Tumor Models in Cancer Research (eBook)

Dr. Beverly A. Teicher is Senior Scientific Director in Cancer Research at Genzyme Molecular Oncology and Genzyme Corporation, Framingham, Massachusetts. She has authored or co-authored more than 400 scientific publications, has edited five books, senior editor for the journal Clinical Cancer Research and is series editor for the Cancer Drug Discovery and Development book series.

Tumor Models in Cancer Research 3
Preface 5
Contents 7
Contributors 11
Part I Introduction 15
Chapter 1: Perspectives on the History and Evolution of Tumor Models 16
1.1 Introduction and Statement of the Problem 16
1.2 Tumor Models for Cancer Drug Development: Where We Were 18
1.2.1 Historical Basis 18
1.2.2 Early Screening Models 19
1.3 Novel Screens Beget Novel In Vivo Model Challenges 21
1.3.1 Non-mammalian Models 21
1.3.1.1 Unicellular 21
1.3.1.2 Multicellular 22
1.3.2 Technology-Intensive Screening 24
1.3.2.1 High-Throughput Screening 24
1.3.2.2 Chemogenomics 25
1.3.2.3 Proteome and Kinome Screens 25
1.3.2.4 Nanotechnology 25
1.3.2.5 RNA Interference 26
1.3.3 In Vitro Models 26
1.4 Tumor Models for Cancer Drug Development: Where We Need to Be 27
1.4.1 “In Vitro” Area Under the Concentration × Time Curve for Target Effect 28
1.4.2 Qualification of Compound for In Vivo Study 28
1.4.3 Initial Rodent Pharmacology and Model Selection 29
1.4.4 Sample Size and Randomization of Animals 29
1.4.5 Correlative Studies 29
1.4.6 Additional Desirable Studies 29
1.5 Conclusion 30
References 31
Part II Transplantable Syngeneic Rodent Tumors 34
Chapter 2: Murine L1210 and P388 Leukemias 35
2.1 Introduction 36
2.2 Role in Drug Screening 36
2.3 Characteristics 37
2.4 Sensitivity to Clinical Agents 38
2.5 Predictive Value 44
2.6 Drug-Resistant Leukemias 46
2.6.1 Resistance to Alkylating Agents 46
2.6.2 Resistance to Antimetabolites 48
2.6.3 Resistance to DNA- and Tubulin-Binding Agents 48
2.7 Conclusions 50
References 52
Chapter 3: Transplantable Syngeneic Rodent Tumors: Solid Tumors in Mice 54
3.1 Introduction 55
3.2 Compatibility! Compatibility! Compatibility! 55
3.3 Compatible But Not Perfect: Inbred Mice and Genetic Drift 55
3.4 Evidence of Tumor–Host Incompatibility and Consequences 56
3.4.1 No-Takes 56
3.4.2 Spontaneous Regressions or Tumors that Fail to Progress to Over 1,000-mg Size 58
3.4.3 Excessive Curability 58
3.4.4 Lack of Invasion and Metastasis 61
3.5 Compatibility Problems Unique to Human Tumors in Immune-deficient Mice 61
3.6 Passage of Tumors in Cell Culture: Maintaining Genotype, Histology, Biologic Behavior, and Drug–Response Characteristic 62
3.7 Cancer: a Cellular Disease, Take-Rate, Feeder Effect, Implications for Cure 63
3.8 Results Tabulation of Chemotherapy Trials: Desired Information from a Tumor Model 72
3.9 Characterization of a Tumor Model and Quality-Control Monitoring 80
3.10 Summary 81
3.11 Methods 82
3.11.1 Tumor Maintenance 82
3.11.2 Origins of Mouse Tumors Used or Discussed 82
3.11.3 Chemotherapy of Leukemias: L1210/0 83
3.11.4 X-Irradiation of Solid Tumors 83
3.11.5 Chemotherapy of Solid Tumors 83
3.11.6 End Points Assessing Antitumor Activity Solid Tumors 84
3.11.6.1 Tumor-Growth Delay (T–C Value) 84
3.11.6.2 Percent Increase Life Span 84
3.11.7 Calculation Tumor-Cell-Kill 84
3.11.8 Activity Rating Solid Tumors 85
3.11.8.1 Activity Rating for Leukemia L1210/0 85
3.11.9 Non-quantitative Determination Antitumor Activity Tumor-Growth Inhibition (T/C Value) 86
References 86
