O adenocarcinoma ductal pancreático (PDAC, pancreatic ductal adenocarcinoma), o tipo mais prevalente de câncer do pâncreas, é uma neoplasia extremamente agressiva e com elevado índice de letalidade. Há uma necessidade premente de identificação de vulnerabilidades no PDAC que possam ser exploradas como alvos terapêuticos, e a utilização de modelos pré-clínicos que recapitulem a complexidade biológica e heterogeneidade clínica da doença é um aspecto central para a realização dessa tarefa. Os xenotransplantes de tecido tumoral derivado de pacientes (PDX, patient-derived tumor tissue xenografts), realizados em camundongos imunodeficientes, replicam com grande similaridade as principais características do tumor original e, assim, constituem uma ferramenta valiosa para o teste de drogas e estudos funcionais. Neste trabalho, 17 amostras cirúrgicas de PDAC humano foram implantadas subcutaneamente em camundongos nude atímicos. Sete tumores (41%) foram enxertados com sucesso e têm sido mantidos em sucessivas gerações de animais receptores. O exame histológico de seis desses xenoenxertos identificou características morfológicas compatíveis com os padrões reconhecidos no PDAC humano, assim como uma consistente similaridade de seu status de diferenciação histológica em relação aos perfis verificados nos tumoresoriginais. O cultivo in vitro de células derivadas de um dos xenotumores resultou em uma nova linhagem de câncer de pâncreas, com morfologia e cinética de crescimento comparáveis às de outras linhagens celulares de câncer pancreático. O potencial tumorigênico dessa nova linhagem foi validado in vivo, com uma consistente formação de tumores após inoculação em camundongos nude. A fim de aproveitar esse recurso para a investigação de potenciais alvos terapêuticos no PDAC, um rastreamento de vulnerabilidades moleculares foi realizado por meio de silenciamento gênico em larga-escala com RNA de interferência (RNAi). Uma biblioteca lentiviral de 4492 shRNAs (short hairpin RNAs), alvejando cerca de 350 genes envolvidos na regulação epigenética, foi empregada para a triagem de genes de suscetibilidade nas células derivadas de PDX, e em outras cinco linhagens tumorais pancreáticas (AsPC-1, BxPC-3, Capan-1, MIA PaCa-2 e PANC-1). Inicialmente, foi realizada uma série de experimentos preliminares, visando à amplificação e controle de qualidade da biblioteca de silenciamento, à produção de vetores lentivirais e à padronização das condições experimentais para a transdução e seleção das células-alvo. Apenas três das linhagens avaliadas (AsPC-1, MIA PaCa-2 e PANC-1) mostraram-se permissíveis à transdução pelos vetores lentivirais, e foram assim utilizadas no screening de alvos epigenéticos. A análise dos dados obtidos nesse ensaio está em curso e os resultados serão utilizados para a definição de potenciais alvos candidatos. Em conclusão, recursos valiosos para apoiar a pesquisa sobre o câncer de pâncreas foram desenvolvidos. A coleção de PDXs estabelecida, bem como a linhagem celular recém-derivada, constituem uma fonte permanente e estável de células de PDAC para análises moleculares e estudos funcionais que busquem elucidar aspectos da doença ainda pouco compreendidos. Adicionalmente, os reagentes gerados e a expertise adquirida com os ensaiosrealizados com a biblioteca de shRNAs contra alvos epigenéticos serão de grande utilidade em futuras investigações para identificar genes com funções importantes na manutenção do fenótipo tumoral, e consequentemente com potencial para serem explorados terapeuticamente. / Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is a highly aggressive and lethal neoplasm. There is a pressing need to identify vulnerabilities in PDAC suited to be exploited as therapeutic targets, and the use of preclinical models recapitulating the biological complexity and clinical heterogeneity of the disease is central to this task. Patient-derived tumor tissue xenografts (PDX), established in immunodeficient mice, replicate with great similarity the main characteristics of the original tumor and thus constitute a valuable tool for drug testing and functional studies. In this work, 17 surgical samples of human PDAC were implanted subcutaneously in athymic nude mice. Seven tumors (41%) were successfully grafted and have been maintained through successive generations of recipient animals. Histological examination of six of these xenografts identified morphological characteristics compatible with the recognized patterns of human PDAC, as well as a consistent similarity of their histological differentiation status in relation to the profiles verified in the original tumors. In vitro culture of cells derived from one of these xenografts resulted in a new pancreatic cancer cell line, with morphology and growth kinetics comparable to those of other pancreatic tumor cells. The tumorigenic potential of this freshly derived cell line was validated in vivo, with a consistent tumor formation following inoculation into nude mice. To take