Download In Vitro and In Vivo Models of Human Cancer

In Vitro and In Vivo Models of
Human Cancer
Barbara Foster
Pharmacology & Therapeutics
CGP 4322
845-1260
[email protected]
Goals for Class
• Have an understanding of in vitro
assays- strengths and limitations
• Appreciation of complexity of working
with animal models
• Resource for your research
• Challenge you to think about the larger
issues
You have a new drug that you
think has an anticancer activity.
What do you do next?
Assay effect of drug on cells in
culture.
Is there a biological response to
the drug?
Death
Proliferation
Static
Growth
Regression
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Measuring changes in the
number of cells
•Cell count
•DNA based
•Respiration
Must understand the basis of the assay and
how the drug of interest could effect
measurements.
Cell Count
Hemocytometer is used to manually count cells
with a microscope.
• Can use dyes to distinguish between live and dead cells.
•Cells are visually assayed.
•Labor intensive.
•Cheap.
Coulter counter counts events.
• Can set parameters to distinguish clumps of cells.
•Can assay large number of cells.
•Fast & easy.
•Requires purchase of expensive equipment.
Markers of Proliferation
Ki 67 is a marker of proliferation.
• Present during all active phases of the cell cycle (G1, S, G2
and mitosis).
•Does NOT tell you what phase of the cell cycle the cells are.
•Absent from resting cells (G0).
•During interphase Ki-67 antigen is detected in the nucleus
and relocates to chromosomes during mitosis.
•Individual cells.
PCNA: Proliferating Cell Nuclear Antigen
.
• Identified as an antigen that is expressed in the nuclei of
cells during DNA synthesis phase of the cell cycle.
•Individual cells.
DNA based
BrdU Incorporation
H3-thymidine Incorporation
•Based on incorporating a labeled nucleotide into
the DNA of dividing cells.
•Individual cells or population based.
•Measures amount of DNA synthesis that has
occurred in a given time frame.
•Does not tell you if the cells are alive or dead,
just that the DNA has be replicated.
Mitochondrial Functional Assay
• Respiration - Assays based on mitochondrial function
(MTT, XTT, MTS, WSTs)
• Mitochondria reduces tetrazolium salts to formazan
crystals resulting in a color change.
• Colorimetric assay determines the amount of the formazan
crystals formed.
• Each cell typically has a linear relationship between cell
number and production of formazan dye which depends on
the respiration of the cells. Must be determined for each
cell type.
• Drugs that affect cellular respiration will alter MTT assay
independent of cell number.
• Performed on cell population.
• Easy, fast and cheap.
Colony Forming Assay
Cell Clonogenic Survival Assay
•Tests the capability of adherent cells to survive and replicate
following drug treatment.
•At 70% confluency treat cells.
•Trypsinize and count cells.
•Replate at low density and allow to grow ~9 days.
•Count colonies.
•Number
•Size
•Accurate, but time consuming.
•Based on ability of a single cell to grow into a colony.
•Not all cells will grow at the low density needed for the assay.
Apoptosis
DNA Laddering
• Method for detecting DNA fragmentation
based on size.
• Utilizes DNA fragmentation that occurs
during apoptosis.
• Cells undergoing apoptosis cleave their
DNA between nucleosomes.
Nucleosomes are ~200bp apart. Run
DNA on agarose gel get pattern of DNA
at ~200bp intervals appearing as a
“ladder”.
• Quantitative on a cell population, not cell
basis.
• Detects late stages of apoptosis.
http://mct.aacrjournals.org/content/2/10/961/F7.large.jpg
Apoptosis
TUNEL: Terminal deoxynucleotidyl transferase
dUTP nick end labeling.
•Method for detecting DNA fragmentation by labeling the
terminal end of nucleic acids.
•Utilizes DNA fragmentation that occurs during apoptosis.
•Quantitative on a cell basis.
•Detects late stages of apoptosis.