Chapter 4: B16 Murine Melanoma: Historical Perspective on the Development of a Solid Tumor Model 90
4.1 Introduction 90
4.2 Historical Context 91
4.3 B16 Melanoma 95
4.4 Conclusions 101
4.5 Dedication 105
References 105
Part III Human Tumor Xenografts 107
Chapter 5: Human Tumor Xenograft Efficacy Models 108
5.1 Introduction 108
5.2 Xenograft Tumor Models for Efficacy Evaluation 111
5.2.1 Immunodeficient Mice 111
5.2.2 Cultured Tumor Cells Vs. Tumor Fragments 112
5.2.3 Subcutaneous Vs. Orthotopic Transplantation 113
5.2.4 Tumor Metastasis 114
5.2.5 Monitoring Tumor Progression and Determining Efficacy 115
5.2.6 Examples of Single Agent and Combination Preclinical Trials 117
5.3 Pros and Cons 121
5.4 Pharmacology and Pharmacokinetic Correlations 122
5.5 Future Perspectives 124
References 126
Chapter 6: Imaging the Steps of Metastasis at the Macro and Cellular Level with Fluorescent Proteins in Real Time 134
6.1 Introduction 134
6.2 The In Vivo Revolution Sparked by Fluorescent Proteins 135
6.2.1 Isolation of Stable High-Level Expression GFP and/or RFP Expressing Tumor Cell Lines 135
6.2.1.1 Production of GFP Retrovirus 135
6.2.1.2 Production of RFP Retroviral Vector 135
6.2.1.3 Production of Histone H2B-GFP Vector 136
6.2.1.4 GFP or RFP Gene Transduction of Cancer Cells 136
6.2.1.5 Establishment of Dual-Color Cancer Cells 136
6.2.2 Imaging Sites of Metastasis 137
6.2.2.1 Patterns of Contralateral and Regional Lung Tumor Metastases Visualized by GFP Expression in Orthotopic Models 137
6.2.2.2 GFP-Expressing Bone Metastases of Lung Cancer in Orthotopic Models 137
6.2.2.3 Prostate-Cancer Bone and Visceral Metastasis Visualized by GFP in Orthotopic Models 137
6.2.2.4 GFP-Expressing Melanoma Bone and Organ Metastasis Models 138
6.2.2.5 GFP-Expressing Brain Metastasis in Orthotopic Models 138
6.2.2.6 GFP-Expressing Experimental Multi-organ Metastases in Nude Mice 138
6.2.3 Whole-Body Fluorescence Optical Tumor Imaging of Tumor Growth and Metastasis 139
6.2.4 Whole-Body Imaging of RFP Pancreatic Cancer Progression 139
6.2.5 Whole-Body Imaging of RFP Prostate Cancer Progression 140
6.2.6 Whole-Body Imaging of GFP Colon Cancer Progression 140
6.3 Imaging Bacterial Targeting of Tumors 141
6.4 Advantages of GFP Imaging 144
6.5 Viral Labeling of Tumors with GFP in Live Animals 146
6.5.1 Selective In Vivo Tumor Delivery of the Retroviral Green Fluorescent Protein Gene to Report Future Occurrence of Metas 146
6.5.2 Telomerase-Dependent Adenovirus to Label Tumors In Vivo for Surgical Navigation 147
6.6 Color-Coded Imaging of the Tumor Microenvironment 148
6.6.1 Color-Coded Imaging of the Tumor–Host Interaction Using Colored Host Mice 148
6.6.1.1 Transgenic GFP Nude Mouse 148
6.6.1.2 Transgenic RFP Nude Mouse 149
6.6.1.3 Transgenic CFP Nude Mouse 150
6.6.2 Color-Coded Imaging in the Tumor Microenvironment 152
6.6.3 Noninvasive Color-Coded Imaging of the Tumor Microenvironment 152
6.7 Imaging the Cell Biology of Metastasis In Vivo 155
6.7.1 Dynamic Imaging of GFP or RFP-Expressing Cancer Cells in Blood Vessels and Lymphatics 155
6.7.2 Imaging Cancer Cell Trafficking in Blood Vessels 156
6.7.3 Imaging Cancer Cell Trafficking in Lymphatic Vessels 157
6.7.4 Determining Clonality of Metastasis Using Color-Coded Cancer Cells 158
6.8 Imaging Lateral Gene Transfer Between Cancer Cells 158
6.8.1 Color-Coded Imaging of Circulating Cancer Cells 158
6.8.2 Color-Coded Imaging of Gene Transfer Between Cancer Cells Interacting In Vivo 160
