Neuropilin-2 (NRP-2), expressed in capillaries and smooth muscle, is involved in a signaling axis featuring both stimulatory and inhibitory pathways. Ligand interaction with NRP-2 determines the effectual pathway: stimulatory VEGF, or inhibitory Semaphorin3. VEGF interaction causes NRP-2 localization with VEGFR-2, while Semaphorin3 (SEMA3) interaction causes NRP-2 to complex with Plexin A. VEGFR-2 interaction induces permeability and angiogenesis, making this an attractive target for anti-angiogenesis treatment. Plexin A interaction induces smooth muscle relaxation, offering a target for treatment of loss of bladder contractility. We aimed to further our understanding of the necessity of NRP-2 in the VEGFR-2-mediated induction of angiogenesis and permeability by performing assays in Nrp-2 knockout (KO) mice. We also tested an inhibitor against Semaphorin3/NRP-2 binding using U87MG cell, a human glioblastoma cell line. Angiogenesis assays were largely inconclusive, but suggested that NRP-2 does play a substantial role in contributing towards vessel growth. Permeability assays showed that NRP-2 plays a significant role in induction of permeability. The Semaphorin3F/NRP-2 inhibitor showed promise as a potential therapeutic, limiting the extent of Semaphorin3-based inhibition of VEGFR-2 pathway. / 2026-02-12T00:00:00Z

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.

According to the CDC, the prevalence of diabetes has increased from 3.3% in 2004 to over 10.1% by 2019 (Prevalence of Diagnosed Diabetes). The United States Department of Agriculture (USDA) recommends that total fat intake should be between 20 - 35% of the total calories an individual consumes in a day and yet the Center for Disease Control and Prevention (CDC) reports that on average, total fat consumption makes up 35.8% of a person’s diet (Dietary guidelines, 2020 & CDC, 2021 respectively). According to Parrish, “dietary fat does not have an immediate effect on blood sugar levels, but consuming a meal high in fat can slow digestion and make it more difficult for insulin to work” (Parrish, 2015). Chronic diabetes can result in hypocontractility of the bladder. Contractility of the bladder is controlled by a multitude of receptors, ligands, and kinases. One receptor our group feels contributes is neuropilin 2. Our group has reported expression of neuropilin 2 in the smooth muscle of the bladder and has shown that expression induces cytoskeleton relaxation. Thus, it is thought that if neuropilin 2 expression is reduced, that potentially that hypocontractility of the bladder can be attenuated. In an in vivo model of diabetes using mice on a high fat diet for 5 months, we observed minimal changes in bladder histology, and variable Nrp2 expression. In silico analysis of data from in vivo and in vitro models of diabetes identified Nrp2 transcriptional induction compared to controls and a connection with multiple differentially expressed genes in the Nrp2 signaling pathway linked with biological processes related to a diabetic pathological state. An in vitro model of diabetes which subjected rat bladder contractile cells to high glucose identified significant cytoskeletal changes, increases in Nrp2 expression, and decreased contractility. Knock-down of Nrp2 using siRNA resulted in increased contractility of smooth muscle cells on collagen gels. These data suggest that Nrp2 signaling is altered under diabetic conditions and could be targeted to attenuate diabetes induced bladder hypocontractility. / 2025-07-06T00:00:00Z

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

Multiple Sclerosis (MS) is an inflammatory disease which causes areas of demyelination in the Central Nervous System (CNS) and affects only humans. Current therapies for MS are focused on anti-inflammatory treatment, which reduce the occurrence and clinical relapses of the disease. However, progressive disability of the disease is related to axonal degeneration. After demyelination, remyelination occurs, which helps repair the demyelinated lesions and protects axons from degeneration. However, this endogenous remyelination is inefficient, and currently there are no therapies available to enhance remyelination. The aim of this thesis was to first characterize a fast and reliable model to study CNS remyelination in vitro, and second to investigate the role of semaphorin 3a (Sema3A) and semaphorin 3f (Sema3F) signaling in CNS remyelination. Various in vivo models have been developed to investigate the pathology of multiple sclerosis, and can be used to test remyelination therapies. However, in vivo models are expensive, animal- and time- consuming. Until now, there has been no well-characterized and robust in vitro model for remyelination study. In this thesis, an ex vivo slice culture system with mouse brain and spinal cord was developed, and characterized by immunofluorescent microscopy and transmission electron microscopy, for CNS remyelination study. Automated (re)myelinating quantification by image pro plus software was developed and validated to provide a fast and reliable way for testing factors that change remyelination efficiency. Two such factors are Sema3A and 3F, which were initially identified as axon guidance cues during development. Sema3A (repulsive) and 3F (attractive) were proved to play a role in oligodendrocyte precursor cell (OPC) migration during development, and hypothesized to be important in remyelination. In this thesis, I investigated the effects and mechanisms for this by adding recombinant SEMA3A or SEMA3F or by knockdown their obligatory receptors Neuropilin (Nrp) 1 and 2, using lentivirus induced miRNAi. Slice culture and primary OPC culture were used to determine the effect on OPC survival, migration, proliferation, differentiation and myelination.