Caspase Assay
•Detects unique proteases activated during apoptosis.
•Quantitative on a cell basis or cell population.
•Depending on the caspase being assayed can detect early
and mid stage apoptosis.
Motility
Wound Healing/Scratch Test
•Scratch a confluent layer of cells and assay the “migration” of
cells into the gap.
•Assay performed over time.
•Need to determine if result of migration (movement of the cells
into the area) or proliferation of cells at the edge.
•Can be used in conjunction with time lapse photography.
Invasion Assay
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Invasion
Transwell migration and invasion
assay
•Can coat the filter with a matrix to determine if the
cell can invade through the matrix.
•Used for chemotaxis – does the cell move towards
a compound.
Competing models of drug action.
Day 0
1
2
3
4
5
Control
1
2
4
8
16
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Cell kill
0.5
1
2
4
8
16
50
1
1.7
3.0
5.3
9.2
16
50
Slow down
IC
50% kill at drug treatment compared to 20%
increase in doubling time (from 1 to 1.25 days):
result in same IC50 (half maximal inhibitory
concentration); concentration that gives half the
maximal inhibitory response.
Dose response assays that assesses total
population growth, growth slow-down and fractional
cell kill may be indistinguishable.
Time as a critical element in
experimental design and
interpretation.
Why use cell culture models?
•Easy.
•Fast.
•Less expensive.
•Homogenous population.
•Easier to molecularly manipulate.
•Lots known about established cell lines.
•Can work out molecular pathways.
Why NOT use cell culture
models?
•Reductionist approach.
•A cell growing as a layer of cells in a dish
does not have the complexity that a cell
growing in vivo has.
•Lacks whole animal physiology and
interacting organ systems.
•Lacks heterogeneity of cancer in patients.
•Often choose the cells based on what we
want to study thereby biasing our studies.
Cells growing in culture lack:
–Whole animal physiology
–Immune system
–Vasculature
–Microenvironment
–Variety of cell types (stromal, immune,
vasculature, epithelial, etc)
Cells in culture can not be used to
evaluate:
–Drug delivery
–Drug metabolism
–Toxicity
You know that your drug has an
anticancer effect on cells in culture.
What do you do next?
Moving your studies in vivo.
Animals in Research
• Use of animals in research setting is a
privilege, not a right.
• Use of animals in research is heavily
supervised and regulated.
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Institute Animal Care and Use
Committee (IACUC)
• Institutional committee that oversees all
animal research on campus.
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The Three A’s of Animal
Research Design
• Adequate: animal numbers
• Appropriate use of animal models to
minimize the number of animals
• Augmentation of animal research
models with cell culture when
scientifically appropriate
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The Three R’s of Protocol
Preparation
• Replacement: Invertebrates, non-living
systems, computer simulations.
• Reduction: minimize numbers of
animals.
• Refinement: reduce or eliminate
unnecessary pain and distress.
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Institute Animal Care and Use
Committee (IACUC)
• Federal legislation defines IACUC.
• IACUC oversees the care and use of animals
to assure that it is humane.
• Committee must have at least 5 members.
Including one member not affiliated with the
institution (lay).
• Appointments to the IACUC are under the
direction of the CEO.
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IACUC Functions - Programmatic
Based on USDA&PHS Regulations/Guide
• Semiannually review of animal program.
• Semiannually Inspect the animal facilities.
• Review and investigate reported concerns.
• Reports and recommendations to the Institutional
Official (IO).
• Protocol review and review of grants for protocol
coverage.
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Models to be covered
•Xenograft
•Spontaneous
•Carcinogen-Induced
•GEMs (Genetically Engineered Mouse models)
–Transgenic
–Knockout
–Regulatable transgenic
Xenograft Models of Cancer
•Human cell lines in Immunocompromised
mouse
•Human tumor grafts in Immunocompromised
mouse
•Mouse cell lines in syngeneic host
•Mouse tumor grafts in syngeneic host
Immunodeficient Mouse Strains
•
•
•
•
Nude Mice.
a hairless mutant discovered in 1962
is immunodeficient
lacks a thymus, which is essential for the production of Tcells, lymphocytes that are essential to the immune system
• does not reject tumor transplantations from other species
• Before discovery of the nude mouse, human tumors were
grafted and grown in immune-privileged sites, such as the
anterior chamber of the eye, the brain and the cheek pouch.