6.8.3 Color-Coded Imaging of Gene Transfer from High- to Low-Metastatic Osteosarcoma Cells In Vivo 160
6.9 Methods 161
6.9.1 Imaging Apparatus 161
6.9.1.1 In Vivo Imaging with an LED Flashlight and Filters 161
6.9.1.2 Simple Light-Box Imaging 161
6.9.1.3 In Vivo Imaging with a Fluorescence Dissecting Microscope 162
6.9.1.4 In Vivo Cellular Imaging with a Variable Magnification Imaging Chamber 162
6.9.1.5 Imaging Chambers Designed for Whole-Body Imaging 163
6.10 Patient-Like Orthotopic Tumor Models 164
6.10.1 Surgical Orthotopic Implantation 164
6.10.1.1 Ovarian Cancer 164
6.10.1.2 Lung Cancer 165
6.10.1.3 Prostate Cancer 165
6.10.1.4 Colon Cancer 165
Colonic Transplantation 165
Intrahepatic Transplantation 166
6.11 Technical Details 166
6.11.1 RFP Retrovirus Production 166
6.11.2 GFP Retrovirus Production 167
6.11.3 RFP or GFP Gene Transduction of Tumor Cell Lines 168
6.11.4 Cell Injection to Establish an Experimental Metastasis Model 168
6.11.5 Surgical Orthotopic Implantation to Establish a Spontaneous Metastasis Model 168
6.11.6 Imaging 169
6.11.6.1 Fluorescence Microscopy 169
6.11.6.2 Fluorescence Stereomicroscopy 169
6.11.7 Chamber Imaging Systems 170
6.11.7.1 Olympus OV100 170
6.11.7.2 INDEC FluorVivo 170
6.11.7.3 UVP iBox 170
6.11.8 Tumor Tissue Sampling 171
6.11.9 Measurement of GFP-expressing Tumor Blood Vessel Length and Evaluation of Antiangiogenetic Agents 171
6.11.10 Immunohistochemical Staining 171
References 172
Chapter 7: Patient-Derived Tumor Models and Explants 176
7.1 Introduction 176
7.1.1 Historical Perspective 176
7.1.2 The Strength of Human Models Derived from Patient Explants 177
7.2 Materials and Methods 178
7.2.1 Establishment of Human Tumor Xenografts from Patient Explants 178
7.2.1.1 Animals 180
7.2.1.2 Tumors 180
7.2.1.3 Tumor Growth Measurements 180
7.2.2 Experimental Design of Drug Testing 180
7.2.2.1 Study Design 180
7.2.2.2 Chemotherapy 181
7.2.2.3 Evaluation Parameters for Tumor Response 182
7.2.3 Molecular Target Characterization of the Freiburg Patient-Derived Tumor Xenograft Panel 182
7.2.4 Clonogenic Assay 183
7.2.4.1 Preparation of Single-Cell Suspensions 183
7.2.4.2 Culture Methods 183
7.2.5 Gene Signatures 184
7.3 Results and Discussion 185
7.3.1 Take Rates and Growth Behavior of Patient Tumor Explants in Nude Mice 185
7.3.2 Comparison of Drug Response of a Tumor Growing in the Nude Mouse and in the Patient 186
7.3.2.1 Cytotoxic Agents 186
7.3.2.2 Targeted Agents 187
7.3.3 Clinically Used Cytotoxic Agents Active in the Freiburg Xenograft Panel 187
7.3.3.1 Cytotoxic Drugs 187
7.3.3.2 Clinically Used Targeted Agents 188
7.3.3.3 Comparison of Freiburg Experience to Other Groups 190
7.3.4 Molecular Characterization of Human Tumor Xenografts for Target-Oriented Drug Discovery 191
7.3.4.1 Significance of Gene Signatures for Anticancer Therapy 191
7.3.4.2 Significance of Tumor Tissue Microarrays 192
7.3.4.3 Target Prevalence in the Freiburg Tumor Collection 193
7.3.5 Assessment of In Vivo Efficacy of Anticancer Agents Drug Discovery Using the Freiburg Xenograft System 194
7.3.6 Selected Examples of Anticancer Agents in Clinical Trials Which Have Been Discovered in a Target-Oriented Approach 197
7.3.7 Possible Future Impact of Patient-Derived Tumor Xenografts and Explants 198
7.4 Summary 198
References 199
Chapter 8: The Pediatric Preclinical Testing Program 203
8.1 Introduction 203
8.2 Selection of Preclinical Models 204
8.3 Molecular Characterization of Tumor Models 207
