Introdução: O adenocarcinoma pancreático é responsável pela maioria das neoplasias pancreáticas e está associado a um prognóstico extremamente pobre tanto devido à alta taxa de diagnósticos em estágio avançado quanto ao elevado índice de recidiva mesmo nos pacientes submetidos à ressecção com intenção curativa. Uma ferramenta que possa predizer adequadamente o prognóstico da doença é fundamental para uma melhor estratificação de risco. Evidências tem mostrado que a resposta inflamatória sistêmica está associada ao prognóstico de diversos tipos de câncer, sendo que a razão neutrófilos/linfócitos (NLR) e suas adaptações e a razão plaquetas/linfócitos (PLR) tem se mostrado promissores para este fim. Objetivo: O objetivo do presente estudo é avaliar o valor prognóstico das razões NLR, NLR derivada (dNLR) e PLR determinados por exames coletados no momento da internação e após tratamento quimioterápico paliativo numa população de pacientes com diagnóstico de adenocarcinoma pancreático, analisando ainda qual o valor de ponto de corte mais adequado para cada parâmetro. Além disso, pretendemos investigar se essas razões podem ter algum valor como fator preditivo de ressecabilidade no adenocarcinoma pancreático. Métodos: Foram coletados dados de pacientes com diagnóstico de adenocarcinoma pancreático confirmado por exame histopatológico atendidos no Hospital de Clínicas de Porto Alegre entre 2003 e 2013. As razões estudadas foram determinadas com base nos hemogramas coletados na internação dos pacientes e após dois ciclos de quimioterapia naqueles que foram submetidos a tratamento paliativo. Resultados: Na análise combinada de todos os pacientes incluídos no estudo, NLR basal, dNLR basal e PLR basal não mostraram evidência de ter impacto prognóstico na sobrevida (P= 0,394, P= 0,152, P= 0,177 respectivamente). No subgrupo de pacientes submetidos a quimioterapia paliativa, NLR, dNLR e PLR calculados pelos exames realizados após 2 ciclos de tratamento mostraram-se fatores prognósticos para sobrevida global (P=0,003, P=0,009 e P=0,001 respectivamente). Os pontos de corte mais adequados encontrados foram 4,11 para NLR (sensibilidade 83% e especificidade 75%), 362 para PLR (sensibilidade 91% e especificidade 62,5%) e 2,8 para dNLR (sensibilidade 87% e especificidade 62,5%). Nenhuma das razões se mostrou estatisticamente significativa como preditor para ressecabilidade (NLR, P=0,88; dNLR, P=0,99; PLR, P=0,64). Conclusões: As razões NLR, dNLR e PLR são úteis como marcadores prognósticos de sobrevida global em pacientes com adenocarcinoma pancreático submetidos a quimioterapia paliativa. Seu uso como preditor de ressecabilidade das lesões pancreáticas não foi demonstrado. / Background: Pancreatic adenocarcinoma is responsible for most of the pancreatic neoplasias and it is associated to an extremely poor prognosis due to diagnosis in advanced stage and the recurrence even among patients treated with curative intention. A prognostic tool is essential for a better risk stratification. Evidence has shown that systemic inflammatory response is associated to the prognosis of a variety of cancers and the neutrophil/lymphocyte ratio (NLR) and adaptations and the platelet/lymphocyte (PLR) ratio seem promising for this purpose. Objetive: The objective of this study is to evaluate the prognostic value of NLR, derived NLR (dNLR) and PLR determined by blood counts collected at hospital admission and after palliative chemotherapy in patients with pancreatic adenocarcinoma, analyzing the ideal cutoff value for each parameter. Also, we intend to investigate if those ratios have some role in predicting the resectability of pancreatic adenocarcinoma. Methods: Data were collected of patients who had diagnosis of pancreatic adenocarcinoma confirmed by histopathologic exam in Hospital de Clínicas de Porto Alegre between 2003 and 2013. The studied ratios were determined by blood counts collected at hospital admission and after two cycles of chemotherapy in patients submitted to palliative treatment. Results: In the combined analysis including all patients, basal NLR, dNLR and PLR did not have prognostic impact in overall survival (P=0,394, P=0,152, P=0,177 respectively). In subgroup analysis of patients submitted to palliative chemotherapy, NLR, dNLR and PLR determined by blood count collected after two cycles of chemotherapy were prognostic for overall survival (P=0,003, P=0,009, P=0,001 respectively). The ideal cutoff values found were 4,11 for NLR (sensibility 83%, specificity 75%), 2,8 for dNLR (sensibility 87%, specificity 62,5%) and 362 for PLR (sensibility 91%, specificity 62,5%). None of these ratios has shown to be able to predict resectability (NLR, P=0,88; dNLR, P=0,99; PLR, P=0,64). Conclusions: NLR, dNLR and PLR are useful as prognostic markers of overall survival in patients with pancreatic adenocarcinoma submitted to palliative chemotherapy. Its use as resectability predictor could not be demonstrated.

