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Model Tumor Systems

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Different mouse strains can be very different ... patterns of toxicity may differ in mice and people ... Mice are often sold very young (soon after weaning) ... – PowerPoint PPT presentation

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Title: Model Tumor Systems


1
Model Tumor Systems
  • Sara Rockwell, Ph.D.
  • Departments of Therapeutic Radiology and
    Pharmacology
  • Yale University School of Medicine
  • New Haven, CT
  • Residents course, February 5, 2009

2
References
  • E. J. Hall and A.J. Giaccia, Radiobiology for the
    Radiologist, 6th edition, Chapter 20
  • S. Rockwell and K. R. Rockwell, Mouse Models for
    Experimental Cancer Therapy, in Sourcebook of
    Models for Biomedical Research
  • R.F. Kallman (ed.) Rodent Tumor Models in
    Experimental Cancer Therapy.

3
Cells in culture as models for tumors
  • Uniform, well defined cell populations
  • Good quantitative endpoints for cell survival
  • Very useful model systems for some studies
  • Examine effects of proliferation, cell cycle
    phase, etc.
  • Examine effects of cellular environment
  • Examine mechanisms of action
  • Examine interactions between drugs and radiation
    and establish the mechanisms underlying such
    interactions
  • Provide insights into effects of sequence, time
    and dose on effects of single agent and combined
    modality treatments

4
What can we learn from cell cultures ?
  • Many things
  • Study biology of cells, how they grow, how they
    interact
  • How they metabolize drugs
  • How they respond to treatment with drugs and/or
    radiation
  • How therapeutic agents interact
  • Example Breast cancer cells in culture, treated
    with Adriamycin, alone or in combination with a
    commonly used herbal medicine called black
    cohosh.
  • Question what happens to tumors and normal
    tissues in vivo?

5
Tumor cell lines in culture differ from tumor
cells in vivo
  • adapted to survive and grow in culture
  • altered proliferation
  • may have been cloned
  • altered gene expression, enzyme activity
  • altered shape and motility
  • altered metabolism
  • altered differentiation
  • altered response to external signals

6
The environment of cells in ordinary cell
cultures differs from that of cells in vivo
  • Limited or no contact with similar cells
  • Limited or no contact with other cell types
  • Oxygen levels are high
  • Nutritional environment artificial and limited
  • pH
  • Cytokines other external signals
  • Temperature, motion, growth surface, etc.

7
Some culture systems are better models for
tumors in vivo
  • Primary cell explants
  • Three dimensional cultures
  • Perfused cultures
  • Physiological growth surfaces
  • Co-cultures containing multiple cell types
  • Tissue and organ cultures
  • None of these fully model tumors in vivo - they
    are all still models, with inherent limitations

8
Spheroids Sutherland et al.
9
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10
Human cells are sometimes needed
  • E.g. in studies of human cytokines, antibodies,
    or gene therapy
  • But remember that cultures of human cells are
    also artificial model systems that do not fully
    model human tumors in vivo
  • Malignant cell lines are heavily selected
  • Normal cells are probably not normal if they
    grow well in culture, even if they have been
    cultured only for a limited period of time

11
Animal tumor models are closer, but still have
limitations as models for human patients
  • Mice are not furry little people
  • Neither are rats
  • Or cats
  • Or dogs
  • Or non-human primates
  • In vivo model systems must also be chosen with
    care to ensure that the model is appropriate for
    the specific question being addressed

12
Large animals with tumors are used only rarely in
experimental cancer therapy
  • Some studies with veterinary patients
  • Generally designed like human clinical trials
  • Have all limitations of human clinical trials
  • Potentially curative regimens
  • Tolerable regimens
  • Clinical grade drugs
  • Oversight as strict as human studies (possibly
    stricter)
  • Multiple layers of oversight IACUC, NIH,
    USDA, AAALAC, FDA

13
Other problems with large animal studies
  • Vet patients more difficult to treat
  • Anesthetized for irradiation
  • Anesthetized for imaging
  • Post procedure care and monitoring
  • Patient variability is greater than in human
    clinical trials
  • Characteristics of tumors may differ from those
    of human tumors of same origin
  • Breast cancer in dogs
  • Grey horse melanoma
  • Dose-limiting toxicities may differ from those in
    people

14
Mice and rats are the most common models
  • Why? Because inbred strains are available
  • Genetically uniform - all identical twins
  • Well defined phenotypes
  • Increasingly well defined genotypes
  • Some have strain-specific tumors at high
    frequencies (sometimes in essentially 100 of
    the animals)
  • Tumors can be transplanted within an inbred
    strain
  • Early passage tumors
  • Serially transplanted tumors