8.3.1 Model Fidelity 209
8.3.2 Primary Screening 210
8.3.3 Response Criteria: Solid Tumors 211
8.3.4 Response Criteria: Acute Lymphoblastic Leukemia Xenograft Models 212
8.3.4.1 Event-Free Survival 212
8.3.4.2 Tumor Growth Delay Value 213
8.3.4.3 Tumor Volume T/C Value 213
8.3.4.4 EFS T/C Value 213
8.4 Data Presentation 214
8.5 Secondary Screening 216
8.6 Combination Drug Testing 217
8.7 Secondary Models 218
8.8 Integrating Molecular Data with Drug Sensitivity 218
8.8.1 Submitting Agents to the PPTP 218
8.9 Closing Remarks 219
References 220
Chapter 9: Imaging Efficacy in Tumor Models 222
9.1 Introduction 222
9.2 Challenges in Preclinical Imaging 223
9.3 Imaging Modalities: Technical Overview and Use in Oncology Models 224
9.3.1 Magnetic Resonance Imaging 224
9.3.2 Computed Tomography 225
9.3.3 Positron Emission Tomography 226
9.3.4 Single Photon Emission Computed Tomography 227
9.3.5 In Vivo Optical Imaging 227
9.3.6 Ultrasound 228
9.4 Imaging End Points in Oncology Models 229
9.4.1 Anatomical Imaging 229
9.4.1.1 Tumor Detection and Staging 229
9.4.1.2 Tumor Burden 230
9.4.2 Functional Imaging 231
9.4.2.1 Imaging Tumor Metabolism and Metabolite Levels 231
9.4.2.2 Cell Proliferation 234
9.4.2.3 Vascular Imaging, Permeability, and Blood Flow 234
9.4.2.4 Tumor Cellularity and Cell Kill 236
9.4.2.5 Receptor Occupancy and Gene Expression 236
9.4.2.6 Tissue Hypoxia and pH 237
9.4.2.7 Apoptosis 237
9.4.2.8 Imaging of Oncogenic Pathways 238
9.4.2.9 Other Imaging Applications in Oncology 238
9.5 Imaging Efficacy in Oncology Models: Future Outlook 239
References 239
Part IV Carcinogen-Induced Tumors 249
Chapter 10: Mammary Cancer in Rats 250
10.1 Introduction 250
10.2 Historical Perspective 251
10.2.1 Induction of Mammary Carcinomas Using MNU 252
10.2.2 Carcinogen Dose and Age of Administration 253
10.2.3 Typical Animal Protocols 254
10.2.3.1 Chemoprevention Protocol 254
10.2.3.2 Therapeutic Protocol 256
10.2.4 Biological Characteristic of Mammary Carcinomas Induced by MNU 256
10.2.5 Value of this Model Relative to Genetically Engineered Models for Mammary Cancer 258
References 258
Part V Disease and Target-Specific Models 261
Chapter 11: Animal Models of Melanoma 262
11.1 Introduction 262
11.2 Xiphophorus Species 263
11.3 South American Opossum 264
11.4 Canine Melanoma 266
11.5 Sinclair Swine 267
11.6 Murine Models 269
11.6.1 Induction with Physical Agents 269
11.6.2 Tumors Arising in Transgenic Mice 271
11.6.3 Spontaneously Arising Murine Melanomas 274
11.6.3.1 Models Employing the B16 Cell Line 274
11.6.4 Tumor Models that Employ Immunodeficient Mice 277
11.6.4.1 Nude Mouse Models 279
11.6.4.2 SCID Mouse Models 280
References 282
Chapter 12: Experimental Animal Models for Investigating Renal Cell Carcinoma Pathogenesis and Preclinical Therapeutic Approac 289
12.1 Introduction 289
12.2 Murine Syngeneic Renal Adenocarcinoma: The Renca Model 290
12.3 Rat Renal Carcinoma Models 296
12.3.1 The Wistar–Lewis Rat Renal Adenocarcinoma 296
12.3.2 The Eker Rat Model 297
12.4 Xenografts of Human RCC Tumor Cell Linesin Immunodeficient Mice 298
References 302
Chapter 13: Animal Models of Mesothelioma 308
13.1 Introduction 308
13.2 Asbestos-Induced Mesothelioma in Animal Models 309
13.2.1 Types of Asbestos Fibers 309
13.2.2 Asbestos-Induced Animal Models of Mesothelioma: General Comments 310
13.2.3 Intraperitoneal Asbestos Injection 311
13.2.4 Intraperitoneal Asbestos Injection: Rats 311
13.2.5 Intraperitoneal Asbestos Injection: Mice 312
