Le cancer épithélial de l’ovaire (EOC) est le plus mortel des cancers gynécologiques. Cette maladie complexe progresse rapidement de façon difficilement décelable aux stades précoces. De plus, malgré une chirurgie cytoréductive et des traitements de chimiothérapie le taux de survie des patientes diagnostiquées aux stades avancées demeurt faible. Dans le but d’étudier l’EOC dans un contexte ex vivo, l’utilisation de modèles cellulaires est indispensable. Les lignées cellulaires d’EOC sont un outil pratique pour la recherche cependant, la façon dont l'expression des gènes est affectée en culture par comparaison à la tumeur d'origine n'est pas encore bien élucidée. Notre objectif était donc de développer et de caractériser de nouveaux modèles de culture in vitro qui réflèteront plus fidèlement la maladie in vivo. Nous avons tout d’abord utiliser des lignées cellulaires disponibles au laboratoire afin de mettre au point un modèle 3D de culture in vitro d’EOC. Des sphéroïdes ont été générés à l’aide de la méthode des gouttelettes inversées, une méthode pionnière pour la culture des cellules tumorales. Nous avons ensuite procédé à une analyse des profils d’expression afin de comparer le modèle sphéroïde au modèle de culture en monocouche et le modèle xénogreffe in vivo. Ainsi, nous avons identifié des gènes stratifiant les modèles tridimensionnels, tant in vivo qu’in vitro, du modèle 2D monocouche. Parmi les meilleurs candidats, nous avons sélectionné S100A6 pour une caractérisation ultérieure. L’expression de ce gène fût modulée afin d’étudier l’impact de son inhibition sur les paramètres de croissance des sphéroïdes. L’inhibition de ce gène a comme effet de réduire la motilité cellulaire mais seulement au niveau du modèle sphéroïde. Finalement, toujours dans l’optique de développer des modèles d’EOC les plus représentatifs de la maladie in vivo, nous avons réussi à développer des lignées cellulaires uniques dérivées de patientes atteintes d’EOC du type séreux, soit le plus commun des EOC. Jusque là, très peu de lignées cellulaires provenant de ce type de cancer et de patientes n’ayant pas reçu de chimiothérapie ont été produites. De plus, nous avons pour la première fois caractérise des lignées d’EOC de type séreux provenant à la fois de l’ascite et de la tumeur solide de la même patiente. / The epithelial ovarian cancer (EOC) is the most lethal of gynecological cancers. This complexe and heterogenous disease progresses rapidly and is almost asymptomatic in early stages. The survival rate of patients with late stage diagnosis remains low albeit cytoreductive surgery and chemotherapy. In order to study the EOC disease in an ex vivo context, the use of different cellular models is necessary. EOC cell lines derived from long-term passages of malignant ovarian cancers are useful tools for molecular and cellular research but it is not clear how culture conditions affect overall gene expression and oncogenic potential as compared to the original tumor. The main goal of this research was to develo and characterize new in vitro model systems that will recapitulate more closely some of the growth conditions encountered by tumor cells in vivo. In order to develop an in vitro tridimensional EOC spheroid model, we have used cell lines previously established in our laboratory. Spheroids were generated using the hanging droplet method, which was innovative for the culture of cancer cells. Comparative gene expression profile analysis of monolayer cultures, 3D spheroids and in vivo xenografts were performed and we have shown that the spheroid transcriptome more closely reflects expression patterns of the in vivo model compared to that of monolayer cultures. Among the best candidates, S100A6 gene over-expressed in the 3D models versus monolayer cultures was chosen for further analysis. To begin to address how S100A6 might affect EOC growth parameters, we have inhibited its expression in our in vitro models. The loss of S100A6 in the spheroid model results in an reduction of cellular migration, which seems to be in line with previous in vivo results published by other researchers. Always with the objective of developing the most relevant to the in vivo disease model systems, we have also succeeded in developing a unique EOC cell lines derived from patients with the most frequently diagnosed serous type of cancer. Very few cell lines derived from this type of cancers and from chemotherapy naïve patients are available. Moreover, we characterize for the first time EOC serous type cell lines derived from the ascites and the solid tumor of the same patient.

