• Typically human cancer cells engrafted into
immuno-deficient mice.
• Problems with immune rejection,
microenvironment, cytokine/hormones.
• Mice lack intact immune system; therefore difficult
to model immunotherapies.
• Some investigators have suggested these to be
“animal culture”, one step from tissue culture.
• https://www.jax.org/news-and-
insights/2006/march/choosing-an-
immunodeficient-mouse-model Google “jackson
labs immunodeficient”

18. Xeno-grafts
• Nude (nu/nu) mice.
• Homozygous for a mutant Foxn1 gene.
• Defective thymic epithelial cells, lack normal
numbers of functional T/B cells.
• Graft rejection due to residual innate lymphoid
cells (NK cells).
• Hairless, easy to visualize/measure
subcutaneous tumors.
• Engraft leukemia poorly.

19. • Rag1 or Rag2 KO.
• Deficient in VDJ recombination; therefore no
mature T/B cells.
• Still make NK cells.
• Difficult to breed.
Xeno-grafts

20. • Scid/Scid – spontaneous recessive mutant involving the
Pkrdc gene.
• Pkrdc gene required for non homologous end joining
(NHEJ; major repair pathway for DNA double strand
breaks).
• NHEJ needed for normal VDJ recombination; no VDJ
recombination, no T/B cells.
• Leaky. Low, but finite ability (0.1% normal) to produce
functional VDJ coding joints.
• Leakiness and spontaneous lymphoma increase with age.
• Used extensively for AML engraftment, concept of cancer
stem cells (SLIC, aka Scid Leukemia Initiating Cells)
originated from these experiments.
Xeno-grafts

21. • “Improved” Scid models.
• Non-obese diabetic (NOD)/Scid.
• NOD developed to model IDDM; immunodeficiency
incompletely characterized, polygenic.
• Defective NK, complement, IL-1 (macrophage activator).
• NOG/Scid—NOD/IL2rg deficient/Scid. IL2rg (“common
gamma chain”) forms IL2/4/7/15/21 receptor.
• Less leaky, less spontaneous lymphoma than Scid.
Xeno-grafts

22. Xeno-grafts
MISTRG mice—RAG KO, IL2RG KO, express 5 human cytokines
Seem to be useful for engrafting human heme malignancies

23. Xeno-grafts
• Historically, malignant cell lines used for xenograft.
• Recent interest in Patient Derived Xenografts (PDX,
Avatars).
• Xenograft from patient primary tumor, usually into Scid or
NOD/Scid mice.
• No passage/selection in vitro.
• Typically used to predict patient drug response in vivo.
• Attractive concept, several commercial entities ($5-50K).
• Unproven; mice without immune systems.
• Clinical trials in progress.
• Perspective: Nature Reviews Cancer 15, 311–316 (2015)

24. Genetically Engineered Mice (GEM)
• Altered mouse germline; mutations are
identical and transmitted to offspring.
• Transgenic
– Tissue specific
– Conditional (w/r/to time, tissue)
• Gene targeted
– “Knock out” KO—gene deletion/inactivation
– “Knock in” KI—gene insertion at specific
target locus

25. Transgenic
FG FG HD
Vav 5’ Vav 3’
NUP98-HOXD13 cDNA
• Plasmid construct: Promoter, Gene, Intron, PolyA signal
• Promoter: Constitutive, Inducible, Tissue specific
• Core Facility; Commercial Facility ($3-20K)
SV40
intron
GH pA
signal

26. Generation of Transgenic Mice
• Generate vector, purify insert.
• Micro-inject fertilized ovum (50-100).
• Implant into pseudopregnant females.
• Tail biopsy to assess integration of transgene (Southern or PCR).
• Typical (good) results– 5-10% of ~80 pups are transgenic.
• If none positive: bad DNA, bad embryos, bad injections, bad
luck, lethal transgene (may need to euthanize prior to birth).
• Time (optimistic) – 3 months