O adenocarcinoma de ducto pancreático é reconhecido mundialmente como uma doença extremamente agressiva a qual apresenta um prognóstico desfavorável para pacientes sem cirurgia de ressecção. Portanto, é fundamental ampliar o conhecimento sobre os mecanismos biológicos envolvidos no câncer pancreático a fim de permitir a identificação de marcadores moleculares e alvos terapêuticos com o intuito de melhorar o diagnóstico precoce e tratamento. Os fatores de transcrição, reconhecidos por serem os efetores finais de vias de sinalização, regulam diversas funções celulares e, alterações na expressão transcricional destes podem contribuir para a transformação celular bem como para a progressão tumoral. Assim, o objetivo do presente estudo foi identificar os reguladores mestres da transcrição, possivelmente envolvidos no câncer pancreático. Para tanto, utilizamos dados de microarranjos para associar os reguladores mestres com o fenótipo tumoral. As análises foram realizadas no ambiente estatístico R utilizando os pacotes RTN, Limma e Survival. O gene TULP3 foi identificado como um regulador mestre da transcrição em amostras de câncer pancreático. O valor prognóstico de TULP3 foi verificado através de análises de sobrevivência em três coortes independentes. Estas análises revelaram que pacientes com adenocarcinoma pancreático, exibindo altos níveis de expressão do gene TULP3, apresentam uma sobrevida global desfavorável. Os altos níveis transcricionais de TULP3 podem desempenhar um papel fundamental na progressão do adenocarcinoma pancreático e conduzir a um resultado clínico desfavorável. Contudo, este estudo destaca a potencial aplicação de TULP3 como um biomarcador de prognóstico clínico para pacientes com adenocarcinoma pancreático. / Pancreatic ductal adenocarcinoma is world-wide recognized as an aggressive disease with poor prognosis in patients who did not undergo resection. Efforts to better comprehend the biological mechanisms of pancreatic cancer are needed to enable the identification of novel molecular markers and therapeutic targets for improving early diagnosis and treatment. Transcription factors are the final effectors of signaling pathways and regulate a number of cellular functions. Changes in expression of transcription factors may contribute to cellular transformation and tumor progression. Thus, the aim of the present study was to identify transcriptional master regulators potentially involved in pancreatic cancer disease. To achieve this goal, we utilized microarray data to associate master regulators with tumor phenotype. Analyses were performed with RTN, Limma, and Survival packages at R environment. We identified TULP3 as a master regulator of transcription in pancreatic cancer samples. TULP3 prognostic value was accessed in three independent cohort analyses. Our data demonstrate that patients with pancreatic cancer, exhibiting high TULP3 transcriptional levels, show a poor overall survival. High levels of TULP3 expression may play an essential role in pancreatic cancer progression and lead to poor clinical outcome. Our results highlight the potential use of TULP3 as a clinical prognostic biomarker for pancreatic adenocarcinoma.

O adenocarcinoma de ducto pancreático é reconhecido mundialmente como uma doença extremamente agressiva a qual apresenta um prognóstico desfavorável para pacientes sem cirurgia de ressecção. Portanto, é fundamental ampliar o conhecimento sobre os mecanismos biológicos envolvidos no câncer pancreático a fim de permitir a identificação de marcadores moleculares e alvos terapêuticos com o intuito de melhorar o diagnóstico precoce e tratamento. Os fatores de transcrição, reconhecidos por serem os efetores finais de vias de sinalização, regulam diversas funções celulares e, alterações na expressão transcricional destes podem contribuir para a transformação celular bem como para a progressão tumoral. Assim, o objetivo do presente estudo foi identificar os reguladores mestres da transcrição, possivelmente envolvidos no câncer pancreático. Para tanto, utilizamos dados de microarranjos para associar os reguladores mestres com o fenótipo tumoral. As análises foram realizadas no ambiente estatístico R utilizando os pacotes RTN, Limma e Survival. O gene TULP3 foi identificado como um regulador mestre da transcrição em amostras de câncer pancreático. O valor prognóstico de TULP3 foi verificado através de análises de sobrevivência em três coortes independentes. Estas análises revelaram que pacientes com adenocarcinoma pancreático, exibindo altos níveis de expressão do gene TULP3, apresentam uma sobrevida global desfavorável. Os altos níveis transcricionais de TULP3 podem desempenhar um papel fundamental na progressão do adenocarcinoma pancreático e conduzir a um resultado clínico desfavorável. Contudo, este estudo destaca a potencial aplicação de TULP3 como um biomarcador de prognóstico clínico para pacientes com adenocarcinoma pancreático. / Pancreatic ductal adenocarcinoma is world-wide recognized as an aggressive disease with poor prognosis in patients who did not undergo resection. Efforts to better comprehend the biological mechanisms of pancreatic cancer are needed to enable the identification of novel molecular markers and therapeutic targets for improving early diagnosis and treatment. Transcription factors are the final effectors of signaling pathways and regulate a number of cellular functions. Changes in expression of transcription factors may contribute to cellular transformation and tumor progression. Thus, the aim of the present study was to identify transcriptional master regulators potentially involved in pancreatic cancer disease. To achieve this goal, we utilized microarray data to associate master regulators with tumor phenotype. Analyses were performed with RTN, Limma, and Survival packages at R environment. We identified TULP3 as a master regulator of transcription in pancreatic cancer samples. TULP3 prognostic value was accessed in three independent cohort analyses. Our data demonstrate that patients with pancreatic cancer, exhibiting high TULP3 transcriptional levels, show a poor overall survival. High levels of TULP3 expression may play an essential role in pancreatic cancer progression and lead to poor clinical outcome. Our results highlight the potential use of TULP3 as a clinical prognostic biomarker for pancreatic adenocarcinoma.