It is well known that most fluid flows observed in nature or encountered in engineering applications are turbulent and involve separation. Fluid flows in turbines, diffusers and channels with sudden expansions are among the widely observed areas where separation substantially alters the flow field and gives rise to complex flow dynamics. Such types of flows are referred to as internal flows since they are confined within solid surfaces and predominantly involve the generation or utilization of mechanical power. However, there is also a vast variety of engineering applications where the fluid flows past solid structures, such as the flow of air around an airplane or that of water around a submarine. These are called external flows and as in the former case the downstream evolution of the flow field is crucially influenced by separation. The present doctoral thesis addresses both internal and external separated flows by means of direct numerical simulations of the incompressible Navier-Stokes equations. For internal flows, the wall-driven flow in a onesided expansion channel and the pressure-driven flow in a plane channel with a single thin-plate obstruction have been studied in the fully developed turbulent state. Since such geometrical configurations involve spatially developing turbulent flows, proper inflow conditions are to be employed in order to provide a realistic fully turbulent flow at the input. For this purpose, a newly developed technique has been used in order to mimic an infinitely long channel section upstream of the expansion and the obstruction, respectively. With this approach, we are able to gather accurate mean flow and turbulence statistics throughout each flow domain and to explore in detail the instantaneous flow topology in the separated shear layers, recirculation regions as well as the recovery zones. For external flows, on the other hand, the flow past a prolate spheroid has been studied. Here, a wide range of Reynolds numbers is taken into consideration. Based on the characteristics of the vortical structures in the wake, the flow past a prolate spheroid is classified as laminar (steady or unsteady), transitional or turbulent. In each flow regime, the characteristic features of the flow are investigated by means of detailed frequency analysis, instantaneous vortex topology and three-dimensional flow visualizations.

Le cancer épithélial de l’ovaire (EOC) est le plus mortel des cancers gynécologiques. Cette maladie complexe progresse rapidement de façon difficilement décelable aux stades précoces. De plus, malgré une chirurgie cytoréductive et des traitements de chimiothérapie le taux de survie des patientes diagnostiquées aux stades avancées demeurt faible. Dans le but d’étudier l’EOC dans un contexte ex vivo, l’utilisation de modèles cellulaires est indispensable. Les lignées cellulaires d’EOC sont un outil pratique pour la recherche cependant, la façon dont l'expression des gènes est affectée en culture par comparaison à la tumeur d'origine n'est pas encore bien élucidée. Notre objectif était donc de développer et de caractériser de nouveaux modèles de culture in vitro qui réflèteront plus fidèlement la maladie in vivo. Nous avons tout d’abord utiliser des lignées cellulaires disponibles au laboratoire afin de mettre au point un modèle 3D de culture in vitro d’EOC. Des sphéroïdes ont été générés à l’aide de la méthode des gouttelettes inversées, une méthode pionnière pour la culture des cellules tumorales. Nous avons ensuite procédé à une analyse des profils d’expression afin de comparer le modèle sphéroïde au modèle de culture en monocouche et le modèle xénogreffe in vivo. Ainsi, nous avons identifié des gènes stratifiant les modèles tridimensionnels, tant in vivo qu’in vitro, du modèle 2D monocouche. Parmi les meilleurs candidats, nous avons sélectionné S100A6 pour une caractérisation ultérieure. L’expression de ce gène fût modulée afin d’étudier l’impact de son inhibition sur les paramètres de croissance des sphéroïdes. L’inhibition de ce gène a comme effet de réduire la motilité cellulaire mais seulement au niveau du modèle sphéroïde. Finalement, toujours dans l’optique de développer des modèles d’EOC les plus représentatifs de la maladie in vivo, nous avons réussi à développer des lignées cellulaires uniques dérivées de patientes atteintes d’EOC du type séreux, soit le plus commun des EOC. Jusque là, très peu de lignées cellulaires provenant de ce type de cancer et de patientes n’ayant pas reçu de chimiothérapie ont été produites. De plus, nous avons pour la première fois caractérise des lignées d’EOC de type séreux provenant à la fois de l’ascite et de la tumeur solide de la même patiente. / The epithelial ovarian cancer (EOC) is the most lethal of gynecological cancers. This complexe and heterogenous disease progresses rapidly and is almost asymptomatic in early stages. The survival rate of patients with late stage diagnosis remains low albeit cytoreductive surgery and chemotherapy. In order to study the EOC disease in an ex vivo context, the use of different cellular models is necessary. EOC cell lines derived from long-term passages of malignant ovarian cancers are useful tools for molecular and cellular research but it