13.2.6 Intrapleural Asbestos Injection 313
13.2.7 Intrapleural Asbestos Injection: Rat 313
13.2.8 Inhalation of Asbestos in Animal Models 314
13.2.8.1 Inhalation Studies in Rats 314
13.2.8.2 Inhalation Studies: Guinea Pig 314
13.2.8.3 Intratracheal Asbestos Administration 314
13.2.8.4 Intratracheal Asbestos Administration: Syrian Golden Hamster 315
13.3 Spontaneous Models of Mesothelioma 315
13.4 Other Agents for Animal Production of Mesothelioma 315
13.4.1 Chemical 315
13.4.2 Man-Made Fibers 316
13.5 Novel Viral-Induced and Transgenic Knockout Models of Mesothelioma 317
13.5.1 SV40 Viral Hamster Models 317
13.5.2 SV40 Transgenic Mouse Models 319
13.5.3 Transgenic Murine Models, Utilizing Nf2, Ink4a/ARF, and P53 Knockout Mice 319
13.6 Orthotopic Transplants and Xenograft 320
13.7 Conclusions 322
References 322
Chapter 14: The Use of Mouse Models to Study Leukemia/Lymphoma and Assess Therapeutic Approaches 326
14.1 Introduction 326
14.1.1 Models of Myeloid Leukemia 328
14.1.2 Models of Acute Lymphocytic Leukemia 331
14.1.3 Chronic Lymphocytic Leukemia 333
14.1.4 HTLV-Related T Cell Leukemia/Lymphoma 335
14.2 Models of Hodgkin’s Lymphoma 335
14.3 Models of Non-Hodgkin’s Lymphoma 337
14.4 Models of Multiple Myeloma 340
14.5 Conclusions 342
References 343
Chapter 15: Spontaneous Companion Animal (Pet) Cancers 353
15.1 Introduction 353
15.2 Overview of Cancer in Companion Animal Species 354
15.2.1 Cancer Incidence and Availability of Veterinary Medical Care 354
15.2.2 Genetic and Molecular Basis of Cancer in Pet Dogs 355
15.3 Potential Opportunities/Advantages of Including Companion Animals with Cancer as Models 358
15.4 Caveats to Inclusion of Pet Dogs with Cancer as Models 362
15.5 The Vested Communities 364
15.6 Study Design Issues Specific to Companion Species Trials 366
15.7 Conclusions 368
References 369
Part VI Genetically Engineered Mouse Models of Cancer 374
Chapter 16: Genetically Engineered Mouse Models of Pancreatic Ductal Adenocarcinoma 375
16.1 Pancreas Anatomy, Physiology, and Development 376
16.2 Histological and Molecular Characteristics of Human PDAC 377
16.3 Modeling PDAC 378
16.4 Transgenic Expression of Oncogenes in the Pancreas 379
16.5 Viral Delivery of Oncogenes 380
16.6 Compound Inducible Mutants 380
16.6.1 Cooperation Between Kras and Tumor Suppressors in PDAC 382
16.6.2 Preclinical Studies 384
16.7 Ongoing and Future Modeling Efforts 386
16.8 Conclusions 388
References 388
Chapter 17: Transgenic Adenocarcinoma of the Mouse Prostate: A Validated Model for the Identification and Characterization of M 394
17.1 Introduction 394
17.2 Generation of the TRAMP Model and Phenotypic Characterization 396
17.3 Gene Expression Profiling Studies 398
17.4 Epigenetic Regulation of Gene Expression 400
17.5 Validating and Elucidating Gene Function Through Additional Genetic Engineering 403
17.6 Testing of Known and Putative Therapeutic Agents 404
17.7 Summary, Conclusions, and Future Directions 411
References 412
Chapter 18: The Utility of Transgenic Mouse Models for Cancer Prevention Research 419
18.1 Introduction 419
18.2 Cancer Prevention in Transgenic Mice: Lessons Learned from Commonly Used Models 420
18.2.1 Mutant Mouse Models for Prostate Cancer Prevention 420
18.2.1.1 PTEN Mutant Mouse Models 420
18.2.1.2 c-Myc Transgenic Mice 421
18.2.1.3 Viral Oncogene Models 421
18.2.2 Mutant Mouse Models for Mammary Cancer Prevention 423
18.2.2.1 TGFa Models 423
18.2.2.2 ErbB-2/HER2/neu Models 423