<p>Pancreatic ductal adenocarcinoma (PDAC) is an incredibly lethal disease with a 5-year survival rate of less than 8 percent in the United States due to a lack of viable treatment options. The failures of chemo- and radiotherapies have been linked to the heterogeneous nature of the tumor microenvironment which forms a hypovascular, immunosuppressive and high coagulation activity tissue. Indeed, PDAC patients have one of the highest rates of thrombosis complications among all cancer types. The expression of two key coagulation factors, Tissue Factor (TF) and Protease Activated Receptor 1 (PAR-1), have been associated with poor patient prognosis and aggressive cancer progression. However, the molecular roles/mechanisms of TF and PAR-1 in PDAC progression are not known. To establish how clotting factors (PAR-1, TF) influence PDAC tumor progression, I utilized a genetically modified mouse model (KPC) where <i>KRas^G12D</i> and <i>TRP53^R172H</i> mutations were specifically introduced into mouse pancreas acinar cells to initiate PDAC progression. Multiple primary mouse PDAC cell lines were generated and characterized. TF and PAR-1 were highly expressed in primary KPC pancreatic lesions, in PDAC tumors, and in KPC-derived cell lines, an expression profile that is also observed in PDAC patient biopsies. In allograft studies, tumor growth and metastatic potential were significantly diminished by shRNA reduction of TF or PAR-1 in cancer cells or by genetic or pharmacological reduction of the coagulation zymogen prothrombin in mice. Notably, PAR-1 deleted KPC cells (KPC-Par-1^KO) failed to generate sizable tumors; a phenotype completely rescued by restoration of PAR-1 expression. To test the significance of targeting PAR-1 in a clinical setting, PAR-1 expression was withdrawn from established tumors to mimic a potential inhibitory effect of PAR-1 on solid PDAC tumors. Removal of PAR-1 from tumors (11 days post injection) yielded a diverse effect on tumor growth which can be categorized into (i) a decline in tumor growth; (ii) continued tumor growth; and (iii) stagnant tumor growth. Immunohistochemistry analysis of KPC2 shCon vs. shPar-1 subcutaneous allograft tumor samples revealed a massive immune cell infiltration in KPC2 shPAR-1 tumors when compared to KPC2 shCon control tumors. Accordingly, KPC-Par-1^KO cells failed to form tumors in immune-competent mice but displayed robust tumor growth in immune-compromised <i>NSG</i> mice, providing the first evidence of a PAR-1 mediated tumor immune evasion pathway operating in PDAC. </p> <p>Together, these results demonstrate that PDAC disease is driven by activation of the coagulation system through tumor cell-derived TF, circulating prothrombin, and tumor cell-derived PAR-1. These studies also highlight a novel mechanism by which thrombin/PAR-1-mediated tumor growth involves suppression of anti-tumor immunity in the tumor microenvironment. <b></b></p>

This study examines the role of innate and adaptive immunity in the immunotherapy based on the combination of the ligands stimulating phagocytosis anchored in the tumour cells membrane and the mixture of TLR agonists. This immunotherapy is primarily focused on the innate immunity activation and induces strong inflammatory infiltration, which neutrophils and NK cells are part of. Therefore, the next aim of this study was to evaluate the anti-tumour activity of neutrophils and NK cells. For examination malignant melanoma and pancreatic adenocarcinoma mouse tumour models were used.

The aim of this thesis is to improve the therapeutic effect of the immunotherapy based on the synergy of TLR agonists with phagocytosis stimulating ligands. Furthermore, this thesis is focused on the information transfer to the specific immunity, as well as it pursues the study of the specific immunity relevance during cancer immunotherapy.