is not clear how culture conditions affect overall gene expression and oncogenic potential as compared to the original tumor. The main goal of this research was to develo and characterize new in vitro model systems that will recapitulate more closely some of the growth conditions encountered by tumor cells in vivo. In order to develop an in vitro tridimensional EOC spheroid model, we have used cell lines previously established in our laboratory. Spheroids were generated using the hanging droplet method, which was innovative for the culture of cancer cells. Comparative gene expression profile analysis of monolayer cultures, 3D spheroids and in vivo xenografts were performed and we have shown that the spheroid transcriptome more closely reflects expression patterns of the in vivo model compared to that of monolayer cultures. Among the best candidates, S100A6 gene over-expressed in the 3D models versus monolayer cultures was chosen for further analysis. To begin to address how S100A6 might affect EOC growth parameters, we have inhibited its expression in our in vitro models. The loss of S100A6 in the spheroid model results in an reduction of cellular migration, which seems to be in line with previous in vivo results published by other researchers. Always with the objective of developing the most relevant to the in vivo disease model systems, we have also succeeded in developing a unique EOC cell lines derived from patients with the most frequently diagnosed serous type of cancer. Very few cell lines derived from this type of cancers and from chemotherapy naïve patients are available. Moreover, we characterize for the first time EOC serous type cell lines derived from the ascites and the solid tumor of the same patient.

Submitted by Emanuel Varela Cardoso (emanuel.varela@ufcg.edu.br) on 2018-07-25T19:08:37Z No. of bitstreams: 1 HUGO CARVALHO AMORIM – DISSERTAÇÃO (PPGEM) 2016.pdf: 4230356 bytes, checksum: 63473c25aa9b0817eed244e6b1fc8738 (MD5) / Made available in DSpace on 2018-07-25T19:08:37Z (GMT). No. of bitstreams: 1 HUGO CARVALHO AMORIM – DISSERTAÇÃO (PPGEM) 2016.pdf: 4230356 bytes, checksum: 63473c25aa9b0817eed244e6b1fc8738 (MD5) Previous issue date: 2016-07-29 / Frutas são alimentos compostos normalmente por 80% a 90% de água, tornando-as extremamente perecíveis. Dessa forma, métodos de conservação devem ser aplicados para prolongar sua vida útil e conservar características de aceitação pelo consumidor. Assim, este trabalho teve como objetivo estudar a transferência de calor durante os processos de resfriamento e congelamento de banana utilizando geometria esferoidal prolata. Para a modelagem matemática, utilizou-se a equação da conservação de energia escrita em coordenadas esferoidais prolatas. A solução numérica da equação governante foi realizada através do método dos volumes finitos com uma formulação totalmente implícita. Um programa computacional foi escrito para simular o processo, através de linguagem computacional do software Mathematica, afim de se obter o histórico de temperatura no centro da banana e o perfil de temperatura, sendo os dados comparados a valores experimentais de congelamento de banana prata com casca, visando obter o coeficiente de transferência de calor convectivo e condutividade térmica da banana. Avaliou-se a influência das dimensões da fruta na cinética de resfriamento e nas frentes de congelamento. Concluiu-se que para a curva de resfriamento da banana, a modelagem pôde prever com bastante exatidão o período de resfriamento mas não o de pós-congelamento. Verificou-se que quanto menor o tamanho do produto, mais rápido é o processo e quanto maior a razão de aspecto de forma, maiores serão os gradientes de temperatura na ponta do produto. As taxas de resfriamento sofreram maior influência quando variou-se as dimensões mantendo um aspecto de forma constante do que variando as dimensões e mudando também o aspecto de forma. Por fim, verificou-se que quanto maior o aspecto de forma, mais não uniforme se dá o processo de transferência de calor na banana. / Fruits are a type of food that are typically made by 80% to 90% water, making them extremely perishable. Thus, conservation methods should be applied to prolong their shelf life and consumer acceptance characteristics. This work aims to study the heat transfer during the cooling and freezing processes of fruits with a prolate spheroid shape. The mathematical modeling consisted of energy conservation equation written in spheroidal prolates coordinates. The numerical solution of the governing equation was performed using the finite volume method with a fully implicit formulation. The process was simulated by computer-based language within the Mathematica software, to obtain the temperature history at the center of the fruit and the temperature profile, the data being compared with experimental values of freezed banana prata, to obtain the transfer coefficient convective-heat and thermal conductivity of the fruit. We evaluated the influence of fruit dimensions of the cooling kinetics and the freezing fronts. It was found that the lower the product size, the faster the process, and the higher the aspect ratio of shape, higher will be the temperature gradient at the top of the product.

