La protéine de stress p8 fut découverte et caractérisée dans notre laboratoire. Cette protéine est surexprimée dans l'adénocarcinome pancréatique et possède un rôle dans la progression tumorale. Pendant ce travail de thèse, nous nous sommes focalisé sur le rôle de p8 dans les phases initiales de la tumorigénèse pancréatique et sur ses mécanismes d'action, impliqués dans la progression tumorale. Nous avons montré que p8 joue un rôle essentiel dans l'apparition des lésions PanINs suite à l'activation de l'oncogène Kras. Au niveau cellulaire, nous avons montré que la protéine p8 protège les cellules tumorales pancréatiques contre le stress de la privation en nutriments via la régulation de l'expression de RelB et IER3. La voie p8, RelB et IER3 est impliquée dans l'inhibition de l'apoptose suite au stress. De plus, nous avons observé que ces trois protéines sont co-exprimées dans les adénocarcinomes humains et leur expression corrèle avec l'évolution de la maladie. Dans un second lieu, nous avons démontré le rôle essentiel de la protéine p8 dans la résistance des cellules tumorales pancréatiques à l'hypoxie et à la privation en glucose. Nous avons identifié la voie p8/Aurora KinaseA, qui en réponse au stress métabolique, réduit l'apparition des dommages à l'ADN en contrôlant l'expression des gènes liés au cycle cellulaire et à la réparation de l'ADN. De plus, nos recherches ont montré que p8 protège les cellules tumorales du stress métabolique en inhibant la mort cellulaire dépendante de l'autophagie. Nous espérons que nos résultats aideront à mieux cibler les cellules tumorales pancréatiques et leur caractère résistant au stress micro-environnemental extrême. / The stress protein p8 was discovered and characterized in our laboratory. Over expressed in pancreatic adenocarcinoma, p8 is involved in tumor progression. During my PhD studies, we focused on the role of p8 in pancreatic cancer development and on its mechanisms of action. First, we demonstrated that p8 is essential for PanIN development following Kras oncogene activation. At the cellular level, we found that p8 protects pancreatic cancer cells upon nutrient starvation stress through the regulation of RelB and IER3 expression. p8, RelB and IER3-dependent cascade inhibits apoptosis after the starvation stress. Furthermore, we showed that these tree proteins are co expressed in human pancreatic adenocarcinoma. One the other hand, our study showed that p8 is involved in pancreatic cancer cells resistance to hypoxia and glucose starvation. We identified a p8/Aurora KinaseA pathway which, in response to such metabolic stress, reduces DNA damage by regulating cell cycle and DNA repair genes expression. Moreover, our studies demonstrated that p8/AURKA path protects cancer cells against metabolic stress by inhibiting autophagy-associated cell death. We expect that our data will help to get new therapeutics against pancreatic cancer.

Les récentes avancées dans notre compréhension de la tumorigenèse pancréatique ont montré que la présence d'un compartiment cellulaire non tumoral : le « stroma » ou « microenvironnement intra-tumoral », avait une incidence directe sur la progression de la maladie. Le but de ce travail était de déterminer le rôle du stroma dans la progression de l'adénocarcinome pancréatique. Pour cela, nous avons étudié le profil d'expression génique spécifique du stroma. Ceci nous a permis de mettre en évidence des gènes impliqués dans la régulation du système nerveux, dénommés « facteurs neurogéniques », pouvant être reliés aux phénomènes de remodelage neural observés dans les adénocarcinomes pancréatiques. Ceux-ci sont associés aux douleurs caractéristiques du cancer du pancréas, aux récidives locales, à l'extension locorégionale. Nous avons alors approfondi notre étude sur l'axe SLIT2/ROBO. Nos résultats montrent qu'au travers la sécrétion de Slit2, le stroma a un impact direct sur le remodelage neural. Ces données peuvent permettre d'ouvrir une nouvelle voie thérapeutique dans le cancer du pancréas, ayant pour but de cibler les conséquences du remodelage neural. / Recent progress in our understanding of pancreatic tumorigenesis had shed light on the non tumoral cell compartment of the tumor, so-called "stroma" or "intra-tumoral microenvironment", in the spread of the disease. The goal of our work was to decipher the role of stroma in the spread of this disease. We could analyze the specific gene expression profile of stroma, leading to the discovery of several genes plausibly linked to neural remodeling, called "neurogenic factors". This neural remodeling is clinically correlated with neuropathic pain and locoregional spread. We have next deepened our analysis on the axis SLIT2/ROBO. We could demonstrate that stromal compartment is able to impact on neural remodeling, through secretion of Slit2. These results provide rationale to investigate the disruption of stromal/neural compartment connexion with Slit2/ROBO inhibitors for treatment of pancreatic cancer reccurrence and pain.