15
Limitations of transplanted tumors must be
remembered in translating results to develop
treatment for cancer in people
  • Artificial model system
  • Inoculation of single focus of malignant cells,
    often at site selected for convenience or to
    minimize stress or injury to host
  • Cells often selected for rapid growth, high
    clonogenicity
  • Sometimes selected for other features
  • Metastatic rate/pattern
  • Response to specific agents
  • Ability to grow in vitro
  • Presence of a specific gene or marker

16
Some considerations in preclinical cancer therapy
studies using animals
  • Activation/metabolism/clearance of drugs and drug
    carriers vary with species and substrain
  • Different mouse strains can be very different
  • Drug metabolism may also vary with husbandry
    (e.g. bedding in rodent cages) and microbiology
  • Pharmacokinetics, biodistribution, clearance may
    be very different in rodents and people
  • Area under curve generally longer in people
  • Maximal attainable peak tumor levels may be lower
  • Can lead to inaccurate predictions of efficacy in
    humans
  • Severity and patterns of toxicity may differ in
    mice and people

17
More considerations in preclinical cancer therapy
studies using animals
  • Good Microbiology is critical to good science
  • Radiation and anticancer drugs are
    immunosupressive - subclinical infections can
    become clinical or lethal after treatment
  • Marrow
  • Gut
  • Lung
  • Subclinical infections can change tumor growth
    and response of tumors to treatment
  • Subclinical infections can change proliferation
    patterns in bone marrow and gut and therefore
    change response to treatment and toxicity of
    treatment
  • Tumors and cell lines can carry and transmit
    pathogens this poses hazards to the mice and to
    people
  • Use of specific pathogen free (SPF) mice is
    critical in experimental cancer therapy.

18
Still more considerations in preclinical cancer
therapy studies using animals
  • Match tumor burden to that in patients
  • Common problem treating unrealistically large
    tumors in mice
  • Growing mice are not good models for adult people
  • Mice are often sold very young (soon after
    weaning)
  • Juvenile mice respond differently to drugs and
    radiation than adult mice proliferation
    patterns in growing tissues hormones
  • Young mice can be unstable models a week of
    difference in age from batch to batch can make a
    huge difference in effect.

19
Still more considerations in preclinical cancer
therapy studies using animals
  • Modeling of clinical treatment regimens requires
    different treatment times in mice
  • Cell proliferation rates (tumors and normal
    tissues)
  • Daily treatment in people doesnt equal daily
    treatment in mice
  • Modeling of tumor and normal tissue responses to
    therapy requires different follow up times in
    mice
  • Mice have a maximum life span of 3 years
  • Mouse tumors have doubling times of days
  • 3 months of follow up in mice equals years of
    follow up in people

20
Human tumor xenografts in immune deficient mice
widely used model for human cancers
  • Only the malignant cells are human
  • Tumor cells have adapted for rapid growth in mice
  • The stroma and vascular bed are mouse
  • The other normal tissues are mouse
  • Pharmacokinetics, biodistribution clearance are
    mouse
  • Activation and metabolism of drug may reflect
    metabolism by mouse cells
  • Hosts have other defects which affect results
  • Nudes thermoregulation
  • SCIDs - DNA repair defects make the vascular bed
    and stroma sensitive to injury and alter the
    balance of direct and indirect tumor cell killing
    from that found in a syngeneic system

21
Special traps with the use of human tumor
xenografts in immune deficient mice to study
human-specific agents (e.g. MAb, siRNA)
  • Only the malignant cells are human
  • Tumor stroma is mouse
  • Vasculature is mouse
  • Normal tissues are mouse
  • Pharmacokinetics, biodistribution, targeting of
    tumor-specific agents will not resemble those in
    a person, because only the tumor will have the
    target
  • High tumor levels
  • Little or no accumulation in normal tissues
  • May have unrealistically large effects on
    xenografts
  • May have little toxicity to normal (mouse)
    tissues
  • Toxicities in the mouse tissues probably will not
    predict toxicities in human patients
  • No way to predict therapeutic ratio in patients

22
Another trap - transplanted tumors in genetically
engineered mice (GEM)
  • 50 generations of inbreeding are needed to
    produce inbred mouse lines
  • Many GEM lines have been inbred only 5-10
    generations from founders with outbred or mixed
    genetic backgrounds
  • GEM may be uniform at the locus of interest,
    because this is verified, but they are not
    uniform at many other critical loci
  • Not homozygous not syngeneic
  • Transplants of tumors arising in GEM are
    problematic
  • Studies using tumor lines transplanted from a
    parental mouse strain are also very problematic

23
Assays of tumor response
  • Tumor cell survival assays
  • TD50 assay
  • Lung Colony Assay
  • Spleen colony Assay
  • Tumor control assay (TCD50 assay)
  • Tumor growth assay

