Tumors and Ly6Chigh Monocytes Fatemeh Zare ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tdx.cat) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. En la utilització o cita de parts de la tesi és obligat indicar el nom de la persona autora. ADVERTENCIA. 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PhD Programme in Biomedicine Programa de Doctorado en Biomedicina Tumors and Ly6Chigh Monocytes Tumores y Monocitos Ly6Chigh Thesis submitted by - Memoria presentada por Fatemeh Zare To qualify for the Doctorate degree by - Para optar al grado de Doctor por la University of Barcelona - Universidad de Barcelona Departamento de Fisiologia y Inmunologia Faculta t de Biologia Universida d de Barcelona Programa de Doctorado de Biomedicina Bienio 2014-2015 Memoria presentada por Fatemeh Zare Para optar al Grado de Doctora por la Universidad de Barcelona Director de Tesis Dr. Antonio Celada Cotarelo Catedratico de Inmunologia Barcelona, Octubre de 2014 ACKNOWLEDGMENTS My PhD has been a lengthy journey and many people have helped me along the way. Firstly I would like to acknowledge my supervisor for all his help and support throughout this time. Prof. Antonio Celada, thank you for encouraging me to work independently as a scientist and for providing me the opportunity to be a part of your group. Prof Roger Gomis, given that for you I was an external student, I wish to especially thank you for your constant support. Dr. Jordi Lloberas, thank you for your wonderful problem solving skills, especially under pressure, during those times when I showed up at your office door with a million questions. My gratitude also goes to Dr. Jaume Comas for teaching me flow cytometry, and developing the cell sorting protocol. I would like to extend my appreciation to all the members of the Macrophage Biology, both past and present, for their technical support and encouragement. Thank you to Juan A. calatayud for our rewarding collaboration and for the many discussions about our work. A million thanks to Dr. Gloria Sans, Dr. Selma Kuznetsova, Lorena V Strella, Juan Torres and Ester Ruiz that I could count on your help always. Many thanks to our lab technician Gemma López for her unending patience and help around the laboratory. I would like to thank my family and friends for their love and support during my studies. I am eternally grateful to my wonderful parents and sister who have always encouraged me in everything and who have been there not only for all the highs , but importantly for all the lows too. Without you I would never have gotten this far. This is for you. TABLE OF CONTENTS 1 INTRODUCTION ...................................................................................... 14 1.1 Cancer .................................................................................................. 15 1.1.1 Cancer and the Immune System .................................................. 16 1.1.2 Tumor Immunology ....................................................................... 16 1.1.3 Cancer Related Inflammation (CRI) ............................................. 17 1.2 Innate and adaptive immune response to tumors ................................ 18 1.3 Immune cells and anti-cancer therapies ............................................... 21 1.4 Cells in the tumor microenvironment .................................................... 23 1.4.1 Inflammatory cell component of tumors ....................................... 24 1.4.2 Tumor-associated leukocytes ....................................................... 25 1.4.3 Macrophages ................................................................................ 26 1.4.3.1 Tumor associated macrophages .............................................. 26 1.4.3.1.1 TAMs phenotype ................................................................ 27 1.4.4 Monocytes .................................................................................... 32 1.4.4.1 Monocyte heterogeneity ........................................................... 33 1.4.4.2 Monocytes sub populations ...................................................... 35 1.4.4.3 Recruitment of monocytes to sites of inflammation ................. 38 1.4.4.4 Ly6Chigh and Ly6Clow monocytes .............................................. 39 1.4.4.5 Monocytes differentiation to macrophages .............................. 42 1.4.4.6 Monocytes recruitment to tumors ............................................. 43 1.5 Monocytes generation .......................................................................... 46 1.6 Animal models ...................................................................................... 48 1.7 Objectives ............................................................................................. 48 2 MATERIALS AND METHODS.................................................................. 49 2.1 Mouse strains ....................................................................................... 50 2.2 Cell lines and culture ............................................................................ 50 2.2.1 MDA-MB-231 cell line ................................................................... 50 2.2.2 C26 cell line .................................................................................. 51 2.3 Mouse bone marrow-derived Ly6Chigh monocytes ............................... 52 2.3.1 Generation of bone marrow-derived Ly6Chigh monocytes ............ 52 Generation of bone marrow-derived Ly6Chigh monocytes from STAT6 wild type and knockout BALB/C mice was carried out in the same manner as mentioned above. .................................................................................................. 52 2.3.2 Enrichment of Ly6Chigh monocytes by cell sorting ........................ 52 2.3.3 In vitro activation of Ly6Chigh monocytes with INF-γ or IL-4 ......... 53 2.3.4 Labeling Ly6Chigh monocytes with DiR tracking dye ..................... 54 2.3.5 Labeling of Ly6Chigh monocytes with PKH26 tracking dye ........... 54 2.4 Animal models ...................................................................................... 55 2.4.1 MDA-MB-231 breast cancer model .............................................. 55 2.4.2 C26 colon carcinoma model ......................................................... 55 2.5 Experimental analysis ........................................................................... 56 2.5.1 Isolation of single cell suspension from tumor tissue ................... 56 2.5.2 Flow cytometry assay (FACS) ...................................................... 57 2.5.3 Isolation of total RNA from tumor tissue ....................................... 59 2.5.4 Real-Time (RT-PCR) .................................................................... 59 2.5.5 In vivo imaging .............................................................................. 60 2.5.5.1 Bioluminescence Imaging ........................................................ 61 2.5.6 Immunohistochemistry analysis .................................................... 61 2.6 Statistical analysis ................................................................................ 62 3 RESULTS ................................................................................................. 63 3.1 In vitro generation of Ly6Chigh monocytes from mice bone marrow ..... 64 3.1.1 Characterization of enriched Ly6Chigh monocytes generated in vitro 67 3.1.2 Staining of Ly6Chigh monocytes .................................................... 69 3.2 Role of Ly6Chigh monocytes in immunodeficient MDA-MB-231 breast cancer model ...........................................................................................................