The heat shock response (HSR) is a robust cellular reaction to mitigate protein damage from heat and other challenges to the proteome. This protective molecular program in humans is controlled by heat shock transcription factor 1 (HSF1). Activation of HSF1 leads to the induction of an array of cytoprotective genes, many of which code for chaperones. These chaperones, known as heat shock proteins (HSPs), are responsible for maintaining the functional integrity of the proteome. HSPs achieve this by promoting proper folding and assembly of nascent proteins, refolding denatured proteins, and processing for degradation proteins and aggregates which cannot be returned to a functional conformation. The powerful ability of the heat shock response to promote cell survival makes its master regulator, HSF1, an important point of research. To garner a better understanding of HSF1, we reviewed the role of the highly dynamic HSF1 protein structure and investigated how HSF1 affects cancer cell behavior and drug response. Cancers can be characterized in part by abhorrent replication, self-sufficient growth signaling, invasion, and evasion of apoptosis. HSF1 has been found to promote proliferation, invasion, and drug resistance in several types of cancer; including lung and ovarian cancer. Ovarian cancer has elevated levels of HSF1, but the role of HSF1 in ovarian cancer behavior had not been previously examined. Researching the role of HSF1 in ovarian cancer is merited, because treatment outcomes are poor due to the high frequency of late stage detection and drug resistance. We hypothesized that HSF1 is important in the malignant growth and drug resistance of ovarian cancer. We have created ovarian cancer cell lines with inducible knockdown of HSF1 to investigate how HSF1 contributes to the behavior of ovarian cancer. This allowed us to examine the behavior of cells in the absence HSF1. Both 2D and 3D spheroid tissue culture models were used to study how HSF1 contributes to the growth and invasion of ovarian cancer cells after treatment with the transforming growth factor β (TGFβ) cytokine. Additionally, we studied how HSF1 reduction modulates the response to multiple therapeutic drugs. Our research shows that HSF1 induces epithelial-mesenchymal transition (EMT) in a 3D growth model. Our work also demonstrates that reduction of HSF1 sensitizes ovarian cancer cells to multiple drugs.

Bien que reconnu comme une étape importante vers une meilleur compréhension de l’évolution des tumeurs, de la morphogénèse des tissus et des tests hauts débits de médicaments, l’utilisation de tests cellulaires in vitro en trois dimensions est toujours limitée et ce surtout par la difficulté d’établir un protocole simple et robuste pour leur formation. Dans ce travail, nous présentons d'abord une nouvelle méthode microfluidique pour la formation des sphéroïdes multicellulaires. Cette technologie des Capsules cellulaire est basée sur l'encapsulation et la croissance des cellules à l'intérieur de micro- sphères creuses, perméable, élastiques. Deuxièmement, nous montrons que ces microcapsules servent de capteurs mécaniques pour mesurer la pression exercée par les sphéroïdes expansion. En imagerie en temps réel multi- photons, on observe en outre que le confinement induit une organisation cellulaire stratifiée, avec un noyau nécrotique, solide et dense, entouré d'un rebord de cellules périphériques hyper-mobiles, qui présentent des propriétés invasives. Troisièmement, nous avons adapté la technologie des capsules cellulaires pour former des tubes creux. Cette géométrie cylindrique nous permet d'étudier l'impact de la libération partielle de confinement (le long de l'axe du tube principal) sur la cinétique de croissance d’agrégats cellulaires pseudo-unidimensionnel (nommé cylindroids). Nos données de microscopie et l’analyse d'images suggèrent un mécanisme