24
Tumor cell survival assays
  • Cell culture assay for tumor cell survival
  • Analogous to assays of cell survival for cells
    grown and treated in culture
  • Technique
  • Prepare treated and control tumors
  • Suspend cells from tumors
  • Count tumor cells
  • Plate cells at low density in cell culture
  • Allow viable clonogenic cells to grow into
    colonies
  • Count colonies, calculate plating efficiency ,
    calculate Surviving Fractions

25
Example Effect of Hypoxia on Radiation Response
of Cells in Solid Tumors
1.0
Hypoxic cells in vitro Hypoxic cells in tumors ?
0.1
Surviving Fraction
0.01
Normally aerated tumors in situ
0.001
Aerobic cells in vitro
0
10
20
30
Dose (Gy)
26
Strengths of cell survival assay
  • Highly quantitative
  • Good precision, reproducibility
  • Not influenced by late host toxicities of
    treatment
  • Measures cell survival directly
  • Can be used to examine subpopulations of tumor
    cells
  • Can be used to probe mechanisms
  • Considers only clonogenic cells (not influenced
    by response of differentiated or non-clonogenic
    cells)

27
Problems with cell survival assay
  • Not applicable to all tumors many tumor cells
    wont clone in vitro
  • Requires preparing a good single cell suspension,
    with high viability can be a technical
    challenge
  • Requires removing cells from tumor
  • Environment of cells changes from that of cells
    remaining in vivo
  • Some cells clonogenic in vivo may not be
    clonogenic in vitro (or vice versa)
  • Limited to relatively low radiation doses
  • Problems in measuring effects of fractionated or
    protracted treatments
  • Cell loss (normal loss from differentiation or
    environment)
  • Apoptosis and rapid cell death after treatment

28
Lung Colony Assay and Spleen Colony Assay
  • Colony formation assays Analogous to cell
    culture assay discussed above and to normal
    tissue assays discussed last session
  • Treat tumors
  • Prepare single cell suspensions
  • Inject cells into tail vein of syngeneic mouse
  • Cells lodge in lung (certain carcinomas and
    sarcomas) or spleen (certain lymphomas) and form
    colonies
  • Allow colonies to grow
  • Kill animal, remove and fix tissue, count
    colonies arising from viable tumor cells

29
Strengths of and problems with these assays
  • Similar to those for cell survival assays in
    culture
  • Additional problems
  • Requires good syngeneic system an immune
    response to the tumor can invalidate the assay
  • Requires good microbiology (Specific Pathogen
    Free SPF host animals)
  • Labor intensive
  • Expensive

30
TD50 Assay
  • Hewitt and Wilson first tumor cell survival
    assay
  • Was used with wide variety of lymphomas and solid
    tumors
  • Less widely used now (mostly because of expense,
    availability of cell culture assays)

31
Basic technique
  • Prepare treated, control tumors
  • Suspend cells from tumor
  • Count cells
  • Inject serial dilutions of cells into one or more
    isolated sites on syngeneic recipient mice
  • Watch for development of tumor at each
    inoculation site
  • Calculate (by probit or logit analysis) number of
    tumor cells required to form tumors in 50 of the
    inoculation sites TD50
  • Use TD50 for treated and control tumors to
    calculate the Surviving fraction for tumor cells
    in treated animals
  • SF TD50 treated cells / TD50 control cells

32
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33
Some things to note about these curves
  • Sigmoidal shape
  • Refects probabilistic endpoint of tumor formation
  • Heterogeneity in tumor inocula, host mice or
    recipient mice decreases the slope of the take vs
    cell number curve
  • Need range of tumor cell inocula
  • Need several mice per point
  • Need long follow up

34
Stengths of TD50 assay
  • Quantitative
  • Not influenced by late toxicities of treatment to
    original host mouse
  • Measures cell survival
  • Can be used to examine subpopulations
  • Can be used to probe mechanisms
  • Considers only clonogenic cells (not influenced
    by response of differentiated or non-clonogenic
    cells)

35
Problems with TD50 Assay
  • Very expensive and labor intensive
  • Requires removing cells from tumor
  • Environment of cells changes
  • Some cells clonogenic in tumor may not be
    clonogenic in site of inoculation (or vice versa)
  • Less precise than other cell survival assays
  • Problems in measuring effects of fractionated or
    protracted treatments
  • Cell loss
  • Apoptosis and rapid cell death

36
Tumor growth delay assay
  • Inoculate tumors into a large number of animals
  • Identical hosts
  • Quantitative injection of tumor cells
  • Site readily accessible for repeated
    measurements
  • 5-20 animals per treatment group
  • Identify each animal
  • Measure tumor volumes beginning when tumors first
    become palpable
  • Daily to weekly
  • Frequency depends on growth rate

