UNIVERSITÀ DEGLI STUDI DI MILANO SCUOLA DI DOTTORATO IN MEDICINA MOLECOLARE CICLO XXVI Anno Accademico 2012/2013 TESI DI DOTTORATO DI RICERCA MED/36 Multimodal molecular imaging for HIF-1α non-invasive assessment in a murine glioma model Dottorando : Alessia LO DICO Matricola N° R09155 TUTORE: Dott.ssa Luisa OTTOBRINI DIRETTORE DEL DOTTORATO: Chiar.mo Prof. Mario CLERICI INDEX INDEX ......................................................................................................................................... I LIST OF ABBREVIATIONS ........................................................................................................ V LIST OF FIGURES .................................................................................................................. VIII SOMMARIO ............................................................................................................................. XII ABSTRACT ............................................................................................................................ XIV INTRODUCTION ........................................................................................................................ 1 1. GLIOMA ................................................................................................................................. 2 1.1 GLIOBLASTOMA ............................................................................................................... 4 1.1.1 CELLULAR COMPOSITION OF GBM ...................................................................... 6 1.1.2 GENETIC OF GBM ................................................................................................... 7 2. HYPOXIA ............................................................................................................................... 9 2.1 HIF-1α .............................................................................................................................. 11 3. HYPOXIA IN GLIOMA DISEASE .......................................................................................... 13 3.1 GLIOMA MODELS ........................................................................................................... 14 4. EFFECT OF DRUG ACTIVITY ON HIF-1α MODULATION ................................................... 16 4.1 HYPOXIA MIMETIC: DFX ................................................................................................ 16 4.2 PI3K PATHWAY INHIBITORS: LY294002 AND BEZ-235 ................................................ 17 4.3 MEK PATHWAY MODULATORS: TRAMETINIB AND PD98059 ..................................... 18 4.4 HIF-1Α MODULATORS: FM19G11 AND DMOG .............................................................. 19 4.5 TEMOZOLOMIDE ............................................................................................................ 19 5. IN VIVO IMAGING ................................................................................................................ 21 5.1 OPTICAL IMAGING ......................................................................................................... 25 5.1.1 BIOLUMINESCENCE ............................................................................................. 27 5.1.2 FLUORESCENCE .................................................................................................. 29 I 5.2 NUCLEAR IMAGING ........................................................................................................ 31 5.3 MAGNETIC RESONANCE IMAGING .............................................................................. 35 6. HYPOXIA AND HIF-1α ACTIVITY IMAGING ........................................................................ 36 AIM OF THE STUDY ................................................................................................................ 42 MATERIALS AND METHODS .................................................................................................. 44 1. IN VITRO EXPERIMENTS .................................................................................................. 45 1.1 CELLS LINES AND REAGENTS...................................................................................... 45 1.2 U251-HRE INFECTION .................................................................................................... 45 1.3 DRUG PREPARATION FOR IN VITRO STUDIES ........................................................... 45 1.4 DFX MODULATION OF LUCIFERASE ACTIVITY ........................................................... 46 1.5 NUCLEAR HIF-1Α DETECTION BY IMMUNOCYTOCHEMISTRY ASSAY (ICC) ............ 46 1.6 NUCLEAR HIF-1Α DETECTION BY TRANSAM KIT ........................................................ 46 1.7 EFFECTS OF LY294002 AND FM19G11 ON LUCIFERASE ACTIVITY IN LIVING AND LYSATED CELLS .................................................................................................................. 47 1.8 EFFECTS OF BEZ235 AND TRAMETINIB TREATMENT ON LUCIFERASE ACTIVITY . 47 1.9 IN VITRO TMZ TREATMENT ........................................................................................... 47 1.10 MTT ASSAY ................................................................................................................... 48 2. IN VIVO EXPERIMENTS ...................................................................................................... 48 2.1 ANIMAL STUDIES ........................................................................................................... 48 2.2 IN VIVO IMAGING STUDY DESIGN ................................................................................ 49 2.3 BLI AND FLI STUDIES ..................................................................................................... 49 2.4 MRI ANALYSIS ................................................................................................................ 50 2.5 PET ANALYSIS ................................................................................................................ 50 2.6 IMMUNOHISTOCHEMISTRY (IHC) ................................................................................. 51 2.7 IN VIVO IMAGING AFTER TEMOZOLOMIDE TREATMENT ........................................... 51 2.8 STATISTICAL ANALYSIS ................................................................................................ 52 II RESULTS AND DISCUSSION ................................................................................................. 53 1. IN VITRO CHARACTERIZATION OF LUCIFERASE ACTIVITY IN RELATION TO .............. 54 HIF-1α MODULATION.............................................................................................................. 54 1.1 DFX MODULATION OF LUCIFERASE ACTIVITY ........................................................... 54 1.2 NUCLEAR DETECTION OF HIF-1α ................................................................................. 56 1.3 EFFECTS OF LY294002 AND FM19G11 ON LUCIFERASE ACTIVITY .......................... 58 1.4 IN VITRO EVALUATION OF HIF-1α REGULATION BY DIFFERENT DRUGS ................ 61 1.5 IN VITRO TMZ TREATMENT ........................................................................................... 66 2. IN VIVO STUDY OF LUCIFERASE ACTIVITY IN RELATION TO INTRATUMORAL HYPOXIA ............................................................................................................................... 69 2.1 BLI ANALYSIS ................................................................................................................ 69 2.2 FLI VALIDATION OF BLI RESULTS ................................................................................ 72 2.3. IN VIVO STUDY OF TUMOR FEATURES BY MRI AND NUCLEAR-BASED IMAGING (PET) ..................................................................................................................................... 73 3. EX VIVO VALIDATION ......................................................................................................... 77 3.1. MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL ANALYSES ................................ 77 4. IN VIVO TEMOZOLOMIDE TREATMENT ............................................................................ 78 CONCLUSIONS ....................................................................................................................... 85 FUTURE PERSPECTIVE ......................................................................................................... 88 BIBLIOGRAPHY ....................................................................................................................... 90 SCIENTIFIC PRODUCTS ...................................................................................................... 105 GRANTS ................................................................................................................................ 106 III IV LIST OF ABBREVIATIONS [64 Cu]ATSM: Copper diacetyl-bis (N4-methylthiosemicarbazone) ATP: Adenosine TriPhosphate BBB: Blood Brain Barrier BL: Bioluminescence BMDC: Bone Marrow Derived Cells CAIX: Carbonic Anhydrase IX cCCD: Cooled Charge-Coupled Device Cdks: Cyclin-Dependent Kinases CK2: Casein Kinase 2 CNS: Central Nervous System CoCl2: Cobalt Chloride CT: Computerized Tomography D2R : Dopaminergic type 2 Receptor DCE-MRI: Dynamic Contrast-Enhanced Magnetic Resonance Imaging DFX: Deferoxamine DMOG: DiMethylOxalylGlycine DNA-PK: DNA-dependent Protein Kinase EGFR: Epidermal Growth Factor Receptor [18 F]FAZA: 18F-FluoroAzomycin Arabinoside