advantage ofthis resource to investigate potential therapeutic targets in PDAC, a screening of molecular vulnerabilities was performed through large-scale gene silencing with RNA interference (RNAi). A lentiviral library containing 4492 short hairpin RNAs (shRNAs), targeting about 350 genes involved in epigenetic regulation, was employed for the search of susceptibility genes in the PDX-derived cells and in other five pancreatic tumor cell lines (AsPC-1, BxPC -3, Capan-1, MIA PaCa-2 and PANC-1). Initially, a series of preliminary experiments were carried out aiming at the amplification and quality control of the silencing library, production of lentiviral vectors and adjustment of the experimental conditions for transduction and selection of the target cells. Only three of the cell lines evaluated (AsPC-1, MIA PaCa-2 and PANC-1) were permissible for transduction by the lentiviral vectors, and were accordingly used in the screening of epigenetic targets. The analysis of data obtained in this trial is ongoing and the results will be used for definition of potential candidate targets. In conclusion, valuable resources to support research on pancreatic cancer have been developed. The established collection of PDXs as well as the newly derived cell line constitutes a permanent and stable source of PDAC cells for molecular analyzes and functional studies seeking to elucidate aspects of this disease that are still poorly understood. Additionally, both the reagents generated and the expertise gained from the RNAi assay against epigenetic targets will have inordinate usefulness in future investigations to identify genes with major functions in maintaining the malignant phenotype, and consequently with the potential to be exploited therapeutically.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with poor prognosis and limited treatment options. Since many patients present with metastatic disease and are thus ineligible for surgical resection, PDAC is almost ubiquitously fatal; new treatment options are therefore needed to combat this disease. A key hallmark of many cancers, including PDAC, is metabolic reprogramming and a shift towards a high glycolytic rate, known as the Warburg effect. This allows cancer cells to generate ATP in the face of hypoxia and to meet the increased metabolic requirements associated with rapid proliferation. We hypothesised that this shift towards glycolytic metabolism has important implications for the regulation of cytosolic Ca2+ ([Ca2+]i) in PDAC, since the plasma membrane Ca2+ ATPase (PMCA), which is critical for maintaining low [Ca2+]i and thus cell survival, is dependent on ATP to extrude cytosolic Ca2+. The relative contributions of mitochondrial vs glycolytic ATP in fuelling the PMCA in human PDAC cell lines (PANC-1 and MIA PaCa-2) were therefore assessed. Moreover, the effects of numerous mechanistically distinct metabolic inhibitors on key readouts of cell death, [Ca2+]i and ATP were investigated. Treatment with glycolytic inhibitors induced significant ATP depletion, PMCA inhibition, [Ca2+]i overload and cell death in both PANC-1 and MIA PaCa-2 cells, while mitochondrial inhibitors had no effect. Subsequently, these experiments were repeated on PDAC cells cultured in media formulated to "switch" their highly glycolytic phenotype back to one more reliant on mitochondrial metabolism. Culture in nominal glucose-free media supplemented with either galactose (10 mM) or alpha-ketoisocaproate (KIC, 2 mM) resulted in a switch in metabolism in MIA PaCa-2 cells, where proliferation rate and glycolysis were significantly decreased, and in the case of cells cultured in KIC, oxidative phosphorylation rate was preserved (assessed using Seahorse XF technology). Following culture of MIA PaCa-2 cells in either galactose or KIC, glycolytic inhibition failed to recapitulate the profound ATP depletion, PMCA inhibition and [Ca2+]i overload observed in glucose-cultured MIA PaCa-2 cells. These data demonstrate that in PDAC cells exhibiting a high rate of glycolysis, glycolytically-derived ATP is important for fuelling [Ca2+]i homeostasis and thus is critical for survival. Finally, using a cell surface biotinylation assay, the keyglycolytic enzymes LDHA, PFKP, GAPDH, PFKFB3 and PKM2 were all found to associate with the plasma membrane in MIA PaCa-2 cells, possibly in a tyrosine phosphorylation-dependent manner. To investigate whether the dynamic membrane-association of glycolytic enzymes provides a privileged supply of ATP to the PMCA in PDAC, the effects of tyrosine kinase inhibitors was assessed on PMCA activity. However, while these inhibited PMCA activity, this occurred without accompanying global ATP depletion. These data indicate that glycolytic ATP is critical for the regulation of [Ca2+]i by the PMCA in PDAC, and that the glycolytic regulation of the PMCA may be an important therapeutic locus. However, further research is required to determine whether membrane-bound glycolytic enzymes regulate its activity.