18.2.2.3 SV40 T-antigen Transgenic Models 424
18.2.2.4 p53-mutant Mouse Models 424
18.2.2.5 MMTV-Wnt-1 Transgenic Mouse Model 425
18.2.2.6 Ras Mutant Models 426
18.2.2.7 c-myc Transgenic Mice 426
18.2.2.8 Cyclin D1 Transgenic Mice 426
18.2.2.9 Inducible Models 427
18.2.2.10 Examples of Dietary or Chemopreventive Studies Using Transgenic Mouse Models of Mammary Cancer 427
Selective Estrogen Receptor Modulators 427
Aromatase Inhibitors 428
Retinoids 428
Tyrosine Kinase Inhibitors 428
Nonsteroidal Anti-Inflammatory Drugs/Cyclooxygenase-2 Inhibitors 429
Energy Balance Interventions 429
18.2.3 Apc-Deficient Models for Intestinal Cancer Prevention Studies 429
18.2.4 Emerging Models of Pancreatic Cancer 430
18.3 Summary and Conclusions 432
References 433
Part VII Metastasis Models 440
Chapter 19: Models for Evaluation of Targeted Therapies of Invasive and Metastatic Disease 441
19.1 Introduction 441
19.2 Therapeutic Strategies for Targeting Metastases 443
19.2.1 Target Identification and Validation 443
19.2.2 Molecular Targets 444
19.2.2.1 Oncogenic Receptor Tyrosine Kinase Signaling Pathways 444
19.2.2.2 HSP90 Chaperone 456
19.2.2.3 Chemokine Receptors 456
19.2.2.4 BCr-Abl: A Paradigm for Tumor-Specific Therapy 457
19.2.2.5 Hedgehog (Hh) 457
19.2.2.6 Wnt Pathway 458
19.2.2.7 Combination Therapies 458
19.2.3 Processes Linked to Metastasis 458
19.2.3.1 Angiogenesis and Hypoxia 458
19.2.3.2 Proteolysis in Invasion and Angiogenesis 460
19.2.3.3 Intravasation and Extravasation 460
19.2.3.4 The Premetastatic Niche 461
19.2.4 Resistance to Therapy 461
19.2.4.1 Cancer Stem-Like Cells 461
19.2.4.2 Dormant Metastases 462
19.2.5 Immunological Approaches 463
19.2.5.1 Antibody-Based Therapies 463
19.2.5.2 Vaccines, Cytokines, and Cell-Mediated Immunotherapy 463
19.2.5.3 Targeting Using Vectors or Peptides with Tumor Selectivity 464
19.3 Detection and Quantitation of Metastases and Determination of Therapeutic Benefit 464
19.4 Animal Models for Evaluating Targeted Therapy of Metastasis 466
19.4.1 Syngeneic Rodent Tumor Models 466
19.4.2 Human Tumor Xenograft Models 466
19.4.3 Organ Colonization and Site-Selective Metastases 468
19.4.3.1 Lung Metastases 468
19.4.3.2 Liver Metastasis 469
19.4.3.3 Brain Metastasis 469
19.4.3.4 Bone Metastasis 469
19.4.4 Spontaneous Metastasis Models 470
19.4.4.1 Orthotopic Implantation Models 471
19.4.4.2 Lymph Node Metastases 471
19.4.5 Transgenic Models 472
19.5 Summary and Conclusions 474
References 475
Part VIII Normal Tissue Response Models 490
Chapter 20: Animal Models of Toxicities Caused by Anti-Neoplastic Therapy 491
20.1 Introduction 491
20.2 Models of Oral Mucositis Induced by Anti-Neoplastic Drugs and Radiation 492
20.2.1 Overview of the Condition 492
20.2.2 The Biology of Mucositis 493
20.2.3 Objectives of Animal Models of Mucositis 494
20.2.4 Current Models 494
20.2.4.1 Screening Models for the Enablement of Pharmaceuticalsand Biologicals 496
Background 496
History of Radiation Model Development in Hamsters 496
20.2.4.2 The Hamster Model for Acute Radiation-Induced Mucositis 496
20.2.4.3 Non-Clinical Endpoints 1
20.2.4.4 The Hamster Model for Fractionated Radiation-Induced Mucositis 500
20.2.4.5 Hamster Models for Chemotherapy-Induced Mucositis Withor Without Concomitant Radiation 500
20.2.4.6 Chemotherapy-Induced Oral Mucositis 500
20.2.4.7 Concomitant Chemotherapy and Radiation 501
20.3 Chemotherapy-Induced Mucositis of the GI Tract 501
20.3.1 Models of Intestinal Mucositis 501
