Exploring mechanisms regulating the heterogeneity of Tumor-Associated Macrophages

Accumulation of macrophages in the tumor microenvironment is associated to poor prognoses in most human cancers. Tumor-associated macrophages (TAMs) represent a heterogeneous and plastic population of cells that contribute to tumor growth, metastatic dissemination, angiogenesis, and immune suppression. Both recruitment of monocytes from the blood and in situ proliferation contribute to the accumulation of TAMs. During the course of tumor progression, the phenotype of the infiltrating TAMs is changing by influences from other cells, extracellular signal molecules and availability of oxygen and nutrients. Therapeutic targeting of TAMs as a monotherapy has limited success, however, experimental studies show promising results when selectively depleting specific subsets of TAMs or changing the function of the TAM population. Yet, the mechanisms whereby macrophage phenotypes are regulated during tumor growth are still largely unknown. In this thesis, we investigated mechanisms underlying the dynamic changes in the TAM population observed during tumor growth. In our first study, overexpression of semaphorin 3A induced the proliferation of anti-tumoral macrophages and at the same time reduced the proliferation of pro-tumoral TAMs resulting in accumulation and activation of CD8 T-cells and NK-cells and restricted tumor growth. In study II, we identified translational regulation of gene expression as an important mechanism regulating the TAM phenotype during tumor growth. By selective inhibition of translational activation, pro-tumoral macrophages were skewed towards an anti-tumoral phenotype. In the third study, we demonstrated a functional difference between macrophages of different ontogeny in a mouse model of glioblastoma. M2-polarized microglia, but not bone-marrow derived macrophages induced the expression of platelet-derived growth factor receptor B (PDGFRB) in glioma cells, enhancing their migratory capacity. In study IV, we showed that Zoledronic acid in combination with interleukin-2 induced the expression of interferon-g by monocytes leading to an up-regulation of TNF-related apoptosis-inducing ligand (TRAIL) on NKcells, inducing their cytotoxicity against tumor cells. In summary, we describe several mechanisms whereby the TAM phenotype may be regulated i.e translational control of gene expression, regulation of proliferation and ontogeny. LIST OF SCIENTIFIC PAPERS I. Wallerius M, Wallmann T, Bartish M, Östling J, Mezheyeuski A, Tobin NP, Nygren E, Pangigadde P, Pellegrini P, Squadrito ML, Pontén F, Hartman J, Bergh J, De Milito A1, De Palma M, Östman A, Andersson J, Rolny C. Guidance Molecule SEMA3A Restricts Tumor Growth by Differentially Regulating the Proliferation of Tumor-Associated Macrophages Cancer Res. 2016 Jun 1;76(11):3166-78 II. Bartish M, Wallerius M, Wallmann T, Masvidal L, Liu H, Joly AL, van Hoef V, Bergh J, Hartman J, Andersson J, Rolny C, Larsson O. RNA Translational Control of the Tumor-Associated Macrophage Phenotype Manuscript III. Wallmann T, Zhang XM, Wallerius M, Bolin S, Joly AL, Sobocki C, Leiss L, Jiang Y, Bergh J, Andersson J, Holland E, Enger P, Swartling F, Uhrbom L, Miletic H, Harris R, Rolny, C. Pro-Angiogenic microglia-induced expression of platelet-derived growth factor receptor beta in glioma cells promotes their migratory capacity Submitted IV. Sarhan D, D'Arcy P, Wennerberg E, Lidén M, Hu J, Winqvist O, Rolny C, Lundqvist A. Activated monocytes augment TRAIL-mediated cytotoxicity by human NK cells through release of IFN-γ Eur J Immunol. 