Murine in Vivo Tumor Models for Investigating the Anticancer Potential of Natural and Synthetic Compounds
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Murine in vivo tumor models for investigating the anticancer potential of natural and synthetic compounds von Carina Atzberger Inaugural-Dissertation zur Erlangung der Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Murine in vivo tumor models for investigating the anticancer potential of natural and synthetic compounds von Carina Atzberger aus Erding München 2018 Aus dem Veterinärwissenschaftlichen Department der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Lehrstuhl für Molekulare Tierzucht und Biotechnologie Arbeit angefertigt unter der Leitung von Univ.-Prof. Dr. Eckhard Wolf Angefertigt an: Fakultät für Chemie und Pharmazie, Lehrstuhl für Pharmazeutische Biologie der Ludwig-Maximilians-Universität München Mentorin: Univ.-Prof. Dr. Angelika M. Vollmar Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Dekan: Univ.-Prof. Dr. Reinhard K. Straubinger, Ph.D. Berichterstatter: Univ.-Prof. Dr. Eckhard Wolf Korreferent/en: Univ.-Prof. Dr. Hermann Ammer Tag der Promotion: 27. Juli 2018 Meiner Familie Table of contents VI TABLE OF CONTENTS I. INTRODUCTION.............................................................................. 1 1. Animal experiments in cancer research ...................................... 2 2. Problems in cancer treatment ....................................................... 6 3. Compounds for in vivo experiments ............................................ 7 3.1. Natural compounds .......................................................................... 7 3.1.1. Neocarzilin A .................................................................................... 8 3.1.2. CA1 .................................................................................................. 8 3.1.3. Jasplakinolide .................................................................................. 8 3.1.4. Latrunculin B .................................................................................... 9 3.2. Synthetic compounds ..................................................................... 10 3.2.1. Ned-19 ........................................................................................... 10 3.2.2. PS89 .............................................................................................. 11 3.2.3. Dinaciclib ........................................................................................ 11 3.2.4. Sorafenib ........................................................................................ 12 3.3. Compounds for combination therapy ............................................. 13 3.3.1. Daunorubicin .................................................................................. 13 3.3.2. Doxorubicin .................................................................................... 14 4. Aim of the thesis .......................................................................... 16 II. MATERIALS, MICE, AND METHODS ........................................... 17 1. Materials ....................................................................................... 17 1.1. Compounds .................................................................................... 17 1.2. Cells ............................................................................................... 17 1.3. Cell culture ..................................................................................... 18 1.4. In vivo experiments ........................................................................ 19 1.4.1. Environmental enrichment ............................................................. 20 1.7 Instruments .................................................................................... 20 1.8 Software ......................................................................................... 21 2. Mice ............................................................................................... 21 2.1. Laboratory mouse strains ............................................................... 21 2.1.1. BALB/c ........................................................................................... 21 2.1.2. C57BL/6 ......................................................................................... 22 Table of contents VII 2.1.3. C57BL/6 albino .............................................................................. 22 2.1.4. Fox Chase SCID ............................................................................ 22 2.1.5. NOD scid gamma (NSG) ................................................................ 22 2.2. Housing .......................................................................................... 23 3. Methods ........................................................................................ 24 3.1. Cell culture ..................................................................................... 24 3.2. Animal experiments ........................................................................ 25 3.2.1. Bioluminescence measurement in living animals - IVIS® spectrum in vivo imaging system ....................................................................... 25 3.2.2. Dose finding tests........................................................................... 26 3.2.3. Pharmacokinetic studies ................................................................ 28 3.2.4. Dissemination assays .................................................................... 28 3.2.5. Ectopic tumor models for the evaluation of tumor growth .............. 30 3.2.6. Patient derived xenograft cell model for monitoring leukemia ........ 31 3.2.7. Orthotopic tumor model for evaluation of metastasis ..................... 33 3.2.8. Statistical analysis .......................................................................... 34 III. RESULTS ...................................................................................... 35 1. Evaluation of murine dose finding tests .................................... 35 1.1. Neocarzilin A .................................................................................. 35 1.2. CA1 ................................................................................................ 36 1.3. Dinaciclib ........................................................................................ 36 1.4. Sorafenib ........................................................................................ 38 1.5. Doxorubicin .................................................................................... 39 1.6. Latrunculin B .................................................................................. 39 1.7. Jasplakinolide ................................................................................ 40 2. Examination of pharmacokinetic studies .................................. 42 2.1. Evaluation of PS89 in pharmacokinetic studies ............................. 42 3. In vivo tumor models to evaluate tumor dissemination ........... 43 3.1. Examination of dissemination of 4T1-luc cells ............................... 43 3.2. Effects of neocarzilin A on dissemination of 4T1-luc cells .............. 46 3.3. Examination of Ril175-luc cell dissemination ................................. 48 3.4. Effects of Cdk5 inhibition on Ril175-luc cell dissemination ............. 49 3.5. Effects of TPC2 on tumor cell dissemination .................................. 52 Table of contents VIII 4. Examination of murine ectopic tumor models .......................... 56 4.1. Preliminary growth experiment with 4T1-luc cells .......................... 56 4.2. Preliminary growth experiment with McA-RH7777 cells ................. 57 4.3. Evaluation of HUH7 cells in a murine ectopic tumor model ............ 58 5. Evaluation of a PDX cell leukemia model for AML-372 ............. 64 6. Tumor metastasis evaluated in an orthotopic liver cancer model ............................................................................................ 67 IV. DISCUSSION ................................................................................. 71 1. Neocarzilin A as a potential natural compound to impede tumor cell dissemination ........................................................................ 71 2. Two-pore channel inhibition as a novel strategy to reduce tumor cell dissemination ............................................................. 72 3. Cdk5 inhibition as a new approach against liver cancer .......... 73 4. Sorafenib resistance in liver cancer cells evaluated in murine tumor models ............................................................................... 74 5. Patient-derived xenograft cells in a mouse model for acute myeloid leukemia ......................................................................... 75 V. SUMMARY .................................................................................... 77 VI. ZUSAMMENFASSUNG ................................................................. 79 VII. REFERENCES .............................................................................. 83 VIII. HOMEPAGES ................................................................................ 97 IX. APPENDIX ..................................................................................... 99 X. ACKNOWLEDGEMENTS ............................................................ 101 Abbreviations X ABBREVIATIONS °C degree Celsius MED minimum effective dose µl microliter mg milligram AML acute myeloid leukemia min minute(s) BCS body condition MTD maximum score tolerated dose