Introduction:Un des problèmes du cancer du Pancréas (CP) est le temps de latence entre la suspicion du CP et la mise en place des traitements. Les méthodes de biopsie liquide pourraient accélérer la mise en évidence d’éléments tumoraux et le diagnostic.Objectif :L’objectif principal de l’étude était de comparer la performance diagnostique de plusieurs techniques de biopsie liquide chez des patients atteint d’un CP résécable d’emblé. L’objectif secondaire était la corrélation avec le taux de récidive post-opératoire.Méthodes:Tout d'abord, nous avons testé 2 méthodes d'enrichissement CTC pour estimer la sensibilité de la détection CTC avec des expériences de cell-spiking de deux lignées de cellules tumorales pancréatiques dans des échantillons de sang de 24 volontaires sains en utilisant la méthode en gradient de densité OncoQuick® et la méthode de sélection négative RosetteSep™. De plus, les mutations KRAS ont été quantifiées dans l'ADN génomique de cellules purifiées par digital droplet PCR (dd-PCR) avec des amorces spécifiques des allèles.Nous avons conçu un essai clinique prospectif (NCT03032913) visant à détecter les cellules tumorales circulantes (CTC), l’ADN tumoral circulant (ADNct) et les onco-exosomes chez les patients atteint de CP et chez les patients d’un groupe témoin. Pour les CTCs : enrichissement et détection de CTCs par la méthode CellSearch©, méthode d’enrichissement de CTCs RosetteSep® et OncoQuick® puis quantification de l’ADN tumoral par dd-PCR. Les exosomes ont été isolés puis caractérisés avec le taux d’expression de Glypican-1. Tous les patients de l’étude ont eu un prélèvement de sang périphérique, les patients du groupe CP ont eu un prélèvement de sang portal peropératoire.Résultats:La sensibilité analytique était de 100 % pour OncoQuick®, quelle que soit la lignée cellulaire, et se situait entre 70 et 100 % pour RosetteSep™. Le taux moyen de récupération des cellules était de 56±23% pour OncoQuick® contre 39±27% pour RosetteSep™ (p<0,001). Les cellules tumorales de la population de cellules sanguines enrichies ont été détectées par dd-PCR après enrichissement par RosetteSep™ et OncoQuick® La détection des allèles K-RAS mutants par ddPCR après enrichissement de RosetteSepTM était 3 à 4 fois plus sensible qu'après OncoQuick®. Ainsi, RosetteSep™ est plus fiable en termes d'efficacité de récupération et de détection des mutants KRAS que OncoQuick®.De février à novembre 2017, 22 patients atteints de CP résécable et 28 patients témoins ont été inclus. Tous les patients ont été détectés positifs par au moins une méthode. Les CTCs ont été détectées chez 9 patients avec la méthode cellsearch (70% dans le sang portal exclusif) et 13 avec la méthode Rosettesep (60%). Les onco-exosomes ont été détecté chez 14 patients sur 22. L’ADNct n’a été détecté que chez deux patients métastatiques. La détection combinée des CTCs et des onco-exosomes était significativement corrélée à la survie sans récidive.Conclusion:Cette étude suggère que la biopsie liquide combinée peut être un outil prometteur à fois diagnostique et pronostique dans le CP à un stade précoce. / Introduction:One of the problems of pancreatic ductal adenocarcinoma (PC) is the latency time between the suspicion of PC and the initiation of treatments, especially neo-adjuvants that require histological evidence. Liquid biopsy methods could be a companion test for diagnosis.Objective :The main objective of the study was to compare the diagnostic performance of several liquid biopsy techniques in patients with resectable pancreatic without neo-adjuvant therapy cancer. The secondary objective was the correlation between the quantification of liquid biopsy parameters and clinic-pathologic features.Methods:First, we tested 2 CTC enrichment methods to estimate the sensitivity of CTC detection with cell spiking experiments of two pancreatic tumour cell lines in blood samples from 24 healthy volunteers using the onco-specific density gradient OncoQuick® and the negative selection enrichment method RosetteSep™. Additionally, KRAS mutations were quantified in genomic DNA of purified cells by digital droplet Q-PCR (dd-PCR) with allele specific primers.We designed a prospective clinical trial (PANC-CTC# NCT03032913) to detect circulating tumour cells (CTC), circulating tumour DNA (ADNct) and onco-exosomes in patients with pancreatic cancer and in patients in a control group using different methods. For CTCs, it was the enrichment and detection of CTCs by the CellSearch© method (reference method), the RosetteSep® and OncoQuick® CTC enrichment method and the quantification of tumor DNA by dd-PCR. Exosomes were isolated and characterized with the expression rate of Glypican-1. All patients in the study had a peripheral blood sample, patients in the PDAC group had a portal blood sample during surgery.Results:Analytical sensitivity was 100% for OncoQuick®, regardless of the cell line, and ranged between 70 and 100% for RosetteSep™. Mean recovery rate of cells was 56±23% for OncoQuick® versus 39±27% for RosetteSep™ (p<0.001). Molecular detection of mutant K-RAS alleles by ddPCR after RosetteSepTM enrichment was 3- to 4-fold more sensitive than after OncoQuick®. Thus, RosetteSep™ is more reliable in terms of recovery efficiency and KRAS mutant detection than OncoQuick®.From February to November 2017, 22 patients with resectable pancreatic cancer and 28 control patients were included. All patients were positive by at least one method. CTCs were detected in 9 patients with the cellsearch method (70% in the exclusive portal blood) and 13 with the Rosettesep method (59%), Onco-exosomes were detected in 14 out of 22(64%) patients in peripheral and/or portal blood. DNAct was detected in only two metastatic patients. The combined detection of CTCs with cellsearch and onco-exosomes was significantly correlated with progression free survival and overall survival when CTC cluster were found.Conclusion: This study suggests that combined liquid biopsy can be a promising tool for both diagnosis and prognosis in early pancreatic cancer.