Dans les conditions normales, les organismes maintiennent une masse cellulaire endocrine stable tout au long de leur vie. En cas d’obésité, la masse de cellules b pancréatiques est capable de maintenir des taux de glucose plasmatique en augmentant la sécrétion en insuline. L’incapacité de ces cellules à fournir de l’insuline entraîne alors l’apparition d’une hyperglycémie et d’un diabète de type II. Cliniquement, la majorité des individus obèses ne développent pas de diabète car les îlots pallient à cette résistance à l’insuline. La preuve de l’adaptation de la masse d’îlots humains à l’obésité, in vivo, n’a pas été clairement décrite et, de plus, peu d’informations existent sur les mécanismes et les types cellulaires impliqués. Actuellement, la mise en évidence de l’augmentation de la masse des cellules b chez les humains obèses repose uniquement sur des études histologiques.But : Au cours de cette thèse, nous avons cherché, dans un premier temps (partie 1), à évaluer la morphologie des îlots pancréatiques et la distribution des cellules a et b chez des donneurs en état de mort cérébrale normaux, en surpoids et obèses. Dans un second temps (partie 2), nous avons étudié l’adaptation au cours du temps des îlots humains à un environnement obésogène. Nous avons montré ainsi que les îlots humains non diabétiques s’adaptent in vivo à l’obésité en modifiant la masse de cellules b, leur fonction et leur expression génique. En suite (partie 3), on a identifié le mécanisme de transdifférenciation des cellules alpha et beta en utilisant la méthode de lineage tracing. Finalement (partie 4), on a déterminé la différence sur l’expression de gène des ilots humains greffé chez les souris sous régime control ou régime riche en graisse en utilisant les puces d’ARN (Illumina).Methodes et Resultats: Des coupes de pancréas humains inclues en paraffines ont été analysées. Les donneurs obèses étaient caractérisés par une augmentation de la masse endocrine totale, de la taille des îlots, de la graisse intra-pancréatique et du ratio b:a dans les îlots, mais avec une diminution du ratio a:b dans les îlots. Au cours de l’étude longitudinale, des souris Rag2-/- non diabétiques ont été greffées sous la capsule rénale avec des îlots humains issus de donneurs en état de mort cérébrale (donneurs non diabétiques ou donneurs avec un dysfonctionnement métabolique déclaré). Les animaux ont été nourris pendant 2 semaines avec soit un régime contrôle soit un régime riche en graisse (high fat diet HFD). Un suivi du poids, du taux des triglycérides, de la glycémie et du C-peptide a été mis en place. Après sacrifice des souris, les greffons et les pancréas endogènes ont été analysés pour le volume endocrine, la distribution des cellules b et a et les mécanismes de régénération des cellules pancréatiques. Après 12 semaines sous régime gras, les souris montraient toutes les caractéristiques typiques de l’obésité, à savoir, une augmentation du poids, un doublement de la graisse abdominale, des triglycérides, de la glycémie et une sensibilité à l’insuline réduite. De plus, l’apparition sur ces animaux d’un doublement rapide de la quantité de C-peptide humain dans le sérum murin nous indique la mise en place d’une compensation fonctionnelle. Une analyse histologique des greffons a permis de mettre en évidence une adaptation de la masse endocrine des îlots avec une augmentation des cellules b. D’autres analyses ont identifié la prolifération et la néogénèse comme les mécanismes responsables de ce doublement de la masse endocrine humaine.Discussion: Ce nouveau modèle animal permet d’étudier, in vivo sur une longue période, l’adaptation des îlots humains à un environnement obésogène murin. Il peut être utilisé