These locations are inconvenient, and the tumors are
eventually rejected.
Immunodeficient Mouse Strains
• SCID Mice.
• mice with severe combined immune deficiency
(SCID) were discovered In 1983
• SCID mice are even more immunodeficient than
nude mice
• lack both T and B lymphocytes
• Tumors from other species are easily transplanted
into SCID mice and will grow without being rejected.
• For certain specific tumors, SCID mice show
improved transplantability over nude mice
Immunodeficient Mouse Strains
• NOD.Cg-PrkdcscidIl2tm1Wjl/Szj Mice
• Commonly known as NOD scid gamma
• Complete null mutation of interleukin-2 receptor
gamma chain
• Lacks mature T and B lymphocytes
• No NK cell activity
• Deficient in cytokine signaling
• Does not produce detectable serum immunogloblin
• Hematopoietic stem cells and peripheral blood
mononuclear cells engraft more efficiently
• Decreased incidence of lymphoma results in longer
lifespan
• Becoming the preferred strain to graft human tissues
into because of better take rates than regular SCIDs
Subcutaneous Implantation
• Implant cells or tissues under the skin
Advantages
-external monitoring of growth
-easy & most commonly used
-human cells
Possible disadvantage
-suboptimal vascular site
-poor take rate
-can be difficult to find tissue that does not grow into
a tumor
-can be an irritant to the animal
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Subrenal Grafting of Tumor
Tissue or Tissue Recombinants
•Use human tissue grafted into an
immuno-deficient host animal
•Combine tissues with various
genetic alterations and evaluate
phenotype
–Example RB-/- epithelium associated
with wildtype stroma and vise versa
Tissue Recombination &
Subrenal Capsule Grafting
• Advantages
– Work with human tumors
– Rescue tissue from embryonic lethal phenotype
– Examine genetic modification in a single tissue
type (epithelium vs stroma)
– Allows targeting of tissue type even when
promoters are not available
– Lentiviral infection of tissues
– Rapid and inexpensive
– Higher take rate than subcutaneous site
– Can recover non-growing, live tissues
Tissue Recombination &
Subrenal Capsule Grafting
• Disadvantages
– Artificial location- Different innervation,
vasculature, anatomy
– Closed system
– Tissue samples are very small
– Technically more difficult
BPH-1 cells grafted alone
Ki67 staining
BPH-1 + UGM (urogenital sinus mesenchyme)
E-cadherin Staining
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Adapted from: Y Wang et al. Cancer Res. (2001) 61(16):6064-72.