<p>Pancreatic cancer is the fourth most lethal cancer and ranks as the eighth most commonly diagnosed cancer worldwide. This is due to its rapid proliferation, strong metastatic potential and its delayed detection. One major risk factor for developing pancreatic cancer is the aggressive inflammatory disease chronic pancreatitis. Chronic inflammation frequently precedes the development of certain pancreatic cancers.</p><p>Inflammation is a protective and necessary process by which the body can alert the immune system of the existence of a wound or infection and mount an immune response to remove the harmful stimuli and start wound healing. The cross-talking of cells of the immune system and infected cells happens through cytokines, soluble proteins that activate and recruit other immune cells to increase the system’s response to the pathogen. Failure to resolve the injury can result in persistent cytokine production that in turn allows a cell that is damaged or altered to survive when in normal conditions it would be killed. Inflammation is thought to create a microenvironment that facilitates the initiation and/or growth of pancreatic cancer cells.</p><p>Cytokines use two important kinases for their signaling: Janus Kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs). The JAKs are activated upon the binding of cytokines to their corresponding receptors. When activated, the JAKs activate STATs through tyrosine phosphorylation. The STATs transduce signals to the nucleus of the cells to induce expression of critical genes essential in normal physiological cellular events such as differentiation, proliferation, cell survival, apoptosis and angiogenesis. STAT3 (a member of the STAT family) is constitutively activated in some pancreatic cancers, promoting cell cycle progression, cellular transformations and preventing apoptosis. Therefore, STAT3 is a promising target for cancer treatment. Novel therapies that inhibit STAT3 activity in cancers are urgently needed. Natural products are a very good resource for the discovery of new drugs against pancreatic cancer.</p><p>Covering more than 70% of the Earths surface, The Ocean is an excellent source of bioactive natural products. Harbor Branch Oceanographic Institute’s Center for Marine Biomedical and Biotechnology Research (HBOI-CMBBR) situated in Florida, aims to find new marine natural products useful in disease prevention and drug therapy. Their current focus is to look for novel treatments for preventing both the formation of new pancreatic tumors and the metastasis of existing tumors.</p><p>The hypothesis of this degree project was that novel inhibitors of STAT3 useful in the treatment of pancreatitis and/or pancreatic cancer could be found from marine-natural products. The first specific aim of this degree project was to set up an assay to identify bioactive marine natural products as inhibitors of inflammation. Furthermore the assay was validated using a commercially available inhibitor of inflammation (Cucurbitacin I). The last aim was to further validate the assay by screening pure compounds and peak library material from the HBOI marine specimen collection.</p><p>At the end of the experimentation time, the assay still was not set-up as there were difficulties in proper cell culture techniques and the cell line did not respond as advertised. While the results were not as expected, the work performed resulted in familiarization with research laboratory practices and increased laboratory skills. Moreover, the results from the assays point to future directions to accomplish this project.</p> / Development of a screening assay for inhibitors of inflammation useful against pancreatic cancer

Background: Among the common malignancies, pancreatic cancer has the shortest long-term survival. The aggressive, rapid, and infiltrative growth pattern of pancreatic cancer, together with the lack of specific symptoms, often leads to late diagnosis. Metastases are frequently found at the time of diagnosis, which prevents curative surgical treatment. Good tumor markers would enable early detection, thus improving the prognosis. Unfortunately, no such markers are available in the clinic. The tumor stroma is defined as the non-malignant cells and the extracellular matrix (ECM) of a cancer. Pancreatic cancer is characterized by an abundant tumor stroma, rich in ECM proteins such as collagens, which have been shown to play important roles in tumor progression. Furthermore, pancreatic cancer cells produce large quantities of ECM