comme un outil dans le décryptage des voies de signalisation impliquées dans l’expansion des cellules b humaines et permettre également l’identification des facteurs prédisposant ces cellules à subir une décompensation. / Under normal healthy conditions, organisms maintain a dynamic endocrinecell mass throughout life. Pancreatic beta cell mass are able to maintain plasma glucose levels increasing insulin secretion in conditions as obesity.Beta cell inability to compensate in insulin demand provokes hyperglycemia and Type 2 Diabetes. Clinically, most obese individuals do not develop diabetes because islets compensate for insulin resistance. Direct evidence that human islet mass adapts longitudinally to obesity in vivo was lacking and, moreover, little information was available on the mechanismsand cell type(s) involved.Current evidence for increased beta cell mass in obese humans (vs lean) is based entirely on postmortem histology.Aim: In this thesis, firstly (Part 1) we performed a descriptive cross sectional study by evaluating the pancreatic islet morphology and alpha and beta cell distribution from our archived human pancreatic sections of obese and normal subjects. Secondly, (Part 2) we explored the longitudinal adaptation of human islets to an obesogenic environment and showed direct evidence that non-diabetic human islets adapt bothendocrine and beta cell mass, function and gene expression to obesity in vivo. Thirdly (Part 3) we performed lineage tracing to determine which cell type alpha or beta give rise to the increase islet mass in obesity. Finally (Part 4) in this diet induced obesity model we developed, we looked at the differential gene expression with Illumina gene chips in a kinetic study on human islets which were laser capture microdissected at 6, 8 and 10 weeks on control or high fat diet.Methods: Archived human pancreatic sections were immunostained for endocrine, beta, alpha, fat. In the obese/immunodeficient mouse model, non-diabetic Rag2–/– mice were transplanted under kidney capsule with human islets from human brain-deceased donors (non-diabetics donors and donors with overt metabolic dysfunction). Animals were fed for 12 weeks with a control or high-fat diet (HFD), and followed for weight, serum triacylglycerol, fasting blood glucose and human C-peptide. After the mice were killed, human grafts and the endogenous pancreas were analyzed for endocrine volume, distribution of beta and alpha cells, and mechanisms of regeneration.Results: The cross-sectional study, performed on archived human paraffin embedded sections of normal weight, overweight, or obese subjects showed that obese donors were characterized by an increased total endocrine mass, bigger individual islet size, increased intrapancreatic fat, increased β to  cell ratio and decreased :β cell ratio in islets. In the longitudinal study, concomitant with the increased weight gain, doubling of abdominal fat, increased serum triacylglycerol and reduced insulin sensitivity in 12 week HFD animals we reported that human islet grafts showed functional compensation, measured as a more than doubling of fasting human C-peptide in mouse serum, and histological adaptation of islet endocrine mass including increased beta cells. Further analysis of the human grafts revealed proliferation and neogenesis as the responsible mechanisms for the doubling of the human endocrine mass.Discussion: This novel model allows, for the first time, longitudinal studies of human islet adaptation to an obese murine environment and may be instrumental in deciphering pathways involved in human beta cell expansion, as well as in helping to identify factors predisposing human beta cells to undergo decompensation.