INTRODUCTION: Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive form of cancer with a high mortality rate, primarily due to lack of effective treatment options. Current therapeutic approaches are limited to surgical resection of the pancreas during early stages of the disease and to the use of non-specific chemotherapeutic drugs such as gemcitabine, neither of which has successfully improved the 5-year survival rate of PDAC. Both the lack of effective treatments and the high mortality of the disease call for the urgent need to develop new therapeutic options. OBJECTIVES: This thesis project focuses on an endogenous inhibitor of the neuropilin 2 receptor (NRP2) called semaphorin 3F (SEMA3F) and its use as a potential new drug in the fight against pancreatic cancer. By binding the transmembrane receptor neuropilin 2 (NRP2), SEMA3F can inhibit angiogenesis and cellular proliferation. Interestingly, given its role as a guidance molecule, it is also a potent mediator of cellular repulsion. All three of these effects will be analyzed in the context of this study. METHODS: Syngeneic pancreatic cancer cells were injected orthotopically in two separate groups of mice. One group involved the use of transgenic Nrp2-/- mice, and served as a way to analyze the absence of the receptor on the vasculature and how that affects the growth of the primary tumor and the formation of metastases in the liver. The other group received intravenous injections of SEMA3F-expressing and control adenovirus, and served to explore the effect of SEMA3F as a potential therapy against the growth of the primary tumor in the pancreas and distant metastases in the liver. RESULTS: We observed a decrease in pancreatic tumor and metastatic growth in the absence of Nrp2 in our transgenic mouse model compared to the WT control. Mice injected with SEMA3F-expressing adenovirus also showed a decrease in primary tumor growth as well as a reduction in the formation of metastases in the liver compared to the control. CONCLUSION: Nrp2 mediates angiogenesis in pancreatic cancer, which facilitates the growth of the primary tumor as well as the formation of metastases. Our results indicate that the anti-angiogenic, anti-proliferative and repulsive actions of SEMA3F could be used to develop an effective treatment option for PDACpancreatic ductal adenocarcinoma. / 2020-07-24T00:00:00Z

Le domaine transmembranaire des récepteurs membranaires est aujourd’hui considéré comme essentiel dans l’activation et la régulation des voies de signalisation sous-jacentes. Ceci est tout particulièrement le cas pour neuropiline-1 et -2 (NRP1/2), et ErbB2, trois récepteurs impliqués dans la croissance tumorale. Notre laboratoire a initialement démontré qu’un peptide ciblant le domaine transmembrane du récepteur NRP1, bloque l’oligomérisation de ce récepteur et provoque ainsi l’inhibition de la prolifération/migration des cellules tumorales et l’angiogenèse in vivo. L’objectif principal de ce travail de thèse était d’élargir cette stratégie aux récepteurs membranaires NRP2 et ErbB2, et ce, dans le contexte du cancer du sein. Mes travaux montrent que ces peptides inhibent la pousse tumorale et les métastases associées dans différents modèles de cancer du sein. Les effets anti-tumoraux peuvent s’expliquer par les propriétés anti-angiogéniques et anti-prolifératives des peptides démontrées in vitro et in vivo. J’ai également disséqué le mécanisme d’action du peptide ErbB2 et montré que le peptide inhibiteur de NRP2 induit des effets secondaires rédhibitoires (promotion des métastases osseuses). Dans l’ensemble, mes recherches valident le potentiel thérapeutique de cette stratégie peptidique et renforce l’idée d’un développement clinique de ces composés. D’une terre inconnue à une terre d’espoir, le cœur de la membrane est incontestablement une nouvelle source d’inspiration pour le développement des médicaments de demain. / The role of transmembrane domains (TMD) in membrane receptor activation and regulation is nowadays appearing as a key step of cell signaling. This has been indeed evaluated for neuropilin-1 and -2 (NRP1/2) and ErbB2 receptors, three membrane receptors whose signaling has clearly been implicated in tumorigenesis. Our team had demonstrated that a synthetic peptide blocking the transmembrane domain of NRP1 blocked NRP1-dependent signaling leading to the inhibition of glioma cell proliferation/migration and tumor associated angiogenesis in vivo. The major goal of this thesis project was to extend this novel strategy to NRP2 and ErbB2 in the breast cancer context. Thus, I was able to demonstrate for the first time that the use of peptides, inhibiting the TMD of these receptors, was able to inhibit tumor growth and related metastases in vivo, in three different breast cancer mouse models that I have developed in the laboratory. These results were supported by in vitro experiments demonstrating anti-proliferative and anti-angiogenic properties of these peptides. Besides, I was able to dissect the mechanism of action of the peptide targeting ErbB2 receptor in vitro and in vivo, and I provided data excluding NRP2 as a target because of an unexpected promotion of bone metastasis. Altogether, my data offer convincing evidences to further develop MTP-ErbB2 and MTP-NRP1 peptides as novel therapeutic compounds for patients suffering metastatic cancers. From terra incognita to the exploration of a world of hope, the heart of the membrane is becoming a new promising estate for drug design.