20.3.1.1 Use of Endoscopy to Assess Chemotherapy-Induced Mucosal Injury 502
20.4 Radiation-Induced Proctitis 503
20.4.1 Overview of the Condition 503
20.4.2 The Biology of Radiation-Induced Proctitis 503
20.4.3 Animal Model of Radiation-Induced Proctitis 504
20.4.3.1 Radiation-Induced Dermatitis 504
Overview of the Condition 504
20.4.4 The Biology of Radiation-Induced Dermatitis 505
20.4.5 Animal Model of Radiation-Induced Dermatitis 505
20.4.6 Bisphosphonate-Related Osteonecrosis of the Jaws 507
20.4.6.1 Introduction 507
20.4.6.2 Rat Model for Bisphosphonate-Associated Osteonecrosis of the Jaws 508
References 509
Chapter 21: Bone Marrow as a Critical Normal Tissuethat Limits Drug Dose/Exposure in Preclinical Models and the Clinic1 512
21.1 Introduction 513
21.2 Blood Cytopenia as a Quantifiable Dose-Limiting Toxicity in the Oncology Clinic 514
21.3 Cancer Therapeutics as Toxicants to Highly Proliferative Hematopoietic Cells 516
21.4 Bone Marrow as a Critical Normal Tissue that Sets Maximum Human Dose/Exposure and Therefore Should Restrict Dose/Exposur 521
21.5 Using Hematotoxicology to Limit Treatment of Mouse Models to Tolerated Human Doses/Exposures 523
21.5.1 Method 1: Treating Mouse Models at Maximum Tolerated Human Dose, Predicted Using CFU-GM Assays 524
21.5.2 Method 2: Treating Mouse Models at Maximum Tolerated Human Dose/Exposure Determined Empirically Using NOD/SCID Mice En 528
21.6 Concluding Thoughts on Improving the Predictive Accuracy of Mouse Efficacy Models Using Human Hematotoxicology Data 536
References 538
Chapter 22: Anesthetic Considerations for the Study of Murine Tumor Models 544
22.1 Overview 544
22.2 Rationale and Requirements for Animal Anesthesia in Cancer Research 544
22.2.1 Humane Reasons 545
22.2.2 To Control Motion 545
22.3 Special Requirements for Anesthesia in Cancer Research 545
22.3.1 Non-Survival Surgery 545
22.3.2 Survival Surgery 545
22.3.3 Functional Studies 546
22.3.4 Controlled Delivery of Anticancer Treatment 546
22.3.5 Assessment of Anesthetic Depth in Rodents 547
22.4 Animal Support 547
22.4.1 Body Temperature 547
22.4.2 Respiration 548
22.4.3 Hydration 549
22.4.4 Analgesia 549
22.4.5 Inhalable Anesthetics 549
22.5 Isoflurane 550
22.5.1 Background About the Drug 550
22.5.2 Anesthetic Properties 551
22.5.3 Typical Applications 551
22.5.4 Required Equipment 552
22.5.5 Protocol for Isoflurane in Mice and Rats 552
22.5.6 Induction of Anesthesia in Rats with Isoflurane, Followed by Injectable Anesthesia 552
22.6 Injectible Anesthetics 553
22.6.1 Ketamine HCl with Xylazine 553
22.6.1.1 Background and Biochemistry 553
22.6.1.2 Anesthetic Properties in Rodents 553
22.6.1.3 Impact of Ketamine on Rodent Physiology 554
22.6.1.4 Anesthesia Protocol of Mice Using Ketamine/Xylazine Anesthesia 554
Preparation 554
Protocol 554
22.7 Pentobarbital 555
22.7.1 Background and Biochemistry 555
22.7.2 Anesthetic Properties in Rodents 555
22.7.3 Physiological Impact of Pentobarbital 556
22.7.4 Applications 556
22.7.5 Protocol for Pentobarbital Anesthesia in Mice 556
22.7.5.1 Preparation 556
22.7.5.2 Protocol 557
22.7.6 Other Injectibles 557
22.8 Summary 557
References 558
Part IX Experimental Methods and Endpoints 560
23.1 Introduction 1
23.2 Ascites Tumors 562
23.3 Solid Tumors 564
23.4 Combination Treatments 568
23.5 Therapeutic Index 576
23.6 In Vivo/Ex Vivo Assay of Primary and Metastatic Disease 577
23.7 In Vivo Resistant Tumors 579
23.8 Drug Penetration into Tumor 580