Clinical Samples of Human Prostate Grafted Subrenal
Before
After 3 Months
Pathology
Gleason Grade 3
Proliferation
Ki67 Staining
PSA Staining
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Renal Grafted
Before
After 3 Months
Pathology
PIN (Prostatic
Intraepithelial Neoplasia)
Basal cells
p63 Staining
PSA Staining
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Kidney
Graft
Kidney
Graft
Graft
Graft
Graft
Kidney
Graft
Graft
Graft
Graft
Nontumorigenic
Kidney
Tumorigenic
Benign Histology
T + E2 Treated
Untreated
Carcinoma * normal glandular 43
structure
Adapted From: WA Ricke et al. International Journal of Cancer, (2006) 118(9): 2123 - 2131
Xenograft Models
• Advantages
–
–
–
–
Human cancer cells
Homogenous population
Minimal variability
Fewer number of animals
needed
– Tighter group of tumor
bearing animals
• Disadvantages
– Does not recapitulate the
complexity of tumor
progression
– Immune compromised host
– Ectopic site
– Can not test prevention
– Not able to study early
events
– Cancer progression limited
Spontaneous Models
• Some strains of laboratory animals are
susceptible to spontaneously
developing certain types of tumors
Spontaneous Models
• Advantages
– May mimic some types of
human diseases
– Can use to study early
disease
– Can use for prevention
– Includes elements of
progression
• Disadvantages
– Variability of disease
progression
– Large animal numbers
needed
– Long time to develop
disease
– Penetrance (Not all animals
get disease)
Carcinogen-Induced
• Animal is treated with a carcinogen to induce
cancer
• Some laboratory animal strains are more
susceptible
• Examples include:
–
–
–
–
–
Lung cancer
Skin cancer
Bladder cancer
Stomach cancer
Prostate cancer
Carcinogen-Induced
• Advantages
• Disadvantages
– Mimics initiation steps of
– Health hazard to
some cancer
investigator
– Can study early events
– Used to id predisposing
conditions
– Can study prevention
– Variability of disease
progression
– Can require large animal
numbers
– Penetrance (all animals may
not get disease)
Genetically Engineered
Mouse (GEM) Models
Transgenic
Knockout
Regulatable
Simple Transgenic Models
• Tissue specific promoter
• Overexpression of gene
• Ectopic expression (inappropriate expression)
–
–
–
–
–
Oncogene
Growth factor
Activated signaling molecules
Dominant Negative molecules
Cell cycle regulators
Endogenous gene is intact.
2007 Nobel Prize in Physiology and Medicine
for discoveries of principles for introducing
specific gene modifications in mice by the use
of embryonic stem cells.
Mario Capecchi
University of Utah
Sir Martin Evans
Cardiff University, UK
Oliver Smithies
UNC-Chapel Hill
Anatomy of a Transgene
Promoter
Ubiqitous
Tissue-specific
Transgene
Start site
–Oncogene
–Growth factor
–Activated signaling molecules
–Dominant Negative molecules
–Cell cycle regulators
pA
Poly-adenylation
site
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Pronuclear Injection
ES Cells
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Pronuclear Injection
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On line Movies from YouTube
Transgenic Technology
Video: Transgenic
Science of Transgenics Part 1 (3:15-5:00) by Satoshi Amagai
http://www.youtube.com/watch?v=ujZHrR1mro8
Science of Transgenics Part 2 PCR screening (0:00-0:47)
http://www.youtube.com/watch?v=EFNtTfhhQ70
Nuclear Injection (1:36-2:23)
http://www.youtube.com/watch?v=Q5-OR87fqac&feature=player_embedded
Video: Stem Cell Transfer
Homologous Recombination 0:50- by Satoshi Amagai
http://www.youtube.com/watch?v=EFNtTfhhQ70
Injection into Blastocyst (1:50-2:30)
http://www.youtube.com/watch?v=N8koAruTRNs&feature=player_embedded
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Chimeras
From Wikipedia, the free encyclopedia
A chimera is an animal that has two or more
different populations of genetically distinct
cells.
Transgenic Models
• Advantages
– May mimic initiation steps of
some cancer
– Can study early events
– Can test genetic lesion that
predispose
– Autochthonous (rising in the
tissue of origin)
– Penetrance usually 100%
– Immune system intact
– Can progress with time
• Disadvantages
– Variability of disease
progression
– Often requires large animal
numbers
– Initiator may be artificial
– Time consuming to
characterize & validate
– Expression influenced by site of
integration
– May produce chimeric offspring
if integrated later in
development.
Knockout Models
Approaches:
• *Homologous recombination
• *Cre-Lox system to KO in a tissuespecific manner
• Dominant Negative Transgenic
(Functional KO)
*Disrupts the endogenous gene.