2013 Jan;43(1):249-57 ADDITIONAL PUBLICATIONS Not included in this thesis Sarhan D, Leijonhufvud C, Murray S, Witt K, Seitz C, Wallerius M, Xie H, Ullén A, Hamenberg U, Lidbrink E, Rolny C, Andersson J, Lundqvist A. Zoledronic acid inhibits NFAT and IL-2 signaling pathways in regulatory T cells and diminishes their suppressive function in patients with metastatic cancer Oncoimmunology. 2017 Jun 14;6(8):e1338238 Christoffersson G, Vågesjö E, Vandooren J, Lidén M, Massena S, Reinert RB, Brissova M, Powers AC, Opdenakker G, Phillipson M. VEGF-A recruits a proangiogenic MMP-9-delivering neutrophil subset that induces angiogenesis in transplanted hypoxic tissue Blood. 2012 Nov 29;120(23)4653-62 CONTENTS 1 General background . 1 1.1 Cancer . 1 1.2 Tumor microenvironment . 1 Tumor angiogenesis . 1 Tumor immunity . 2 1.3 mRNA abundance and RNA translational regulation . 3 2 Tumor-Associated Macrophages . 4 2.1 Macrophage origin and maintenance during homeostasis . 5 2.2 TAM phenotypes . 5 M1and M2 macrophages . 5 TAM phenotypes during tumor growth . 6 TAM recruitment during tumor progression . 9 In situ proliferation of TAMs . 9 2.3 TAM functions . 10 Angiogenesis . 10 Invasion and metastasis . 11 Immune suppression . 12 3 Translational regulation in the immune system with a focus on macrophages . 13 4 Therapeutic implications . 15 5 Aims of the thesis . 17 6 Results and discussion . 19 6.1 Summary of the main findings . 19 6.2 Study I . 20 6.3 Study II . 24 6.4 Study III . 28 6.5 Study IV . 31 6.6 Conclusions, significances and further perspectives . 32 7 Acknowledgements . 35 8 References . 39 LIST OF ABBREVIATIONS 4E-BP 4E binding protein bFGF basic fibroblast growth factor BM Bone marrow BMDM Bone marrow-derived macrophage CBR Carbonyl reductas CCL Chemokine (C-C motif) ligand CCR Chemokine (C-C motif) receptor CNS Central nervous system CSF Colony stimulating factor CTL Cytotoxic T lymphocytes CX3CR1 CX3C chemokine receptor 1 CXCL Chemokine (C-X-C motif) ligands EGF Epidermal growth factor eIF Eukaryotic initiation factor FcgR Fcg receptor GBM Glioblastoma multiforme GO Gene-ontology HIF Hypoxia-inducible factor hnRNP Heterogeneous nuclear ribonucleoprotein IFN Interferon IL Interleukin IPA Ingenuity Pathway Analysis IRF Interferon regulatory factor IkBa NF-kB inhibitor NFKBIA LPS Lipopolysaccharides MAPK Mitogen-activated protein kinase MDSC Myeloid-derived suppressor cells MEF Mouse embryonic fibroblasts Met-tRNAi Methionyl initiator transfer RNA MHC Major histocompatibility complex MMP Matrix metalloproteinases MMTV Mouse Mammary Tumor Virus MNK MAPK interacting protein kinase MRC Mannose receptor C-type mRNA Messenger RNA NK Natural killer NP1 Neuropilin 1 PBMC Peripheral blood mononuclear cell PD-1 Programmed cell death-1 PD-L1 Programmed death ligand 1 PDAC Pancreatic ductal adenocarcinoma PDGF Platelet-derived growth factor PDGFR Platelet-derived growth factor receptor PHD Prolylhydroxylase domain PIC Pre-initiation complex PyMT Polyoma middle T antigen ROS Reactive oxygen speices SEMA Semaphorin shRNA Short hairpin RNA TAK Transforming growth factor beta-activated kinase TAM Tumor-associated macrophage TAMM Tumor-associated macrophages and microglia TEM Tie2-expressing monocyte TGF Transforming growth factor TLR Toll-like receptor TNF Tumor necrosis factor TRAIL TNF-related apoptosis-inducing ligand uPA Urokinasetype plasminogen activator VEGF Vascular endothelial growth factor ZA Zoledronic acid