de croissance par pointe et la prouvent la génération d’une contrainte radiale. La combinaison des configurations sphériques et cylindriques tend vers l'image globale du confinement qui déclenche la motilité cellulaire et l'invasion par la périphérie de l'agrégat cellulaire tandis que la prolifération des cellules est inhibée dans le noyau lorsque la pression augmente. Quatrièmement, nous utilisons l’alginate comme moule pour concevoir des coquilles et tubes multicouches perméables. En particulier, une légère adaptation du protocole nous permet d'ancrer une fine couche de Matrigel (utilisé comme une membrane basale artificielle) sur la paroi interne de l'alginate. Par l'utilisation de ces capsules sphériques décorés de Matrigel, nous montrons que les monocouches sphériques fermés de cellules épithéliales, ou des kystes, peuvent être facilement conçus avec des tailles qui sont imposées par la taille des capsules. De même, les capsules tubulaires décorées de Matrigel sont utilisées pour la formation des organoïds cultivés à partir de cellules extraites des cryptes du côlon de la souris. Enfin, notre technologie offre une nouvelle voie pour produire dans les tests cellulaires in vitro utiles pour développer de nouvelles thérapies anticancéreuses ou des approches d'ingénierie tissulaire et d'étudier l'interaction entre la mécanique et de la croissance dans les agrégats cellulaires in vitro. / Although recognized as an important step towards better understanding of tumor progression, tissue morphogenesis and high throughput screening of drugs, the use of three dimensional in vitro cellular assays is still limited, especially due to the difficulty in establishing simple and robust protocols for their formation. In this work, we first present a novel microfluidics-assisted method for multicellular spheroids formation. This Cellular Capsules technology is based on the encapsulation and growth of cells inside permeable, elastic, hollow micro-spheres. Second, we show that these microcapsules serve as unique mechanical sensors to measure the pressure exerted by the expanding spheroids. By multiphoton live imaging, we additionally observe that confinement induces a layered cellular organization, with a dense, solid, necrotic core surrounded by a rim of hyper-motile peripheral cells, which exhibit enhanced invasive properties. Third, we adapt the Cellular Capsules technology to form hollow tubes. This cylindrical geometry allows us to investigate the impact of partial confinement release (along the main tube axis) on the growth kinetics of pseudo-one dimensional cellular aggregates (named cylindroids). Our microscopy data and image analyses suggest a tip-growing mechanism and evidence radial stress generation. The combination of the spherical and cylindrical configurations leads to the overall picture that confinement triggers cell motility and invasion at the periphery of the cellular aggregate while cell proliferation is inhibited in the core as pressure builds up. Fourth, we use alginate as a template to design multilayered permeable shells and tubes. In particular, slight adaptation of the protocol allows us to anchor a thin layer of Matrigel (used as an artificial basement membrane) to the alginate inner wall. Using these Matrigel-decorated spherical capsules, we show that closed spherical monolayers of epithelial cells, or cysts, can be readily engineered with sizes that are imposed by the size of the capsules. Similarly, Matrigel-decorated tubular capsules are shown to be convenient for the formation of organoids grown from cells extracted from the cypts of mouse colon. Finally, our technology offers a new avenue to produce in vitro cell-based assays useful for developing new anti-cancer therapies or tissue engineering approaches and to investigate the interplay between mechanics and growth of in vitro cellular assemblies.