Helicoverpa armigera et Epiphyas postvittana, deux insectes nuisibles pour l'agriculture, ont développé des résistances contre la plupart des insecticides connus. Les lipides étant les constituants majeurs de leur régime alimentaire, les fonctions digestives des lipases deviennent alors une cible privilégiée pour l'élaboration de nouveaux insecticides. L'identification récente de gènes codant pour l'expression de protéines potentiellement actives et inactives apparentées aux lipases pancréatiques (PLRP) dans le tractus digestif de ces deux insectes et dont le niveau de transcription varie en fonction de leur régime alimentaire a ouvert un nouveau champ de recherche. Dans le but de contribuer à cette thématique, nous avons construit cinq lipases recombinantes d'E. postvittana (EpLIPs) et testé leur expression dans trois systèmes différents (E.coli, P.pastoris et bacculovirus). Le tractus digestif de Helicoverpa armigera a été étudié par chromatographie échangeuse d'ions et les différentes protéines séparées ont été testées en spectrométrie de masse et sur pHstat pour l'activité. Les résultats obtenus ont permis de mettre en évidence, pour la première fois, la présence à la fois d'une lipase active et d'une lipase inactive dans le tractus digestif d'un lépidoptère. Par ailleurs, la caractérisation biochimique d'un mutant GPLRP2-Δ β9 a été faite dans le but de comprendre l'effet de cette boucle, partiellement délétée dans les lipases d'insectes, dans la spécificité du substrat. Nous avons pu montrer que cette boucle β9 est essentielle pour la stabilisation de la chaîne acyle durant la réaction de lipolyse. / Helicoverpa armigera and Epiphyas postvittana, two major pest crops, have developed resistances against most of the known insecticides. Lipids being a major component of insect diet, digestive function of lipase are a target of choice for new insecticide design. The recent identification of active and inactive pancreatic lipase related protein (PLRP) genes in those two insects midgut, with a level of transcription depending on the diet, opened the field of insect digestive lipase study. In order to contribute to this thematic, we built five recombinants lipase from E. postvittana (EpLIPs) and tested their expression in three different systems (E.coli, P.pastoris and bacculovirus). Protein structure prediction of EpLIPs allowed us to develop some functional hypothesis enlightening the role of inactive lipase in lipid transport. H. armigera midgut contents were separated through a one step purification chromatography and the different fractions were tested for activity and mass spectrometry. The results obtained gave the first evidence of the presence of both an active and an inactive lipase in lepidopteran midgut. In addition to this work, a biochemical characterisation of a β9 GPLRP2 mutant was carried out to understand the effect of this loop, partially deleted in insect lipase, in substrate specificity. The result shows that β9 loop is essential for stabilizing the leaving acyl chain during the lipolysis reaction.

<div>Pancreatic cancer is the present third leading cause of all cancer-associated deaths with a under 9% 5-year survival rate. Aggressive tumor progression and lack of early detection technique lead to the fact that most patients are diagnosed at terminal stage - pancreatic ductal adenocarcinoma (PDAC). Despite that numerous therapeutic approaches have been introduced, most options cannot advance to or fail at the clinical trials. It has been suggested that previous failure is due to insufficient understanding of PDAC tumor microenvironment (TME). Human PDAC is composed of severely fibrotic tissue (i.e., desmoplasia) that harbors a variety of malignant cells (e.g., pancreatic stellate cells, cancer-associated fibroblasts, macrophages, etc.), excessive extracellular matrices (ECM), as well as abnormal expression of growth factors, cytokines, and chemokines. Multiple cell-cell and cell-ECM interactions jointly result in a stiffened, hypoxic, and fluid pressure-elevated PDAC tissue. The resulting pancreatic TME not only physically hinders penetration of therapeutics, but also dynamically interacts with the residing cells, regulating their behaviors.</div><div><br></div><div>Increasing tumor tissue stiffness in PDAC is not only a passive outcome from desmoplasia, but an active environmental factor that promotes tumor survival, growth, and invasion. However, traditional in vitro cell culture systems such as two-dimensional (2D) culture plate and animal models are not ideal for mechanistic understanding of specific cell-matrix interactions. Therefore, dynamic hydrogels have been introduced as a category of advanced biomaterials that exhibit biomimetic, adaptable, and modularly tunable physiochemical property. Dynamic hydrogels can be precisely engineered to recapitulate a variety of aspects in TME, from which to investigate the role of dynamic tumor-stroma interaction in PDAC progression. The goal of this dissertation was to exploit synthetic polymers (i.e., poly(ethylene glycol) (PEG)) or natural ECM (i.e., gelatin and hyaluronic acid (HA)) as precursors to prepare the dynamic cancer-cell laden gels. The design utilized the orthogonal thiol-norbornene photopolymerization to prepare the primary homogenous xxvi</div><div><br></div><div>gel network. Next, through further functionalizing gel precursors with phenolic derivatives, enzymatic reaction (i.e., tyrosinase) or flavin mononucleotide (FMN)-mediated photochemistry could be harnessed to manipulate the dynamic changes of substrate mechanics. Experimentally, a computational model and the associated validation were presented to investigate the process of gel stiffening. Finally, these techniques were integrated to prepare cell-laden gels with spatial-temporally tunable properties that were instrumental in exploring the synergistic effects of dynamical matrix stiffening and presence of HA in promoting epithelial-mesenchymal transition (EMT) in PDAC cancer and stromal cells.</div>

OBJECTIVE: Cardiovascular disease (CVD) remains the leading cause of death globally. Reducing high blood cholesterol, which is a dominant risk factor for CVD events, is an essential goal of medical treatment. Statins are known as first‐choice agents. However, clinical trials report that some statins increased the risk for type 2 diabetes (T2D). Our objective was to investigate the effect of different statins on insulin secretion and content from pancreatic β-cells after chronic and acute exposure and determine the underlying mechanisms. METHODS: The effects of simvastatin, lovastatin and pitavastatin on GSIS and content were studied in clonal pancreatic β-cells (INS-1 832/13) cultured in high glucose (12 mM). Insulin content and secretion were measured after chronic and acute incubation of statins using homogenous time-resolved fluorescence (HTRF) insulin assay kit (Cisbio). Intracellular Ca2+ was measured using fura-2 AM (Invitrogen). RESULTS: Simvastatin (25-200 nM) and lovastatin (50-200 nM) significantly inhibited GSIS and depleted insulin content in a dose-dependent manner after 72-hour exposure. When the secretion level was normalized for content, the inhibitory effect was not observed. Simvastatin (200 nM) also increased the amplitude of intracellular Ca2+ oscillations at low glucose, but this was not reflected in the amplitude of oscillatory insulin release. In contrast, pitavastatin (25-200 nM) did not affect GSIS and only decreased insulin content at the highest dose tested. CONCLUSION: Inhibition of GSIS by simvastatin and lovastatin could be due to depletion of insulin content. Decreased Ca2+ sensitivity may also contribute to inhibition of GSIS by simvastatin. Pitavastatin had less inhibitory effect on GSIS and insulin content as compared to simvastatin and lovastatin indicating that not all lipophilic statins have a detrimental impact on GSIS. We suggest that statins may have differential mechanistic effects on β-cells some of which may contribute to the risk of T2D.

Les pertes de la fonction et de la masse des cellules beta pancréatiques jouent un rôle central dans le diabète de type 2 (DT2). Les beta-arrestines 1 et 2 (ARRB1 et ARRB2), sont impliquées dans la sécrétion et/ou la survie des cellules beta pancréatiques.Dans une première étude, afin de caractériser précisément la fonction d’ARRB1 dans les cellules beta pancréatiques, nous avons eu pour objectif de générer des souris invalidées spécifiquement dans ces cellules en utilisant le système Cre/lox sous le contrôle du promoteur Ins1. Des études avaient été publiées à partir des deux lignées Ins1Cre-/+ et Arrb1f/f. Nous avons généré et travaillé sur les souris Arrb1f/f :Ins1Cre-/+. Le phénotype des souris Arrb1f/f :Ins1Cre-/+ était faible et surtout non reproductible comparé aux souris Arrb1f/f :Ins1Cre-/- utilisées comme témoins. Le faible niveau d’expression d'Arrb1 dans les cellules beta et le manque d'anticorps spécifique pour l'immunocytochimie ont rendu difficile la vérification de l'absence d'expression de ARRB1 dans ces cellules. Après séquençage du gène modifié Arrb1 des souris “floxées“, nous avons pu montrer que l'insertion du premier site loxP avait induit un décalage du cadre de lecture introduisant un codon stop et, par conséquent, la non-expression du gène Arrb1. Étant donné que les souris Arrb1 “floxées“ utilisées comme témoins étaient déjà knockout (KO), le projet utilisant ces souris a dû être arrêté.Notre équipe a rapporté l'implication d'ARRB2 dans la régulation de la masse des cellules bêta pancréatique, mais son rôle dans la signalisation du récepteur du Glucagon-Like Peptide-1 (GLP-1R), une cible thérapeutique majeure du DT2, n'avait pas encore été exploré.Nous avons montré, dans une deuxième étude, une meilleure tolérance orale au glucose ainsi qu’une augmentation de la sécrétion d’insuline chez les souris Arrb2 KO par rapport aux souris témoins sur les îlots en présence des concentrations physiologiques circulantes de GLP-1. Ceci est corrélé à une production d’AMPc et un recrutement de la PKA plus élevés dans les cellules beta Arrb2 KO. A l’inverse, l’activation des kinases ERK1/2 est