The aim of this thesis was to study how to increase effectiveness of cancer immunotherapy based on synergy of compounds stimulating phagocytosis and TLR agonist. Tumor microenvironment was modified by enzymes, which catalised conversion of lactate to pyruvate or acetate. It was monitored effect of enzymes on tumor size, survival of experimental mice and cytotoxicity on tumor cells.

Les ultrasons focalisés de haute intensité (HIFU) permettent la destruction de tissus biologiques par élévation de la température. Cette technique reconnue est utilisée actuellement dans le monde médical afin de traiter certaines masses tumorales. Le projet décrit dans ce document détaille la mise au point et l'utilisation de deux systèmes thérapeutiques indépendants, ayant pour objectif principal le traitement peropératoire puis extracorporel des tumeurs hépatiques. Dans un premier temps, un système chirurgical existant, destiné au traitement des métastases hépatiques et en cours d'évaluation clinique, a été utilisé. La mise en place d'une modalité de traitement par focalisation électronique a permis d'augmenter le volume de coagulation initial et ainsi faciliter la procédure opératoire. Basée sur les conclusions de cette première étude, une seconde version de sonde peropératoire a été modélisée puis développée. La géométrie du transducteur utilisé a permis une modification de la forme des lésions produites. Les performances de cette sonde de traitement ont été évaluées numériquement puis validées lors d'expérience in vitro et in vivo. L'efficacité, la simplicité et la reproductibilité des traitements réalisés sur le foie ont conduit à une possible application du dispositif aux cancers du pancréas. Après étude numérique et évaluation de la faisabilité in vitro, une validation sur modèle animal a été entreprise. L'ensemble des résultats obtenus au cours de ces différents traitements peropératoires a permis d'envisager la faisabilité d'un dispositif de traitement par voie extracorporelle. Une étude théorique a donné lieu à la réalisation d'un prototype expérimental. Après calibrations et étalonnages, des résultats in vitro préliminaires ont été obtenus / High intensity focused ultrasound (HIFU) allows the destruction of biological tissue by temperature increase. This technique is commonly used in the medical world for treating certain types of tumor masses. The project described in this document details the development and use of two independent therapy systems, with the main objective to treat intraoperatively and extracorporeally liver tumors. As a first step, an existing surgical system, intended to treat liver metastases and under clinical evaluation, was used. The establishment of a treatment modality based on electronic focusing has contributed to increase the coagulation volume and thus simplify the operative procedure. Based upon the findings of this first study, a second version of intraoperative probe was modeled and developed. The geometry of this new transducer allowed to change the shape of produced ablations. The performance of this probe were evaluated numerically and then validated with in vitro and in vivo studies. The effectiveness, simplicity and reproducibility of the treatments performed in the liver led to a possible application of the device to pancreatic cancer. After numerical study and in vitro feasibility assessment, animal model validation was also undertaken. All the results obtained during the peroperative treatments was used to study the feasibility of an extracorporeal treatment. A theoretical study has led to the development of an experimental prototype. After calibration, preliminary in vitro results were obtained

<p dir="ltr">Pancreatic ductal adenocarcinoma (PDAC) remains one of the most deadliest cancer and despite advancements in cancer therapy, remain highly refractory to treatment, largely due to its desmoplastic tumor microenvironment (TME) characterized by complex interactions among cancer cells and stromal components. Particularly, the PDAC associated coagulation system due to leaky tumor vasculatures plays a pivotal role in reshaping the PDAC stroma and its pathogenesis. Understanding the intricate interplay between tumor cells, stromal cells, and the elevated coagulation pathway elements, including tissue factor, thrombin, and fibrin, is essential for developing effective therapeutic strategies. To address these challenges, this research proposes the engineering of a novel PDAC-associated coagulation system using a microfluidic technology, known as coagulation-on-tumor-microenvironment-on-chip (cT-MOC). The study aims to integrate key coagulation pathways in cT-MOC to investigate pivotal interactions in the PDAC stroma: <i>i)</i> thrombin-protease-activated receptors (PARs) mediated promotion of PDAC fibrosis via activation of cancer-fibroblast cross-talk; <i>ii)</i> in-depth analysis of transport and mechanical properties of collagen-fibrin microstructure; <i>iii)</i> inhibited drug delivery in reprogrammed PDAC stroma due to pronounced fibrin deposition on collagen. By leveraging innovative microfluidic technologies and comprehensive experimental approaches, the research endeavors to provide a novel platform that bridges traditional <i>in vitro</i> and <i>in vivo</i> models to overcome the challenges posed by the desmoplastic TME and enhance therapeutic strategies for treatment by targeting the coagulation at the PDAC TME.</p>