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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 5 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 15 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 32 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. 25 Institute Animal Care and Use Committee (IACUC) • Institutional committee that oversees all animal research on campus. 26 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 27 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. 28 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. 29 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. 30 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 36 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 40 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 41 Renal Grafted Before After 3 Months Pathology PIN (Prostatic Intraepithelial Neoplasia) Basal cells p63 Staining PSA Staining 42 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 52 Pronuclear Injection ES Cells 53 Pronuclear Injection 54 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 55 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 59 Knockout Transgenic Models 60 Black mouse White mouse Black mouse Chimeric mouse Black mouse Black nontransgenic mouse Agouti Transgenic mouse Black mouse 61 Cre-Lox Recombination Isolated from bacteriophage P1 Cre = causes recombination loxP = locus of crossover P1 floxed = flanked by loxP 62 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. 63 Clone with exon 1 removed, but retains selection markers. Does not survive selection. 64 Tissue Specific Knockout 65 On line Movies from YouTube Transgenic Technology Homologous Recombination Knockout/Knockin (0:50-6:07) http://www.youtube.com/watch?v=EFNtTfhhQ70 66 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 69 Tetracycline Regulated System tet Transactivator reverse tet-dep Transactivator 70 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 74 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 76 Human vs. Mouse Prostate Histology Human Stroma-thick bundles of smooth muscle Mouse Stroma- loose connective tissue with 77 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/ 80 81