diminuée indiquant un recrutement majeur des ERK1/2 par ARRB2 au GLP-1R. En parallèle, j’ai montré que les taux de ARRB1 et ARRB2 des îlots pancréatiques sont altérés par des conditions diabétogènes et diabétiques. Mes résultats démontrent clairement un rôle critique de ARRB2 dans la signalisation du GLP-1R. Un défaut d’expression de la protéine pourrait participer au déficit des mécanismes de compensation de la masse fonctionnelle des cellules beta conduisant au DT2. / The loss of function and mass of pancreatic beta-cells play a central role in type 2 diabetes (T2D). Beta-arrestin 1 and 2 (ARRB1 and ARRB2) are involved in insulin secretion and/or beta-cell survival. In a first study, in order to characterize the role of ARRB1 in beta-cells, we aimed to invalidate the Arrb1 gene specifically in these cells using the Cre/lox system under the control of the Ins1 promoter. Studies had been published with both Ins1Cre-/+ and Arrb1f/f lines. We generated Arrb1f/f:Ins1Cre-/+ mice. The phenotype of Arrb1f/f :Ins1Cre-/+ mice was weak with a lack of reproducibility compared to Arrb1f/f :Ins1Cre-/- mice used as controls. The low expression level of Arrb1 in beta-cells and the lack of specific antibody for immunocytochemistry made it difficult to verify the absence of expression of ARRB1 in these cells. After sequencing the modified Arrb1 gene of the “floxed” mice, we observed that the insertion of the first loxP site induced a shift in the reading frame introducing a stop codon and, consequently, the non-expression of the Arrb1 gene. Since the “floxed“ Arrb1 mice used as controls were already knockout (KO), the project using these mice was stopped.Our team has reported the involvement of ARRB2 in the regulation of beta-cell mass, but its role in Glucagon-Like Peptide-1 (GLP-1) receptor signaling, a major therapeutic target for T2D, remained to be explored. In a second study, we showed a better glucose tolerance and an increase in insulin secretion from isolated islets in Arrb2KO compared to control mice in the presence of physiological circulating concentrations of GLP-1. This was correlated with higher cAMP production and PKA activation in Arrb2KO beta-cells. By contrast, the activation of ERK1/2 kinases was decreased indicating a major recruitment of ERK1/2 by ARRB2 to GLP-1R. In parallel, we showed that the expression levels of ARRB1 and ARRB2 in pancreatic islets were altered in diabetogenic and diabetic conditions. My results clearly demonstrate a critical role of ARRB2 in GLP-1R singaling which could impact the function, maintenance and plasticity of beta-cell mass in response to GLP-1. A lack of expression of ARRB2 could participate in the deficit of compensatory mechanisms of the functional beta-cell mass leading to T2D.

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

O adenocarcinoma de ducto pancreático (PDAC) é a quarta causa de morte em decorrência de neoplasias nos países ocidentais. Atualmente, a cirurgia ressectiva é a única possibilidade de cura para a doença, porém, a recidiva tumoral acontece em menos de um ano após a intervenção cirúrgica, mesmo com a quimioterapia adjuvante. A terapia fotodinâmica (PDT) é uma alternativa promissora no tratamento do câncer. No entanto, pouco se sabe sobre o uso da PDT em tumores pancreáticos. Portanto, o objetivo deste trabalho foi avaliar a eficiência da PDT com o azul de metileno (MB) como fotossensibilizador (MB-PDT) em induzir a morte de linhagens de PDAC humanas (AsPC-1, Panc-1, MIAPaCa-2 e BxPC-3) e estudar a contribuição de vias de necrose regulada nos efeitos citotóxicos da terapia sobre estes modelos. Os resultados obtidos mostraram que a MB-PDT foi capaz de induzir a morte massiva das células de PDAC. Além disso, eles indicaram que há dois perfis de susceptibilidade entre as quatro linhagens estudadas quando submetidas a MBPDT com 4,5 J/cm2 de energia e 6min de irradiação. De acordo com os dados apresentados, a diferença nas sensibilidades das linhagens à terapia não está associada à diferenças na capacidade de incorporação do MB ou na localização sub-celular do fotossensibilizador nas diferentes células, uma vez que a localização é, predominantemente, lisossomal em todas elas. Adicionalmente, mostrou-se que as linhagens menos susceptíveis ao tratamento, MIAPaCa-2 e Panc-1, apresentam níveis significativamente menores de RIPK3 e MLKL, dois dos componentes do necrossomo, essenciais para a execução da necroptose. Além disso, foi visto que a MB-PDT induz um aumento de fosforilação de MLKL em AsPC-1, demonstrando a ativação da necroptose após a terapia nestas células, mas não em MIAPaCa-2 (menos responsiva à terapia com 4,5 J/cm2 deenergia e 6min de tempo de irradiação). Ainda, a inibição da via de sinalização necroptótica diminuiu significativamente as porcentagens de morte das células mais susceptíveis (BxPC-3 e AsPC-1), não alterando a resposta de Panc-1 e MIAPaCa-2, corroborando a ativação e importância da necroptose para a citotoxicidade da MB-PDT. Finalmente, neste trabalho foi mostrado que o aumento do tempo de irradiação, mantendo-se a quantidade total