Impact of aldehyde dehydrogenase isotypes on xenograft and syngeneic mouse models of human primary glioblastoma multiforme von Friederike Constanze Lämmer Inaugural-Dissertation zur Erlangung der Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Impact of aldehyde dehydrogenase isotypes on xenograft and syngeneic mouse models of human primary glioblastoma multiforme von Friederike Constanze Lämmer aus Leipzig München 2016 Aus dem Zentrum für Klinische Tiermedizin der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Institut für Tierpathologie Arbeit angefertigt unter der Leitung von: Univ.-Prof. Dr. Kaspar Matiasek Angefertigt am Institut für Allgemeine Pathologie und Pathologische Anatomie der Technischen Universität München Mentor: Univ.-Prof. Dr. Jürgen Schlegel Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians Universität München Dekan: Univ.-Prof. Dr. Joachim Braun Berichterstatter: Univ.-Prof. Dr. Kaspar Matiasek Korreferenten: Priv.-Doz. Dr. Ivica Medugorac Univ.-Prof. Dr. Johannes Hirschberger Tag der Promotion: 6. Februar 2016 Contents Abbreviations vii 1 Introduction 1 1.1 Human Glioblastoma multiforme .................... 1 1.2 Models in GBM reasearch........................ 4 1.2.1 Mouse models in GBM research................. 6 1.2.2 Canine Glioblastoma multiforme ................ 6 1.3Theoryofcancergrowth......................... 7 1.4 Aldehyde dehydrogenase superfamily .................. 10 1.4.1 ALDH1A1 ............................. 10 1.4.2 ALDH1A3 ............................. 11 2 Objective 12 3 Materials and Methods 13 3.1 Material .................................. 13 3.2Methods.................................. 16 3.2.1 Collection of GBM patients data ................. 16 3.2.2 Canine GBM samples . ...................... 17 3.2.3 Immunohistochemistry analysis ................. 17 3.2.4 Analysis of ALDH isoforms on the mRNA level......... 19 3.2.5 Mouse experiments ........................ 23 3.2.6 Culture of cell line LN18, U87-MG, Gl261, C6,andF98 ..... 29 3.2.7 Cell pellet ............................. 29 3.3 Summary of samples ........................... 29 4 Results 30 4.1 Human PGBM cohort analysis ...................... 30 4.1.1 Immunohistochemistry . ................... 30 4.1.2 Double immunoflorescence for ALDH1A1 and ALDH1A3 with Ki67 41 4.1.3 MGMT methylation status and expression of different ALDH1 isoforms .............................. 41 4.1.4 ALDH expression by RT-PCR ................... 44 4.2 Comparative analysis of ALDH in a xenograft and a syngeneic model for GBM .................................. 47 4.2.1 U87-MG xenograft model ..................... 48 4.2.2 Gl261 syngeneicmodel...................... 48 4.3 Evaluation of ALDH isoforms in rat GBM cells............. 50 v Contents 4.4 Immunohistochemistry analysis of ALDH expression in canine GBM .53 5 Discussion 55 5.1 Analysis of the distribution of ALDH in human PGBM ......... 55 5.1.1 Primary PGBM .......................... 55 5.1.2 Secondary PGBM ......................... 59 5.1.3 Conclusion of human PGBM analysis .............. 59 5.2 Evaluation of the syngeneic and the xenograft PGBM mouse model . 59 5.2.1 ALDH expression in mouse models ............... 60 5.3 Evaluation of rat GBM cells....................... 61 5.4 Evaluation of the canine GBM model.................. 61 5.5 Conclusion of animal model analysis .................. 62 6 Outlook 64 7 Summary 65 8 Zusammenfassung 66 9 Supplemental Data 68 10 Danksagung 92 vi Abbreviations AB antibody ALDH aldehyde dehydrogenase cDNA complementary deoxyribonucleic acid CSC cancer stem cell DEAB N,N -diethylaminobenzaldehyde DNA deoxyribonucleic acid dNTPS deoxynucleotide DSF disulfiram EDTA ethylenediaminetetraacetic acid disodium salt solution FFPE formalin fixed paraffin embedded GAPDH glyceraldehyde 3-phosphate dehydrogenase GBM glioblastoma multiforme GFAP glial fibrillary acidic protein H&E hematoxylin and eosin stain IDH isocitrate dehydrogenase IFC immunoflorescence IgG immunoglobulin G IHC immunohistochemistry MES mesenchymal 6 MGMT O -methylguanine DNA methyltransferase MOS medium overall survival MRI magnetic resonance imaging mRNA messenger ribonucleic acid NAD nicotinamide adenine dinucleotide PBS phosphate buffered saline PCR polymerase chain reaction PGBM primary glioblastoma multiforme PML promyelocytic leukemia gene PN proneural RA retionoic acid RAS retionoic acid signaling RFS relapse-free survival RNA ribonucleic acid ROS reactive oxygen species RT-PCR reverse transcriptase PCR SDS sodium dodecyl sulfate SGBM secondary glioblastoma multiforme vii Contents TMZ temozolomide TUM Technische Universität München UK United Kingdom USA United States of America WHO World Health Organisation viii 1 Introduction 1.1 Human Glioblastoma multiforme Glioblastoma multiforme (GBM) is the most common adult glioma with a medium overall survival (MOS) of 15 months (450 days) (Hau et al., 2007). The incidence of GBM in the USA is 3.19 in 1000000 (Vigneswaran et al., 2015). The median age at diagnosis is 64 years. Men are 1.6 times more often affected than women (Ver- haak et al., 2010). The reason for this gender-specific preponderance is unclear. 