proteins, especially the basement membrane (BM) protein type IV collagen. All epithelial cells are anchored to a BM, which must be degraded in order for an in situ cancer to become invasive. Matrix metalloproteinases (MMPs) are enzymes involved in BM degradation. In this thesis, the tumor stroma of pancreatic cancer is studied, focusing on the BM proteins type IV and type XVIII collagen, with the aim to clarify if the stroma could be a source of novel tumor markers for this form of cancer. Additionally, the role of type IV collagen produced by the cancer cells is studied. Methods: Expression patterns of type IV and type XVIII collagen, MMPs involved in collagen degradation, and collagen receptors (integrins) were studied by immunoflourescence in both normal and pancreatic cancer tissue, and in pancreatic cancer cell lines. Circulating plasma levels of type IV and type XVIII collagen and conventional tumor markers (TPS, Ca 19-9, CEA and Ca 125) were measured in controls and pancreatic cancer patients at the time of diagnosis and after treatment. The role of cancer cell produced type IV collagen was studied in human pancreatic cancer cell lines by functional blocking of integrin receptors (integrin a1, a2 and b1) and integrin-binding sites on type IV collagen, and by siRNA-induced down-regulation of type IV collagen synthesis. Proliferation was analyzed by a luminescence based cell viability assay, migration by time-lapse microscopy, and apoptosis by M30-neoepitope detection. Results: MMPs involved in BM degradation were upregulated in pancreatic cancer tissue. The expression of type XVIII collagen shifted from a general BM expression pattern in normal tissue, to mainly being found in the tumor vasculature in pancreatic cancer. Type IV collagen, on the other hand, remained highly expressed in the vicinity of the cancer cells. The a1, a2, and b1 integrin receptors were highly expressed at the cancer cell surface. Both down-regulation of type IV collagen synthesis and blocking the integrin/type IV collagen interaction decreased cell proliferation and migration. The proliferative capacity was rescued by the addition of exogenous type IV collagen. Furthermore, the circulating levels of both type IV and type XVIII collagen were increased in pancreatic cancer patients at the time of diagnosis compared to controls. After treatment, the levels were normalized for type XVIII collagen, whereas the levels of type IV collagen remained high after surgery. High postoperative levels of type IV collagen were associated with short overall survival. A similar association to short survival was found for preoperative type XVIII collagen levels. No such associations to survival could be detected for the conventional markers.   Conclusion: The results of this thesis show that type IV and type XVIII collagens can serve as tumor markers for pancreatic cancer with advantages compared to conventionally used markers. Additionally, evidence is provided of an autocrine loop, involving type IV collagen and its integrin receptors, with importance for retaining a proliferative and migratory phenotype in pancreatic cancer cells.

Microarray technologies enable scientists to identify co-expressed genes at large scale. However, the gene expression analysis does not show functional relationships between co-expressed genes. There is a demand for effective approaches to analyse gene expression data to enable biological discoveries that can lead to identification of markers or therapeutic targets of many diseases. In cancer research, a number of gene expression screens have been carried out to identify genes differentially expressed in cancerous tissue such as Pancreatic Ductal Adenocarcinoma (PDAC). PDAC carries very poor prognosis, it eludes early detection and is characterised by its aggressiveness and resistance to currently available therapies. To identify molecular markers and suitable targets, there exist a research effort that maps differentially expressed genes to protein interactions to gain an understanding at systems level. Such interaction networks have a complex interconnected structure, whose the understanding of which is not a trivial task. Several formal approaches use simulation to support the investigation of such networks. These approaches suffer from the missing knowledge concerning biological systems. Reasoning in the other hand has the advantage of dealing with incomplete and partial information of the network knowledge. The initial approach adopted was to provide an algorithm that utilises