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Neuropilin-2 (NRP2) is a single transmembrane receptor and was first found in the nervous system to play a role in axon guidance. Interestingly, NRP2 was also found on many tumor cells and various studies showed that NRP2 is associated with a poor prognosis in different cancers and is involved in migration and therapy resistance. We investigated the prognostic potential of NRP2 in the pancreatic ductal adenocarcinoma (PDAC) and found out that in contrast to other kinds of cancer a high expression of NRP2 is associated with a longer cancer specific survival. We hypothesized that this effect could be either triggered through an expression of different interaction partners of NRP2. Both semaphorine 3F and VEGFs can bind to NRP2 but have different effects on cancer cells. Semaphorine 3F was found to have a great potential as a cancer inhibitor in pancreatic cancer whereas VEGFs are often associated with a worse prognosis. Both compete for the binding to NRP2. Furthermore, we found high expression of NRP2 in tumor-associated macrophages (TAMs) in PDACs. Until now, NRP2 expression and function is poorly analyzed in the immune system. Therefore, we next focused on the investigation of NRP2 in the immune system during cancer progression. We used LysM:cre-NRP2LoxP/LoxP (conditional knock-out of NRP2 in macrophages) and Vav:cre-NRP2LoxP/LoxP (conditional knock-out in all immune cells) for our experiments. We showed that NRP2 is upregulated during the differentiation/maturation of macrophages. Next, we injected LLC cells subcutaneously to analyze the effect of NRP2 knock-out in macrophages (LysM:cre) or in the all immune cells (Vav:cre). No difference was detected in tumor size, but the vascularization was impaired in both mouse models. Different tumor models with extended tumor growth times and metastasis should be performed next to proof the importance of NRP2 in immune cells during tumor progression. Due to the broad expression of NRP2 in the immune system we used the Vav:cre-NRP2LoxP/LoxP mouse to investigate the role of NRP2 during an immune response. We used a mild allergic inflammation model of the lung and analyzed the different immune cell populations. Interestingly, T cells and eosinophils were reduced during the inflammation indicating, that the conditional knock-out of NRP2 is inhibiting the immune response. We further analyzed the role of NRP2 in T cells and found out, that the expression of NRP2 is very different in the various T cell populations. CD8+ T cells express ca. 10 times as much mRNA for NRP2 compared to CD4+ T cells. Also, the CD4 subpopulation showed a diverse expression of NRP2. Th2 and Th17 express a lot of NRP2 and Treg and Th1 very low levels. These results suggest an important role of NRP2 in certain cells. The knock-out of NRP2 in Th2 cells leads to an upregulation of IL-13, IL-5 and IL10. We first showed the importance of NRP2 during an immune response and found interesting regulations in immune cell populations and important cytokines. More work needs to be done to understand the functions of NRP2 during an immune response.:INDEX OF ABBREVIATIONS IV TABLE OF FIGURES VI LIST OF TABLES VIII 1 INTRODUCTION 1 1.1 Neuropilins 1 1.1.1 Neuropilin-2 3 1.1.2 Neuropilin-2 in cancer 5 1.2 Pancreatic ductal adenocarcinoma 6 1.3 The immune system 8 1.4 Neuropilin-2 in the immune system 9 1.5 Mouse models 11 2 METHODS 12 2.1 NRP2 related survival study in human pancreatic ductal adenocarcinoma (PDAC) using a Tissue Micro Array (TMA) 12 2.2 Role of NRP2 in the immune system using different mouse models 12 2.2.1 Mouse lines 12 2.2.2 LysM:cre-NRP2LoxP/LoxP 13 2.2.3 Vav:cre-NRP2LoxP/LoxP 14 2.2.4 Genotyping 14 2.3 Cell culture 15 2.3.1 Cell lines 16 2.3.2 Primary culture 16 2.3.3 Isolation and differentiation of bone marrow derived macrophages (BMDM) 16 2.3.4 Isolation of primary T cells 17 2.3.5 Isolation of T lymphocytes with positive and negative selection 18 2.3.6 Isolation of lymphatic and/or splenic CD8+ cells 18 2.3.7 Isolation of lymphatic and splenic native CD4+ cells 18 2.3.8 Differentiation and culture of naïve CD4+ cells 19 2.4 Lewis lungs carcinoma model 20 2.5 Immunohistochemistry 20 2.5.1 Paraffin embedded tissue 20 2.5.1.1 Fixation 21 2.5.1.2 Cutting 21 2.5.1.3 Hydration 22 2.5.1.4 H&E 22 2.5.2 Evaluation of necrotic areas 22 2.5.3 PAS-Staining 22 2.5.4 CD31 Staining 23 2.5.5 Fluorescent staining on frozen tissue against CD31 and CD206 24 2.5.6 Evaluation of fluorescence CD31 and CD206 staining with ImageJ/Fiji 24 2.5.7 Evaluation of the amount of M2-macrophages in LLC tumors 25 2.6 Mild allergic model of the lung 25 2.7 Quantitative Real-Time PCR (qPCR) 26 2.7.1 RNA isolation 26 2.7.2 cDNA synthesis 27 2.7.3 qPCR 27 2.8 Flow cytometry 28 3 RESULTS 30 3.1 Neuropilin-2 as a prognostic marker in PDAC 30 3.2 Effects of NRP2 deficiency on macrophages and role of NRP2 in immune cells during tumor progression in mice 35 3.2.1 Effects of NRP2 depletion on macrophages 35 3.2.2 Role of a conditional knock-out of NRP2 in immune cells during tumor progression 38 3.3 Role of NRP2 knock-out in immune cells during inflammation 45 3.3.1 Mild allergic model of the lung 45 3.3.2 Analysis of NRP2 deficient T cells 49 4 DISCUSSION 55 4.1 Neuropilin-2 as a prognostic marker in PDAC 55 4.1.1 Sema3F as a potential important factor in PDACs 56 4.1.2 NRP2b as a potential anti-tumorigenic splice variant 57 4.1.3 VEGF-C as a prognostic marker in PDACs 59 4.1.4 Prognostic potential of co-expression of NRP2 and VEGF-C in PDACs 59 4.2 Effects of NRP2 deficiency on macrophages and the role of NRP2 in immune cells during tumor progression in mice 60 4.2.1 Effects of NRP2 depletion on macrophages 61 4.2.2 Analysis of NRP2 deficient macrophages on LLC growth 62 4.2.3 Influence of NRP2 deficiency in hematopoietic stem cells on bones and blood cells 63 4.2.4 Effects of NRP2 deficient immune cells on tumor pathology 64 4.2.5 The role of NRP2 on the vascularization in LLC tumors 65 4.3 The role of NRP2 in during an inflammatory response 68 4.4 Conclusion 72 5 SUMMARY 73 REFERENCES 74 ACKNOWLEDGMENTS/DANKSAGUNG 85 VERSICHERUNG 87 |