Au moins 60% des patients atteints de cancer vont recevoir de la radiothérapie (RT). L’efficacité de la radiothérapie dans le traitement du cancer est limitée par le phénomène de radiorésistance des cellules cancéreuses et par la toxicité des radiations sur les tissus sains. La découverte de nouveaux agents radiosensibilisants et radioprotecteurs permettrait de surmonter ces difficultés. Les modèles cellulaires 3D tels que les sphéroïdes, émergent motivés par le besoin de modèles précliniques plus proches des tumeurs in vivo. L’objectif du projet est d’optimiser un système microfluidique pour tester facilement et à faible coût des agents thérapeutiques avec la RT sur des sphéroïdes dans un contexte de repositionnement. Le système microfluidique développé permet la formation de 336 sphéroïdes homogènes en deux jours avec intervention minimale de l’utilisateur. Les sphéroïdes sont répartis dans 16 chambres de culture séparées par un système de valve magnétique pour éviter des effets bystander entre sphéroïdes irradiés et non-irradiés. Une nouvelle technique d’irradiation a été développée permettant d’exposer un système à 4 doses de radiation différentes. En tout, 4 doses de radiation et 4 concentrations d’agents thérapeutiques peuvent être testées par système. En utilisant cette approche, l’efficacité de trois agents avec la RT a été évaluée avec des tests de survie clonogénique. Nous avons démontré que le Talazoparib, un inhibiteur de PARP, radiosensibilise les cellules de sarcome de tissus mous (STS) cultivés en sphéroïdes à 2 Gy. Le système développé permet d’évaluer le potentiel d’agents thérapeutiques avec la RT et contribue à l’adoption des sphéroïdes comme modèle préclinique. / At least 60% of cancer patients will receive radiotherapy (RT) as part of their treatment. The efficacy of radiotherapy in the treatment of cancer is limited by the phenomenon of radioresistance of cancer cells and by the toxicity of radiation on healthy tissues. The discovery of new radiosensitizers and radioprotectors is essential to overcome these challenges. 3D cellular models such as spheroids emerge motivated by the need for better preclinical models. The objective of the project was to optimize a microfluidic system for easy, fast and low-cost testing of therapeutic agents with RT on spheroids. The developed microfluidic system allows the formation of 336 homogeneous spheroids in two days with minimal user intervention. The spheroids are distributed in 16 culture chambers separated by a magnetic valve system to avoid bystander effects between irradiated and unirradiated spheroids. A new irradiation technique has been developed to expose a system with 4 different radiation doses. In total, 4 radiation doses and 4 concentrations of therapeutic agents can be tested per system. Using this approach, the efficacy of three agents with RT was evaluated with clonogenic assays. Radiosensitizing properties of Talazoparib, a PARP inhibitor, on soft tissue sarcoma (STS) cells cultured as spheroids at 2 Gy were demonstrated. The developed system enables the evaluation of therapeutic agents with RT and contributes to the wide adoption of spheroids as a preclinical model.

Lung cancer leads in mortality among all types of cancer in the US and Non-small cell lung cancer (NSCLC) is the major type of lung cancer. Immuno-compromised mice bearing xenografts of human lung cancer cells represent the most common animal models for studying lung cancer biology and for evaluating potential anticancer agents. However, orthotopic lung cancer models based on intrapulmonary injection of suspended cancer cells feature premature leakage of the cancer cells to both sides of the lung within five days, which generates a quick artifact of metastasis and thus belies the development and progression of lung cancer as seen in the clinic. Based on intrapulmonary inoculation of multicellular spheroids (MCS), we have developed the first orthotopic xenograft model of lung cancer that simulates all four clinical stages of NSCLC progression in mice over one month: Stage 1 localized tumor at the inoculation site; Stage 2 multiple tumor nodules or larger tumor nodule on the same side of the lung; Stage 3 cancer growth on heart surface; and Stage 4 metastatic cancer on both sides of the lung. The cancer development was monitored conveniently by in vivo fluorescent imaging and validated by open-chest anatomy, ex vivo fluorescent imaging, and histological studies. The model enjoys high rates of postoperative survival (100%) and parenchymal tumor establishment (88.9%). The roughness of the inoculated MCS is associated negatively with the time needed to develop metastatic cancer (p=0.0299). In addition, we have constructed a co-culture MCS that consisted of A549-iRFP lung cancer cells and WI38 normal human fibroblast cells. The pro-proliferation effect and the high expression of α-smooth muscle actin (α-SMA) by the co-cultured WI38 cells indicated their transformation from normal fibroblasts to cancer-associated fibroblasts (CAFs). The morphology of the co-culture MCS features a round shape, a tight internal structure, and quicker development of roughness. The large roughness value of co-culture MCS suggests that small co-culture MCS could be inoculated into mice lung with a small needle to reduce the surgical trauma. Taken together, a new orthotopic model of NSCLC has been developed, which would facilitate future development of medications against lung cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal form of cancerwith very few available treatment options of which none has great effect.Cancer cells and stromal cells such as stellate cells which exist in abundancein PDAC interact by crosstalk, resulting in a tumorigenic collective response.With the help of a previously developed 3D co-culture spheroid model theeffect of a CRISPR/cas9 knockout of the cellular communication cetworkfactor 1 (CCN1) gene together with gemcitabine (GEM) treatment has beeninvestigated in terms of Panc1 cell viability and gene expression. Spheroidsconsisting of wild-type and knockout cell lines, each identified by westernblots were cultured, imaged and treated. Viability assays and RNA extractionfollowed by PCR showed that the viability of the cancer cells in the spheroidswere higher for the cells with CCN1 knockout. Cancer cells were also coculturedwith stellate cells with the goal of investigating the effect of thecellular crosstalk on chemoresistance. / Pankreatisk duktal adenokarcinom (PDAC)  är en ytterst dödlig form av cancermed få tillgängliga behandlingsalternativ, varav ingen är särskilt effektiv.Cancerceller och stromala celler så som de stellatceller som rikligt förekommeri PDAC interagerar med varandra genom överhörning, vilket leder till en effektsom hjälper tumören att proliferera. Effekten av en CRISPR/cas9 knockoutav genen CCN1 tillsammans med behandling med gemcitabin vad gällercellviabilitet och genuttryck studerades med hjälp av en tidigare utveckladfleratig sfäroidmodell. Sfäroider, bestående av vildtypceller och knockoutcellerlinjersom identifierades med western blots, odlades, fotades och behandlades.Viabilitetstester och extraktion av RNA följt av PCR visade att viabilitetenav cancerceller i sfäroiderna var högre för de celler som var knockout.Cancerceller samodlades även med stellatceller med målet att undersökaeffekten av cellernas överhörning på motståndet mot kemoterapi.