a network-centric approach to pancreatic cancer, by re-constructing networks from known interactions and predicting novel protein interactions from structural templates. This method was applied to a data set of co-expressed PDAC genes. To this end, structural domains for the gene products are identified by using threading which is a 3D structure prediction technique. Next, the Protein Structure Interaction Database (SCOPPI), a database that classifies and annotates domain interactions derived from all known protein structures, is used to find templates of structurally interacting domains. Moreover, a network of related biological pathways for the PDAC data was constructed. In order to reason over molecular networks that are affected by dysregulation of gene expression, BioRevise was implemented. It is a belief revision system where the inhibition behaviour of reactions is modelled using extended logic programming. The system computes a minimal set of enzymes whose malfunction explains the abnormal expression levels of observed metabolites or enzymes. As a result of this research, two complementary approaches for the analysis of pancreatic cancer gene expression data are presented. Using the first approach, the pathways found to be largely affected in pancreatic cancer are signal transduction, actin cytoskeleton regulation, cell growth and cell communication. The analysis indicates that the alteration of the calcium pathway plays an important role in pancreas specific tumorigenesis. Furthermore, the structural prediction method reveals ~ 700 potential protein-protein interactions from the PDAC microarray data, among them, 81 novel interactions such as: serine/threonine kinase CDC2L1 interacting with cyclin-dependent kinase inhibitor CDKN3 and the tissue factor pathway inhibitor 2 (TFPI2) interacting with the transmembrane protease serine 4 (TMPRSS4). These resulting genes were further investigated and some were found to be potential therapeutic markers for PDAC. Since TMPRSS4 is involved in metastasis formation, it is hypothesised that the upregulation of TMPRSS4 and the downregulation of its predicted inhibitor TFPI2 plays an important role in this process. The predicted protein-protein network inspired the analysis of the data from two other perspectives. The resulting protein-protein interaction network highlighted the importance of the co-expression of KLK6 and KLK10 as prognostic factors for survival in PDAC as well as the construction of a PDAC specific apoptosis pathway to study different effects of multiple gene silencing in order to reactivate apoptosis in PDAC. Using the second approach, the behaviour of biological interaction networks using computational logic formalism was modelled, reasoning over the networks is enabled and the abnormal behaviour of its components is explained. The usability of the BioRevise system is demonstrated through two examples, a metabolic disorder disease and a deficiency in a pancreatic cancer associated pathway. The system successfully identified the inhibition of the enzyme glucose-6-phosphatase as responsible for the Glycogen storage disease type I, which according to literature is known to be the main reason for this disease. Furthermore, BioRevise was used to model reaction inhibition in the Glycolysis pathway which is known to be affected by Pancreatic cancer.

Das Pankreaskarzinom bleibt trotz verbesserter Diagnose- und Therapiemöglichkeiten weiterhin eine Krankheit mit einer sehr schlechten Prognose und Lebenserwartung nach Diagnosestellung. Eine innovative Therapiemöglichkeit stellt eine Gruppe von Histondeacetylase-Inhibitoren dar, die einen direkten Einfluss auf die Regulation der Genexpression in Tumorzellen haben. Das Ziel der vorliegenden Arbeit bestand darin, die Wirkung des HDAC-Inhibitors 4-Phenylbutyrat auf Pankreaskarzinomzellen in-vitro und vor allem in-vivo zu untersuchen. Neben dem Einfluss auf die Zellproliferation in-vitro und in-vivo wurde in-vivo im subkutanen und orthotopen Tumormodell der Einfluss auf Tumorwachstum, Zellproliferation, Nekroseausbreitung, Regulation des Connexin 43 und Histonacetylierung im Tumorgewebe untersucht. Die Untersuchungen zeigen, dass 4-PB durch seinen hemmenden Effekt auf das Wachstum von Xenografttumoren und auf die Proliferation von Pankreastumorzellen sowie durch seine fördernde Wirkung auf die Expression von Connexin 43, Acetylierung von H4 und Bildung eine Pseudokapsel, ein potentiell wirksames Medikament bei der experimentellen Behandlung des Pankreaskarzinoms ist.