On line Movies from YouTube
Transgenic Technology
Video: Stem Cell Transfer
Homologous Recombination 0:50- by Satoshi Amagai
http://www.youtube.com/watch?v=EFNtTfhhQ70
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Knockout Transgenic Models
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Black mouse
White mouse
Black mouse
Chimeric mouse
Black mouse
Black nontransgenic mouse
Agouti Transgenic mouse
Black mouse
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Cre-Lox Recombination
Isolated from bacteriophage P1
Cre = causes recombination
loxP = locus of crossover P1
floxed = flanked by loxP
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Positive selection for genetic manipulation.
Transiently transfect with Cre to remove the
selection markers.
Negative selection for clones that retain the
selection markers.
PCR screen clones for both exon 1 & 2.
Gene deletion.
Floxed gene for tissue-specific knockout.
Clone with exon1 and selection markers removed. Does not survive selection.
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Clone with exon 1 removed, but retains selection markers. Does not survive selection.
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Tissue Specific Knockout
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On line Movies from YouTube
Transgenic Technology
Homologous Recombination Knockout/Knockin (0:50-6:07)
http://www.youtube.com/watch?v=EFNtTfhhQ70
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Inducible Systems
• Requires a drug or hormone to induce
gene expression
• Can be used with Cre to obtain a
regulated knockout
• Examples include:
– Tet On/Off (Tetracycline)
– RU486 (Progesterone)
– Ecdysone (Insect steroid hormone)
Ideal Inducible System
•
•
•
•
•
•
Tightly regulated
Not leaky- low basal level
Induce to high level of expression
Induce only in presence of inducer
Induce nontoxic
Nonphysiological- not affect expression of
endogenous genes
• Reversible expression
Conditional Transgenic
General Scheme
CBS= cognate binding site
Pmin= minimal promoter
pA= polyadenylation signal
Pspec= specific promoter
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Tetracycline Regulated System
tet Transactivator
reverse tet-dep Transactivator
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Inducible Systems
• Advantages
• Disadvantages
– Control when gene is
expressed
– Can rescue an embryonic
lethal gene
– Allows for normal
development of the organ
– Requires bi- or trigenics to get
tissue specific expression
– Large breeding program
– Expensive
– Can be dependent on
expression level of regulator
How to pick a model
• Recapitulates key features of human
disease
• Suitable for the question to be asked
– Consider mode of action of treatment
– Mechanism of action of transgene
– Effect on transgene expression
Know your model
• Key features
–Strengths
–Weaknesses
• Strain background can make a
difference on disease progression
Strain Background Affects
TRAMP Phenotype
Wild-type
C57Bl/6
C57Bl/6 x FVB
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Recognize differences between
model and human disease
•
•
•
•
Anatomical differences
Functional differences
Physiological differences
Examples:
– Reproductive Tract
– Bone turnover
– Metabolism/sensitivity to drugs
Anatomy of the Prostate Gland
Human vs. Mouse
Human
One gland encapsulated in
fibromuscular sheath
Mouse
Four lobes with no capsule
http://media.wiley.com/CurrentProtocols/TX/tx1603/tx1603-fig-0002-1-full.gif
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Human vs. Mouse Prostate Histology
Human
Stroma-thick bundles of smooth muscle
Mouse
Stroma- loose connective
tissue with
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thin layer smooth muscle
What do you measure
• When start experiment
– How often can you treat?
– What stage of disease does therapy target?
– Does treatment have memory?
– Only target disease present at time of treatment
– Can animals be continuously enrolled on
protocol or do all animals need to be treated at
same time (biological therapy)?
What do you measure
• When to end experiment
– Will disease continue to progress
• Miss window of observed response if wait too long
– How rapidly do the animals need to be
processed- Can you do all the tissue collection?
• What do you measure
– How evaluate for an effect
http://web.ncifcrf.gov/researchresources/mmhcc/
Available Strains
Newly Accepted Strains
Strain Submission Forms
Suggest a Strain
General Information
MMHCC Home
MMHCC Home Page http://emice.nci.nih.gov/
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