95 % of all GBM develop de novo (primary glioblastoma multiforme, PGBM), while 5 % evolve out of low grade gliomas, for example astrocytomas, and are named sec- ondary glioblastoma multiforme (SGBM) (Figure 1.1). Analysis of GBM genomics revealed multiple tumour suppressor and oncogenes that are inactive and active, respectively, during tumour progression and de novo formation (Figure 1.2). For example, in low grade gliomas and SGBM, mutations of isocitrate dehydrogenase (IDH1 and IDH2) are found most frequently, whereas PGBM never have these muta- tions. Genetic differences between SGBM and PGBM tumours are used in diagnostics (Nobusawa et al., 2009) and are relevant for the selection of the therapy. With im- munohistochemistry (IHC), the expression of IDH1 in SGBM and low grade GBM can be detected easily (Figure 1.3). Despite various efforts and combined therapy (Hau et al., 2007; Stupp et al., 2009), PGBM is still an incurable disease. At the moment, the standard therapy for patients (a) T1 sequence PGBM with (b) T2 sequence PGBM contrast agent Figure 1.1: Magnetic resonance imaging from a PGBM patient, transversal sections. Tumour in the right hemisphere. 1 1 Introduction Anaplastc Diffuse Astrocytoma Astrocytoma WHO II WHO III $QDSODVWLF 6HFRQGDU\ 2OLJRDVWURF\WRPD IDH 2OLJRDVWURF\WRPD *OLREODVWRPD :+2,, mutated :+2,,, :+2,9 $QDSODVWLF 2OLJRGHQGURJOLRPD 2OLJRGHQGURJOLRPD :+2,, 1HXURQDOSURJHQLWRU :+2,,, FHOO &ODVVLFDO 6XEW\SH IDH wild type 3ULPDU\ 0HVHQ *OLREODVWRPD FK\PDO :+2,9 6XEW\SH 3URQHXUDO 6XEW\SH Figure 1.2: Development of PGBM and SGBM. IDH mutation as difference of low grade, SGBM and PGBM. (a) IDH1 positive sample of SGBM patient (b) IDH1 negative sample of PGBM patient Figure 1.3: IHC, 200 x, IDH1. a) secondary glioblastoma multiforme, positive for the R132H point mutation, b) primary glioblastoma multiforme, negative for the R132H point mutation. Used for diagnosis of PGBM and SGBM. 2 1 Introduction below the age of 70 years consists of: surgical resection, external beam irradiation five times a week for six weeks, as well as oral temozolomide (TMZ) daily. Relapse occurs frequently within approximately 7 months (210 days) (Alifieris and Trafalis, 2015) (Figure 1.4). This is due to the fact that a complete microscopic resection can never be achieved because of the diffuse infiltration of PGBM. If re-irradiation and re-resection of recurrent tumours are not eligible, other medications like carboplatin, irinotecan, carmustine, etoposide, or vincristine are used in clinical trials (Mrugala et al., 2012; Pallud et al., 2015). Even investigatory approaches like convection enhanced delivery, stem cell treatment, immunotherapy, and gene therapy might be available in the future to elongate the survival of PGBM patients (Arshad et al., 2015; Stangeland et al., 2015). For the choice of medication, it is essential to have prognostic markers to predict the course of the disease, or to evaluate developed medications. At the moment there is only one stable predictive marker defined for PGBM therapy. The success of PGBM 6 treatment with TMZ is depends on the methylation status of the O -methylguanine DNA methyltransferase (MGMT) promoter (Hegi et al., 2008; Quillien et al., 2012; 6 Stupp et al., 2009). TMZ is an alkylating agent, targeting the O -position of guanine. This leads to DNA double strand breaks and finally results in apoptosis. MGMT, 6 however, removes methyl adducts at the O -guanine and works therefore as a DNA repair enzyme, antagonising TMZ.IftheMGMT promoter is methylated, the enzyme is not produced and the alkylating agents can lead to DNA double strand breaks (Hegi et al., 2004). Patients with a high MGMT promoter methylation benefit more from a TMZ therapy and have a longer MOS than patients with low MGMT promoter methylation. Histopathologically, PGBM are very heterogeneous (Bonavia et al., 2011). Mod- ern pathology combines morphological patterns with molecular markers to manifest (a) prae operation (b) post operation (c) secondary PGBM Figure 1.4: Magnetic resonance imaging from a PGBM patient, sagittal sections. a) before surgical resection, b) after surgical resection, c) 4 months after surgical resection, secondary PGBM visible. 3 1 Introduction diagnosis. However, PGBM grow infiltrative, lead to neovascularisation and necrosis, and show an enhanced mitotic