Pancreatic cancer is one of the most lethal cancers and despite all research efforts the last 50 years, there are still no effective therapy for this terrible disease. Until quite recently most research in the field of pancreatic duct adenocarcinoma (PDAC) was focused on the tumor cells and mechanisms essential for their proliferation and survival. However, the tumor does not only consist of tumor cells, rather a combination of tumor cells and numerous stroma cell types, i.e. the tumor microenvironment. The tumor cells have developed the ability to activate the surrounding cells to produce factors important for the progression of the tumor. Cancer associated fibroblasts (CAFs) are the major stroma component and as much as 70% of the total PDAC tumor mass consists of these cells. In this thesis I have investigated the mechanisms involved in the cross-talk between tumor cells and CAFs and distinguished the local and systemic effects of this communication. Tumor derived IL-1α was identified as an important factor creating the inflammatory profile seen in CAFs. In PDAC patients, IL-1α was detected in 90% of the tumors and high expression was associated with poor clinical outcome. Moreover, the PDAC tumors had elevated expression levels of many inflammatory factors that were induced in CAFs by the tumor derived IL-1α in vitro. Consequently, this high expression of inflammatory factors in CAFs will attract immune cells including tumor associated macrophages (TAMs), dendritic cells (DCs), and CD8+ T cells. This indicates an immune suppressive role of CAFs, protecting the tumor cells by acting as decoy targets for immune cells homing into the tumor. The inflammatory factors produced in the PDAC microenvironment did not only affect the infiltrating immune cells, but had also systemic effects that included decreased levels of blood DCs in PDAC patients. Furthermore, these myeloid and plasmacytoid DCs were partly activated and had a semi mature phenotype and impaired immunostimulatory function. Low levels of blood DCs were direct associated with poor patient prognosis and the same was seen for low expression of ICOSL by the DCs. The findings presented in this thesis indicate an essential role for the cross-talk between tumor cells and stroma in the production of tumor  promoting factors. Treatment of PDAC patients with drugs that target the IL-1α signaling pathway could prevent the communication between these cells, thus reduce the amount of inflammatory factors both locally and systemically. Altogether, our findings support the idea that neutralization of the IL-1α signaling molecule could be a promising therapy for pancreatic cancer. The findings presented in this thesis indicate an essential role for the cross-talk between tumor cells and stroma in the production of tumor promoting factors. Treatment of PDAC patients with drugs that target the IL-1α signaling pathway could prevent the communication between these cells, thus reduce the amount of inflammatory factors both locally and systemically. Altogether, our findings support the idea that neutralization of the IL-1α signaling molecule could be a promising therapy for pancreatic cancer. / Mindre än 5% av patienterna som drabbas av cancer i bukspottkörteln förväntas överleva i mer än fem år. De typiska symtomen kommer sent och sjukdomen framskrider snabbt. Några av de riskfaktorer som identifierats är tobaksrökning, fetma och typ 2 diabetes. Forskningen har hittills siktat in sig på tumörcellerna och de mekanismer de använder för att överleva och föröka sig. Men en tumör innehåller också normala kroppsceller och vid bukspottkörtelcancer kan så mycket som 70 procent bestå av i sig ofarliga bindvävsceller. Miljön i tumören skapas av samspelet mellan dessa celltyper. De cancerceller som är bäst på att utnyttja omgivningen för sin tillväxt fortlever och för sina egenskaper vidare. En sådan egenskap är att kunna manipulera bindvävsceller till att producera signalsubstanser och tillväxtfaktorer som gynnar tumören. Mekanismerna bakom denna kommunikation har studerats och ett viktigt fynd var att tumörcellerna producerar signalämnet interleukin 1-alpha (IL-1a). Detta protein upptäcktes i 90 procent av de undersökta tumörerna, och var kopplat till dålig prognos hos patienterna. Signalen via IL-1a sätter igång tillverkningen av substanser som behövs för nybildning och tillväxt av blodkärl, i sin tur en förutsättning för att tumören ska leva vidare och växa. Proteinet stimulerar också celldelning i tumören, bidrar till att lura kroppens immunförsvar och underlättar spridning av dottertumörer till andra delar av kroppen. När vi slår ut signaleringen kan tumörcellerna inte längre påverka bindvävscellerna lika effektivt, och således minskar förekomsten av flera faktorer som gynnar tumörtillväxten. IL-1a kan därför vara en lovande kandidat att utforska vidare för framtida som ett läkemedel mot bukspottkörtelcancer.