de energia aplicada no tratamento, melhora a eficiência da MB-PDT em induzir a morte das células que apresentam limitações para executar a necroptose, sugerindo que mais de uma via de morte esteja sendo ativada após a terapia e que o tempo de irradiação atuaria modulando esta ativação. Complementarmente, foi mostrado que os tempos maiores de irradiação aumentam o estresse oxidativo intracelular que é acompanhado por uma diminuição significativa do conteúdo intracelular de glutationa reduzida (GSH), indicando, preliminarmente, que a ferroptose pode estar sendo acionada após os protocolos mais longos de irradiação. Coletivamente, os resultados apresentados neste trabalho confirmam a eficiência da MB-PDT no tratamento de diferentes linhagens de PDAC, indicando que a necroptose está sendo ativada e contribuindo para a citotoxicidade da terapia sobre as células que não apresentam resistência à esta via de morte. Ainda, eles demonstram que o aumento do tempo de irradiação pode transpor a barreira de resistência de algumas linhagens à terapia, provavelmente por induzir a ativação de outras vias de necrose regulada, mostrando a importância da otimização do protocolo de tratamento no aumento da eficiência da MB-PDT sobre os tumores de pâncreas. Finalmente, os resultados confirmam a MB-PDT como alternativa eficaz no tratamento do PDAC, apresentando um amplo espectro de atuação sobre subtipos tumorais resistentes à vias clássicas de morte celular, uma característica importante no contexto de uma terapia anti-cancer. / Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of death due to neoplasms in western countries. Currently, resective surgery is the only therapetical approach to cure this disease, but tumor´s recurrence occurs less than one year after the surgery, even with adjuvant chemotherapy. Photodynamic therapy (PDT) is a promising alternative for the cancer treatment. However, the efficacy of PDT to treat pancreatic tumors as well as the mechanisms involved in the induction of tumorigenic cell death remain unclear. For this purpose, in this study, we set out to evaluate the efficacy of PDT using methylene blue (MB) as a photosensitizer (MB-PDT), in inducing death of human PDAC derived cell lines (AsPC-1, Panc-1, MIAPaCa-2 and BxPC-3) and to deeper investigate the contribution of necroptosis to the cytotoxic effects of the therapy. We observed that MB-PDT was able to induce massive death of PDAC cells. Moreover, our results indicated that upon MB-PDT (4.5 J/cm2 energy and 6min of irradiation time), there were two susceptibility profiles among the four cell lines studied. Data also showed that this differential profile of cell response was neither associated with the differences in the MB incorporation capacity nor with the subcellular location of the photosensitizer, since the localization was predominantly lysosomal in all of tested cell lines. In addition, less susceptible cells, MIAPaCa-2 and Panc-1, showed significantly lower levels of RIPK3 and MLKL, two of the necrosome components, essential for triggering necroptosis. Furthermore, while MB-PDT (4.5 J/cm2 and 6min of irradiation) has been able to increase MLKL´s phosphorylation levels, an essential step in necroptosis induction, in AsPC-1cells, less responsive MIAPaCa-2 cells presented no variations on the phosphorylation state of this pseudokinase. Moreover, pharmacological inhibition of the necroptotic signaling pathway significantly decreased cell death percentages of the most susceptible cells (BxPC-3 andAsPC-1), without altering the response of Panc-1 and MIAPaCa-2, corroborating that activation of necroptosis was strongly involved in the cytotoxicity of MB-PDT. Finally, this work showed that increasing the irradiation time improved the efficacy of MB-PDT in killing cells which display limitations to perform necroptosis, suggesting that the irradiation time would be modulating the degree of oxidative stress generated and this stimuli would in turn, be responsible for triggering other regulated cell death pathways in a RIKP3 and MLKL independent way. Indeed, this increase in oxidative stress was accompanied by a significant decrease in GSH, a global indicatior of less antioxidant cell capacity, preliminarily pointing at the induction of ferroptosis by longer irradiation protocols. In summary, we demonstrated that MB-PDT is able to induce cell death in different PDAC cell lines and that different regulated cell death mechanisms are being activated upon MB-PDT induction. Furthermore, it was demonstrated that increased irradiation time may overcome the resistance barrier of some cell lines, probably inducing the activation of other regulated cell death pathways, showing the importance of optimizing the irradiation protocol in order to maximize the efficacy of the therapy. Finally, our observations point MB-PDT as an alternative and effective therapy for pancreatic cancer treatment, displaying a broad-spectrum action on tumors displaying different resistance mechanisms to classic cell death pathways, a desired property for improving an anticancer therapy.

