The Role of Sema3A In Inflammation Mediated Tumor Progression Master Thesis KTH Royal Institute of Technology Stockholm, June 2015 Emma Nygren Supervisor Charlotte Rolny, Cancer Center Karolinska Co-Supervisors Majken Wallerius, Cancer Center Karolinska Tatjana Wallmann, Cancer Center Karolinska Examiner Peter Nilsson, KTH SciLife Laboratory Abstract In the tumor microenvironment there are many different cell types present and among these, immune cells display a large proportion. Central players in the tumor immunity are macrophages that come in two different phenotypes, the M1 and M2 macrophages. M1 polarized macrophages are tumor suppressive while M2 polarized macrophages support tumor growth. The factors that contribute to the skewing of macrophages from one phenotype to another are under investigation. Interestingly, our lab has identified Immune Semaphorin 3A (Sema3A) as a participating player in regulating the accumulation of anti-tumoral M1 macrophages leading to a suppression of tumor growth. In light of these data this thesis has focused on the role of endogenous Sema3A in the tumor microenvironment. A tumor cell line expressing shRNA against Sema3A mRNA was generated using lentiviral mediated gene therapy. This knockdown cell line showed 72 % lower mRNA expression compared to control and was evaluated in vivo by monitoring tumor progression in female BALB/c mice. The immune cell composition of the tumors was analyzed using flow cytometry. The results from the in vivo experiment show that endogenous Sema3A has a limited effect on tumor progression. A slight shift to a more tumor supportive immune profile was observed in the knockdown tumors. Moreover, a virus for transducing cells to overexpress Sema3A under a suitable promoter for systemic delivery was generated. 1 Sammanfattning Många olika sorters celler är närvarande i tumörers mikromiljö och immunceller utgör en stor andel av dessa. Makrofager är centrala spelare i tumörimmunförsvaret och dessa kan indelas i olika aktiveringsgrader eller fenotyper, M1 eller M2 makrofager. M1 polariserade makrofager är tumörsuppressiva medan M2 makrofager bidrar till tumörtillväxt. De faktorer som reglerar skiftningen mellan M1 och M2 fenotyperna är under utredning. Vårt labb har identifierat att Immunsemaforinen 3A (Sema3A) spelar en roll i att reglera ackumuleringen av antitumorala M1 makrofager vilket leder till hämmad tumörtillväxt. Med denna information som bakgrund har detta examensarbete fokuserat på Sema3As roll i tumörmikromiljön. Med hjälp av lentivirusmedierad genterapi skapades en tummörcellinje som uttrycker shRNA mot Sema3AmRNA. Denna cellinjes visade 72 % lägre Sema3A mRNA uttryck jämfört med kontroll och utvärderades sedan in vivo genom att följa tumörtillväxten i BALB/c mushonor. Immuncellsammansättningen i tumörerna analyserades sedan med hjälp av flödescytometri. Resultaten från in vivo experimentet visar att endogent Sema3A har en begränsad effekt på tumörutvecklingen. En något mer tumörgynnande immunprofil observerades i de tumörer där Sema3Auttryck var minskat. Utöver detta skapades också ett lentivirus för att transducera celler så att de överuttrycker Sema3A under en passande promotor för systemisk tillförsel. 2 Table of Contents Abstract .................................................................................................................................. 1 Sammanfattning .................................................................................................................. 2 Table of Contents ................................................................................................................ 3 1. Introduction ..................................................................................................................... 4 1.1 The project aims and hypothesis .................................................................................... 4 1.2 Cancer as a general concept .............................................................................................. 5 1.3 Summary of key immune system aspects ..................................................................... 7 1.4 The link between the immune system and cancer .................................................... 7 1.5 The role of macrophages in cancer ................................................................................. 8 1.6 Immune Semaphorins as regulators .............................................................................. 9 2. Techniques and models ............................................................................................. 10 2.1 Lentiviral mediated gene therapy ................................................................................ 10 Packaging of virus particles ............................................................................................................... 11 Harvest and use of generated virus ............................................................................................... 12 2.3 Gateway cloning ................................................................................................................. 12 2.4 The use of shRNA for gene knockdown ...................................................................... 14 2.5 The cell line and mouse model ...................................................................................... 15 2.6 Flow Cytometry .................................................................................................................. 16 3. Results ............................................................................................................................. 17 Overexpression of Sema3A in tumor cells ....................................................................... 17 Knockdown of Sema3A in tumor cells ............................................................................... 19 4. Discussion ....................................................................................................................... 24 4.1 Knockdown of endogenous Sema3A expression in 4T1 tumor cells ................ 24 4.2 Technical evaluation ........................................................................................................ 24 4.3 Future prospectives .......................................................................................................... 25 Acknowledgements .................................................................................................................. 26 5. References ...................................................................................................................... 27 Appendix 1 – Materials and Protocols ....................................................................... 30 Gateway cloning ........................................................................................................................ 30 shRNA ............................................................................................................................................ 30 Transfection ............................................................................................................................... 31 Transduction .............................................................................................................................. 31 mRNA-expression ..................................................................................................................... 31 Proliferation Assay ................................................................................................................... 32 In Vivo experiment ................................................................................................................... 32 Flow cytometry .......................................................................................................................... 32 Appendix 2 – Vector Maps ............................................................................................. 34 3 1. Introduction This master thesis was performed in the field of cancer biology and tumor immunology. In this section an introduction to the subjects that are central is presented along with a description of the aims and hypothesis of the project. 1.1 The project aims and hypothesis The importance of immune cells in the tumor microenvironment has been increasingly acknowledged over the recent decades (1) and there is now an interest of developing immune therapies against cancer. To do this, there is a need to understand the mechanisms behind tumor immunology and therefore it is important to study the interactions between tumor cells and immune cells. The understanding of these mechanisms can be used to develop new therapy strategies to treat cancer. The protein Immune Semaphorin 3A (Sema3A) has been identified to be involved in some of the mechanisms that underlie tumor immunology. The overall aim of this thesis project was to investigate the role of Sema3A in breast cancer progression. The work was divided into two technical sub aims listed below: 1. Cloning of Sema3A into a lentiviral Phosphoglycerate kinase (PGK) promoter vector to produce high quality concentrated virus for systemic delivery. 2. Knocking down of Sema3A in 4T1 tumor cells in order to study the contribution of endogenous Sema3A to tumor progression. The motivation for the first aim was that in order to evaluate the therapeutic potential of Sema3A, it had to be cloned under a suitable promoter. When using lentiviruses systemically (injected into the blood stream)