27. Evaluation of Transgenic Mice
• Import potential founders
• Breed potential founders (How many? Which ones?)
• If offspring all negative: bad luck (chimeric founder),
mis-genotyped, germ cell/embryonic lethality.
• Transmission rate should be >75%.
• Euthanize F1 positive mouse, collect tissues, assess
RNA expression.
• Sequence RNA to verify no mutations.
• Want >1 founder, decrease possibility that phenotype is
due to integration effect.
• Time (optimistic) – 3 months

28. Conditional Transgenic
• Estrogen Receptor fusion protein (ie, Myc-ER
fusion).
– Fusion protein in inactive conformation until ligand
(estrogen, Tamoxifen) added.
• Tet-on/off.
– Express transgene under control of Tet operon.
– Express rTa or tTa under control of tissue specific
promoter.
– Turn on or off with Tet(Dox) cycline.
• NB—these are bad for teeth
– Two transgenes.

29. Gene Targeting (KO/KI)
• Homologous Recombination in ES cells in vitro
• Targeting vector construction:
– Targeting arms
– Selectable marker
– Counter-selection

30. Germline
mNUP98
ApaI
SstI SstI
pNHKI
ApaI SstI
5’NUP98/
HOXD13
Neo
3’NUP98
mNUP98 Exon
hNUP98 Exon
LoxP
pTK-Neo
Vector
hHOXD13 Exon
mNUP98 Intron
TK
20 Kb 18 Kb
NsiI
NsiI
Amp
R
Ori LacZ
Targeting Construct
12
12

31. Southern
Screen
J1 EF BJ 73 190 191 136
23 kb —
23 kb —
23 kb —
ApaI
SstI SstI
20 Kb 18 Kb
NsiI
12
SstI
Neo
12
SstI
SstI

32. Gene Targeting
Core
Facility
PI
Lab

33. Gene Targeting (KO/KI)
• Chimeric mice (by coat color)
• Go germline!! (presume germ cells are also
chimeric)
• Screen germline pups (by coat color and DNA).
• Stable allele can now be bred.
• Realistically, 1 yr timeline.
• CRISPR now allows much simpler targeting of
loci in ES cells in vitro

34. • Cre-Lox system from lambda phage.
• Cre recombinase recognizes sequence specific loxP
sites.
• Many cell-type specific or inducible Cre transgenic mice
available (ex: CD19-Cre (B cell); LcK-Cre (T cell); Mx1-
Cre (inducible)).
Conditional GEM using Cre-Lox

35. Combinations
• KI/KO with gene transduction/transplantation of HSC.
• Transgenic with chemical or retroviral insertional
mutagenesis.
0.00
20.00
40.00
60.00
80.00
100.00
120.00
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58
Weeks
Percent
Survival

36. What’s the best model?
What’s the question?
• Gene/pathway discovery: Maybe RIM. Have built in
“tag” lacking in chemical mutagenesis. But NGS costs
suggest re-evaluation.
• Proof of causation: Probably GEM— can start with
primary cells. Problem with cell lines is they have many
uncharacterized abnormalities. Human IPS cells??
• Disease progression: Probably GEM—watch disease
evolve in situ –normal tissue microenvironment.
• Pre-clinical therapeutic: GEM or xenograft.

37. Pre-clinical therapeutic
Model Pros Cons
GEM • Natural microenvironment
• Intact immune system
• Genetically homogeneous
• Transferable and reproducible
• Not human cancer
• Not human
pharmacokinetics
• May be difficult to generate
large numbers of animals
Xeno • Human cancer
• Easy to generate large
numbers of animals
• Transferable and reproducible
• Evolution/selection in plastic
• Foreign microenvironment
• Lack immune system
• Not human
pharmacokinetics
PDX • Human cancer
• Primary cancer
• Easy to generate large
numbers of animals
• Expensive
• Not easily transferable
• Foreign microenvironment
• Lack immune system
• Not human
pharmacokinetics

38. Conclusions
• Clearly delineate your goal (s).
• Read the literature (but not exhaustively or
exclusively).
• Consult with senior investigators.
• Begin your studies.
• Iterative cycles of 1-3 are OK.