37
  • At specific volume, randomize into treatment and
    control groups
  • Treat
  • Continue monitoring growth until each tumor
    reaches a defined size
  • Calculate time for each tumor to grow from the
    treatment volume to this size
  • Calculate growth delays induced by treatments

38
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41
Strengths of assay
  • Clinically relevant endpoint but it measures
    the success of failed treatments because all
    tumors must regrow for the assay to be valid
  • Need little prior knowledge of treatment efficacy
    to plan experiments
  • Relatively precise endpoint (if done well large
    numbers of similar tumors frequent, precise
    measurements, etc. )
  • Can be used with fractionated and protracted
    treatment regimens

42
Problems with assay
  • Uses non-curative treatments - all treatments
    fail
  • Requires syngeneic tumor/host system or
    immunosuppressive effects of treatment will
    influence the results
  • Cannot be used with tumors that metastasize early
  • Labor intensive and expensive
  • Toxicities to host can limit assay
  • Lethal toxicities of some drugs preclude using
    doses that perturb tumor growth
  • Drug toxicities may alter tumor growth
    indirectly, complicating interpretation of data
  • An amazing amount of really bad, misinterpreted
    tumor growth data can be found in the literature

43
Some problematic variations of tumor growth
studies (discussed only as a warning, not as a
recommendation for taking these shortcuts)
  • Kill all mice at a predetermined time, measure
    tumors (Classic chemotherapy approach gives
    volumes as T/C)
  • Kill all mice when the control tumors get as big
    as the animal care committee will let them grow
  • For relatively successful treatments, treated
    tumors may still be small or even undetectable
  • Cannot distinguish control from slow recurrence
    or slow growth
  • Measure time until tumors kill mice
  • Gives growth delay as T/C
  • For solid tumors, causes of death vary in
    different mice
  • Inhumane

44
Tumor control assay
  • Called by various names
  • Tumor control dose 50 (TCD50 assay)
  • Tumor cure dose 50 assay
  • Effective dose 50 (ED50)
  • Basic approach to determine the dose of
    radiation (alone or in combination with other
    agents) that controls 50 of a population of
    identical tumors

45
TCD50 Protocol
  • Inoculate tumors into a large number of animals
  • Quantitative injection
  • Site readily accessible for repeated measurements
  • 5-20 animals per treatment group
  • Identify each animal
  • Measure tumor volumes beginning when tumors first
    become palpable
  • Measurement frequency depends on growth rate
  • Daily to week
  • At specific time or volume, randomize into
    treatment and control groups

46
TCD50 protocol, continued
  • Treat with range of doses ranging from doses
    where 100 recurrence is expected to doses where
    100 control is expected
  • Continue monitoring the volume of each tumor
    until
  • It grows to a predetermined volume and is deemed
    not cured
  • It regresses completely and has been gone for a
    long enough time that it is statistically
    unlikely to recur (time depends on tumor usually
    gt 3 months). It is then considered controlled
  • Develop dose-response curves proportion of
    tumors cured as a function of dose
  • Calculate TCD50s for using probit or logit
    analyses
  • Compare TCD50s for different regimens

47
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48
Comments on TCD50s
  • Last slide showed data on probit scale
  • On a linear scale, the curve has a sigmoidal
    shape
  • Refects the probabilistic endpoint
  • Heterogeneity in treatments, tumors or mice
    decreases the slope of the tumor control vs dose
    curves
  • Uniformity of mice, tumors, and treatment
    regimens is essential for good data

49
Strengths of assay
  • Clinically relevant endpoint
  • Viewed as best assay by some regulatory
    agencies and clinical trial groups
  • Applicable to radiation dose ranges and treatment
    intensities of clinical interest
  • Can be used with fractionated and protracted
    treatments
  • Can be relatively precise if experiment is done
    right (large numbers of animals, well matched
    tumors, etc)

50
Problems with assay
  • Requires syngeneic tumor/host system
  • Requires non-metastatic tumors
  • Requires good microbiology in animal colony
  • Exceedingly labor intensive and expensive
  • Each TCD50 requires 50 animals, most of which
    will be followed for months
  • Requires prior information on treatment agents
    used to chose curative doses for assay
  • Requires cures to obtain any information
  • Therefore if combining radiation with drug that
    does not cure tumors alone, cannot measure effect
    of drug alone
  • Therefore Usually cannot be used to determine
    whether effects are additive, synergistic, etc

51
Conclusions
  • In vitro and in vivo models can provide valuable
    insights into the therapeutic responses of solid
    tumors
  • All model systems and all assays have limitations
    none are perfect models for human cancer
    patients
  • Watch for poorly designed studies, misinterpreted
    studies, and erroneous conclusions as you read
    the literature

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