Introdução: Os benefícios do emprego do selante de fibrina no reforço de anastomoses pancreatico-jejunais, após ressecção duodenopancreática, visando a redução da incidência de fístula pancreática pós operatória (FPPO), ainda são questionáveis. Objetivo: Avaliar a influência do emprego do selante de fibrina na anastomose pancreatico-jejunal, após duodenopancreatectomia, na incidência de fístula, bem como suas consequências clínicas e os custos hospitalares. Metodologia: Estudo retrospectivo de 62 pacientes consecutivos submetidos a duodenopancreatectomia, divididos em dois grupos: 31 pacientes utilizando o selante de fibrina (GCS) e 31 pacientes sem o emprego de selante (GSS). As variáveis estudadas foram agrupadas em epidemiológicas, clínicas, laboratoriais, com destaque para a incidência de fístula pancreática, classificada segundo a definição do International Study Group on Pancreatic Fistula, suas complicações pós operatórias catalogadas segundo a classificação de Clavien e suas repercussões na assistência e nos seus custos avaliados pelo método de absorção com rateio simples de todas as despesas, exceto a despesa com medicamentos, tratada de forma separada. Resultados: Os grupos foram homogêneos para os parâmetros epidemiológicos, clínicos, e laboratoriais e não foram registradas diferenças significativas na comparação da evolução pós operatória e dos indicadores assistenciais hospitalares. Por outro lado, os custos hospitalares foram mais elevados no GCS, em relação ao GSS (p<0,0001). Conclusão: O emprego do selante de fibrina, no reforço da anastomose pancreatico-jejunal, em pacientes submetidos a duodenopancreatectomias, nas condições estudadas, não melhorou os resultados clínicos e assistenciais e ainda aumentou os custos hospitalares. / Introduction: The benefits of fibrin sealant employment in strengthening pancreatico-jejunal anastomosis after duodenopancreatic resection, reducing the incidence of pancreatic fistula postoperative (PFPO) are still questionable. Objective: To evaluate the influence of the use of fibrin sealant in pancreatico-jejunal anastomosis after pancreaticoduodenectomy in the incidence of fistula and its clinical consequences and hospital costs. Methodology: A retrospective study of 62 consecutive patients who underwent pancreaticoduodenectomy, divided into two groups: 31 patients using fibrin sealant (GCS) and 31 patients without the sealant employment (GSS). The variables were grouped into epidemiological, clinical, laboratory, especially the incidence of pancreatic fistula classified as defined by the International Study Group on Pancreatic Fistula, their postoperative complications cataloged according to Clavien rating and its repercussions on care and its costs assessed by the absorption method with simple apportionment of all expenses except the expenditure on medicines, treated separately. Results: The groups were homogeneous for clinical, epidemiological and laboratory parameters and no significant differences were recorded in the comparison given postoperative progress and hospital assistance indicators. Moreover, hospital costs were higher in GCS, with respect to GSS (p <0.0001). Conclusion: The use of fibrin sealant in pancreatojejunal anastomosis after pancreaticoduodenectomy, in the studied conditions, did not improve the results of care and also increased hospital costs