23.9 Conclusion 587
References 588
Chapter 24: Tumor Cell Survival 596
24.1 Introduction 597
24.2 Selection of Tumor–Host Systems 598
24.3 Cell Survival Assays 600
24.3.1 Implications of Clonogenic Cell Survival 600
24.3.2 Measuring Clonogenicity 600
24.3.2.1 Identifying Clonogenic Cells 601
24.3.2.2 Motion Artifacts 603
24.3.2.3 Cell Density Problems 603
24.3.2.4 Colony Density Problems 604
24.3.2.5 Counting the Colonies 604
24.3.3 Tumor Cell Suspensions 605
24.3.3.1 Preparing the Suspension 605
24.3.3.2 Counting the Cells 606
24.3.3.3 The Importance of Single-Cell Suspensions 606
24.3.4 Scheduling Problems in Clonogenic Assays 607
24.4 Analysis of Cell Survival Data 609
24.5 Conclusions 611
References 611
Chapter 25: Apoptosis In Vivo 614
25.1 Introduction 614
25.2 Recognition and Quantification of Apoptosis 615
25.2.1 Morphological Assessment In Vivo 615
25.2.2 Quantification of Apoptosis In Vitro 616
25.3 Apoptosis in Tumor Biology 617
25.3.1 The Role of Apoptosis in Tumor Development 617
25.3.2 Genetic Regulation of Apoptosis 618
25.4 Apoptosis in Cancer Therapy 620
25.4.1 Response of Normal Tissue to Cytotoxic Therapy 620
25.4.2 Apoptosis in Tumors Responding to Cytotoxic Therapy 621
25.4.3 In Vivo Imaging of Apoptosis 624
25.5 Summary and Conclusions 625
References 625
Chapter 26: Transparent Window Models and Intravital Microscopy: Imaging Gene Expression, Physiological Function and Therapeuti 630
26.1 Introduction 630
26.2 Chronic Window Preparations 633
26.2.1 Procedures 635
26.2.1.1 Rabbit Ear Chamber 635
26.2.1.2 Dorsal Skin Chamber Preparation 636
26.2.1.3 Mammary Fat Pad Chamber Preparation 637
26.2.1.4 Cranial Window Preparation 637
26.2.1.5 Angiogenesis Gel Assay and Tissue Engineered Vessel Model 638
26.3 Acute (Exteriorized) Preparations 641
26.3.1 Procedures 641
26.3.1.1 Mesentery 641
26.3.1.2 Liver Tumor Preparation 641
26.3.1.3 Pancreatic Tumor Preparation 642
26.3.1.4 Mammary Fat Pad Tumor Preparation 643
26.4 In Situ Preparations 643
26.4.1 Procedures 644
26.4.1.1 Corneal Pocket Assay 644
26.4.1.2 Chick Chorioallantoic Membrane 644
26.4.1.3 Tail Lymphatics 645
26.4.1.4 Ear Model 645
26.5 Intravital Microscopy and Image Analysis 647
26.5.1 Intravital Microscopy Work Station 647
26.5.1.1 Conventional Single-Photon Microscopy 647
26.5.1.2 Multiphoton Laser-Scanning Microscopy 647
26.5.1.3 Optical Frequency Domain Imaging 647
26.5.2 Tumor Growth and Regression 649
26.5.3 Vascular Parameters 649
26.5.3.1 Angiogenesis and Hemodynamics 649
Single-Photon Microscopy Procedure 650
Multiphoton Laser-Scanning Microscopy Procedure 650
26.5.3.2 Vascular Permeability 651
Single-Photon Microscopy Procedure 651
Multiphoton Laser-Scanning Microscopy Procedure 651
26.5.3.3 Leukocyte Endothelial Interactions 652
Single-Photon Microscopy Procedure [92] 652
Multiphoton Laser-Scanning Microscopy Procedure [80] 652
26.5.4 Extravascular Parameters 653
26.5.4.1 Interstitial pH Measurements 653
26.5.4.2 Interstitial and Microvascular pO2 Measurements 654
26.5.4.3 Tissue Nitric Oxide Distribution 654
26.5.4.4 Interstitial Diffusion, Convection, and Bindings 655
Single-Photon Fluorescence Recovery After Photobleaching Procedures 655
Multiphoton Fluorescence Recovery After Photobleaching Procedures [101] 656
Multiphoton Fluorescence Correlation Spectroscopy Procedures [102] 656
26.5.4.5 Gene Expression: Promoter Activity via GFP Imaging 657
26.6 Novel Insights 658
26.7 Future Perspectives 660
References 660
Index 669