Als països desenvolupats, una de cada cinc persones morirà a causa del càncer. S'ha descrit que les cèl·lules canceroses presenten modificacions en els glicans presents a la superfície cel·lular i aquesta glicosilació anòmala podria reflectir-se en les glicoproteïnes de secreció. Per aquest motiu es planteja l'estudi de la glicosilació de dues proteïnes de secreció en situació normal i tumoral: la ribonucleasa pancreática humana (RNasa 1) i l'antigen prostàtic específic (PSA).La RNasa 1 és una glicoproteïna secretada majoritàriament pel pàncreas. S'ha desenvolupat un mètode immunològic per a detectar els nivells de RNasa 1 en sèrum. Malgrat la millora de la sensibilitat, respecte d'estudis anteriors, no s'han observat diferències significatives entre la concentració de RNasa 1 en sèrum de pacients control sans, afectats de neoplàsia pancreàtica, de pancreatitis o d'altres patologies.L'estudi de les estructures glucídiques de la RNasa 1, mitjançant assaigs immunològics, permet observar diferències importants en la glicosilació entre la situació normal i tumoral: Els antígens sialilats sLex i sLea només apareixen en la RNasa 1 de medi de cultiu de cèl·lules d'adenocarcinoma pancreàtic Capan-1 i MDAPanc-3 i l'antigen fucosilat Ley només apareix en la RNasa 1 de pàncreas de donant. S'ha purificat la RNasa 1 secretada per la línia MDAPanc-3, cosa que ha permès seqüenciar-ne les estructures glucídiques i comparar-les amb les de la RNasa 1 purificada del medi de les cèl·lules Capan-1 i de pàncreas de donant, corroborant els resultats abans esmentats.L'antigen prostàtic específic (PSA) és una glicoproteïna secretada principalment per la pròstata. Els seus nivells sèrics s'utilitzen actualment com a marcador del càncer de pròstata, però la seva especificitat no permet diferenciar clarament una situació benigna d'una maligna.La purificació i caracterització glucídica del PSA secretat per les cèl·lules de carcinoma prostàtic LNCaP mostren diferències molt clares amb la glicosilació que presenta el PSA purificat de plasma seminal de donant. Principalment, el PSA present en situació tumoral, purificat de les cèl·lules de carcinoma prostàtic, no conté àcid siàlic, però presenta nivells més alts de fucosilació que el PSA en situació normal. El PSA purificat de plasma seminal de donant sí que conté àcid siàlic. Aquests resultats s'han obtingut mitjançant assaigs immunològics amb detecció per lectines i s'han corroborat per seqüenciació glucídica. D'acord amb les estructures glucídiques que millor diferencien el PSA de situació normal i tumoral, s'ha portat a terme la caracterització glucídica de mostres biològiques que contenen PSA. S'han desenvolupat diferents assaigs immunològics de detecció per lectines o associats a l'activitat sialiltransferasa, amb un enriquiment previ en PSA per immunoadsorció indirecta o cromatografia per interacció tiofílica. Els resultats dels diferents assaigs permeten concloure que el PSA del sèrum de pacients de neoplàsia prostàtica presenten un contingut en àcid siàlic similar al del plasma seminal de donant, encara que són lleugerament menys sialilats. Aquests resultats s'adiuen amb els determinats sobre mostres de PSA purificat.La separació del PSA per electroforesi bidimensional mostra diverses formes amb pI àcid en el PSA de plasma seminal, explicades per la presència d'àcid siàlic. Es detecten formes de pI més bàsic que el teòric per al PSA en el secretat per les cèl·lules LNCaP, que correspon a formes pPSA. Al sèrum de pacients de neoplàsia prostàtica s'hi observen formes sialilades.Les proteïnes de secreció, RNasa 1 i PSA, es troben alterades a nivell glucídic en situació tumoral, cosa que podria ser d'utilitat per a finalitats diagnòstiques.

Electronic medical record databases (e.g. the Clinical Practice Research Datalink, CPRD) are increasingly used in epidemiological research. The CPRD has two formats of data: coded, which is the sole format used in almost all research; and free-text (or ‘hidden’), which may contain much clinical information but is generally unavailable to researchers. This thesis examines the ramifications of omitting free-text records from research. Cases with bladder (n=4,915) or pancreatic (n=3,635) cancer were matched to controls (n=21,718, bladder; n=16,459, pancreas) on age, sex and GP practice. Coded and text-only records of attendance for haematuria, jaundice and abdominal pain in the year before cancer diagnosis were identified. The number of patients whose entire attendance record for a symptom/sign existed solely in the text was quantified. Associations between recording method (coded or text-only) and case/control status were estimated (χ2 test). For each symptom/sign, the positive predictive value (PPV, Bayes' Theorem) and odds ratio (OR, conditional logistic regression) for cancer were estimated before and after supplementation with text-only records. Text-only recording was considerable, with 7,951/20,958 (37%) of symptom records being in that format. For individual patients, text-only recording was more likely in controls (140/336=42%) than cases (556/3,147=18%) for visible haematuria in bladder cancer (χ2 test, p<0.001), and for jaundice (21/31=67% vs 463/1,565=30%, p<0.0001) and abdominal pain (323/1,126=29% vs 397/1,789=22%, p<0.001) in pancreatic cancer. Adding text records reduced PPVs of visible haematuria for bladder cancer from 4.0% (95% CI: 3.5–4.6%) to 2.9% (2.6–3.2%) and of jaundice for pancreatic cancer from 12.8% (7.3–21.6%) to 6.3% (4.5–8.7%). Coded records suggested that non-visible haematuria occurred in 127/4,915 (2.6%) cases, a figure below that generally used for study. Supplementation with text-only records increased this to 312/4,915 (6.4%), permitting the first estimation of its OR (28.0, 95% CI: 20.7–37.9, p<0.0001) and PPV (1.60%, 1.22–2.10%, p<0.0001) for bladder cancer. The results suggest that GPs make strong clinical judgements about the probable significance of symptoms – preferentially coding clinical features they consider significant to a diagnosis, while using text to record those that they think are not.