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Molecular and Functional Characterization of P2Y12 Expression in Tumor-Associated Macrophages

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Molecular and Functional Characterization of P2Y12 Expression in Tumor-Associated Macrophages Aus der Klinik für Dermatologie, Venerologie und Allergologie Universitätsklinikum Mannheim der Medizinischen Fakultät Mannheim der Ruprecht-Karls-Universität Heidelberg (Direktor: Prof. Dr. med. S. Goerdt) Molecular and Functional Characterization of P2Y12 Expression in Tumor-associated Macrophages INAUGURALDISSERTATION zur Erlangung der Doktorwürde der Naturwissenschaftlich-Mathematischen Gesamtfakultät der Ruprecht-Karls-Universität Heidelberg Vorgelegt von Loreen Kloss DISSERTATION submitted to the Combined Faculty of Natural Sciences and Mathematics of the Ruperto Carola University Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by Loreen Kloss, M.Sc. Born in: Herrenberg, Germany Oral examination: June 17th, 2019 Molecular and Functional Characterization of P2Y12 Expression in Tumor-associated Macrophages Referees: Prof. Dr. rer. nat. Viktor Umansky PD Dr. med. Astrid Schmieder Table of Contents Table of Contents Abstract ...................................................................................................................................... V Zusammenfassung ................................................................................................................... VII 1. Introduction ....................................................................................................................... 1 1.1 Macrophages ............................................................................................................... 1 1.1.1 Origin and maintenance of macrophages ............................................................ 1 1.1.2 General and tissue-specific functions of macrophages ....................................... 2 1.1.3 Macrophage polarization and activation ............................................................. 4 1.1.4 Macrophages in diseases ..................................................................................... 6 1.2 Tumor-associated macrophages (TAM) ...................................................................... 8 1.2.1 TAM promote tumor growth and metastasis ...................................................... 9 1.2.2 TAM promote angiogenesis ............................................................................... 10 1.2.3 TAM support an immunosuppressive tumor microenvironment ...................... 12 1.2.4 TAM as therapeutic targets in cancer immunotherapy ..................................... 13 1.3 Malignant melanoma ................................................................................................ 14 1.3.1 Epidemiology, risk factors and pathophysiology ............................................... 14 1.3.2 Melanoma immuno-microenvironment ............................................................ 16 1.3.3 Melanoma immunotherapy ............................................................................... 18 1.4 Purinergic receptors .................................................................................................. 19 1.4.1 Classification and function ................................................................................. 19 1.4.2 Purinergic signaling regulates inflammation and immunity .............................. 21 1.4.3 Purinergic receptors in the tumor microenvironment ...................................... 23 1.5 The purinergic receptor P2Y12 .................................................................................. 25 1.5.1 Structure and signaling of P2Y12 ....................................................................... 25 1.5.2 P2Y12 expression in platelets ............................................................................. 26 1.5.3 P2Y12 expression in microglia ............................................................................ 27 1.5.4 P2Y12 expression in leukocytes ......................................................................... 29 1.6 Aim of the study ........................................................................................................ 31 2. Materials .......................................................................................................................... 32 3. Methods ........................................................................................................................... 39 3.1 Cell culture methods.................................................................................................. 39 3.1.1 Cell lines .............................................................................................................. 39 ___________________________________________________________________________ I Table of Contents 3.1.2 Cryopreservation of cells .................................................................................... 40 3.1.3 Production of lentivirus for transduction of eukaryotic cell lines ...................... 40 3.1.4 Generation of transgenic cell lines ..................................................................... 41 3.1.5 Isolation of CD14+ cells from human peripheral blood ...................................... 41 3.2 In vitro Assays ............................................................................................................ 42 3.2.1 BrdU Cell Proliferation Assay ............................................................................. 42 3.2.2 Transwell migration assay with pBM ................................................................. 42 3.2.3 Transwell migration assay with transgenic Raw 264.7 cells .............................. 43 3.2.4 Adhesion of U937 to HUVECs ............................................................................. 43 3.2.5 U937 Transmigration Assay ................................................................................ 44 3.3 Molecular biology ...................................................................................................... 44 3.3.1 Cloning of hsP2Y12 and mmP2Y12 .................................................................... 44 3.3.2 RNA isolation and cDNA synthesis ..................................................................... 45 3.3.3 Polymerase chain reaction (PCR) ....................................................................... 46 3.3.4 Quantitative Real-time PCR (qRT-PCR) ............................................................... 46 3.3.5 Microarrays ........................................................................................................ 47 3.3.6 In situ hybridization ............................................................................................ 47 3.4 Biochemical methods ................................................................................................ 47 3.4.1 Generation of an anti-human P2Y12 antibody .................................................. 47 3.4.2 Cell lysates and protein determination .............................................................. 48 3.4.3 SDS-PAGE ............................................................................................................ 48 3.4.4 Western blot analysis (WB) ................................................................................ 49 3.4.5 Enzyme-linked immunosorbent Assay (ELISA) ................................................... 49 3.4.6 Proteome Profiler Mouse Cytokine Array .......................................................... 49 3.4.7 Immunocytochemistry (ICC) ............................................................................... 50 3.4.8 Immunohistochemistry (IHC) ............................................................................. 50 3.4.9 Immunofluorescence (IF) ................................................................................... 51 3.4.10 Sequential staining ............................................................................................. 51 3.4.11 Fluorescence-activated cell sorting (FACS) ........................................................ 51 3.5 Mouse experiments ................................................................................................... 52 3.5.1 Mice .................................................................................................................... 52 3.5.2 B16F10 melanoma model .................................................................................. 52 3.5.3 Isolation of murine macrophages ...................................................................... 53 3.5.4 Generation of bone marrow-derived macrophages (BMDM) ........................... 53 ___________________________________________________________________________ II Table of Contents 3.6 Statistical analysis ...................................................................................................... 53 4. Results .............................................................................................................................. 54 4.1 Characterization of P2Y12 expression in human pBM in vitro .................................. 54 4.1.1 P2Y12 is strongly induced in MDI-treated pBM ................................................. 54 4.1.2 MDI induces a M2-like macrophage phenotype in pBM ................................... 55 4.1.3 Generation of an anti-P2Y12 peptide antibody and characterization of its specificity in transgenic U937 cells .................................................................... 58 4.1.4 P2Y12 is expressed in MDI-treated pBM and its induction can be inhibited with mifepristone ......................................................................................................
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