Le cancer du poumon non-à-petites-cellules (CPNPC) représente 85% des cas de cancer du poumon. Cependant, les modèles utilisés de culture cellulaire en deux dimensions (2D) représentent partiellement les caractéristiques physiopathologiques du CPNPC. Notre objectif est de réaliser un modèle in vitro plus représentatif des caractéristiques de ce type de cancer. La culture tridimensionnelle (3D) dans laquelle les cellules forment un sphéroïde, est considérée comme un modèle plus fidèle aux tumeurs retrouvées chez les patients grâce à la structure et aux interactions intercellulaires du modèle. Pour mieux représenter le microenvironnement tumoral, nous intégrons une lignée cellulaire de fibroblastes dans le sphéroïde de CPNPC, afin d’imiter l’interaction entre cellules cancéreuses et cellules stromales. En effet, bien que les fibroblastes représentent un faible pourcentage des cellules au sein des tumeurs de CPNPC, elles jouent un rôle prépondérant dans la biologie tumorale et la réponse aux médicaments. Nous avons testé différentes lignées de fibroblastes et différents ratios de co-culture afin de déterminer les conditions optimales de notre modèle de co-culture 3D. Après 7 jours de co-culture, les cellules cancéreuses démontrent un potentiel migratoire plus élevé lorsque mesuré dans des chambres de Boydens. Cet effet n’est dépendant ni de la prolifération, ni d’un changement de phase dans le cycle cellulaire. Nous avons caractérisé la localisation des fibroblastes au sein du sphéroïde par des expériences de microscopie à fluorescence. L’expression de la protéine α-SMA du cytosquelette a aussi été déterminée par immunofluorescence. Nous avons par la suite établi un modèle de co-culture sur 24 jours afin de maximiser la communication entre les cellules cancéreuses et les fibroblastes. Ce modèle de co-culture long terme a par la suite été analysé selon son contenu en cytokines, chimiokines et métabolites. Enfin, nous avons réalisé un criblage de médicaments au jour 24 de notre co-culture long terme afin d’évaluer une réponse thérapeutique des cellules cancéreuses, dans des conditions plus semblables au microenvironnement tumoral. / Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases. However, the two-dimensional (2D) cell culture models used represent partially the pathophysiological characteristics of NSCLC. Our goal is to develop an in vitro model that is more representative of the characteristics of this type of cancer. The three-dimensional (3D) culture, in which the cells form a spheroid, is considered to be a model more faithful to the tumors found in patients due to the structure and intercellular interactions of the model. In order to better represent the tumor microenvironment, we are integrating a fibroblast cell line into the spheroid of NSCLC to mimic the interaction between cancer cells and stromal cells. Although fibroblasts represent a small percentage of cells in NSCLC tumors, they play a major role in tumor biology and drug response. We tested different fibroblast cell lines and co-culture ratios to determine the optimal conditions of our 3D co-culture model. After 7 days of co-culture, the cancer cells show a higher migratory potential when measured in Boydens chambers. This effect is not dependent on proliferation or change of phase in the cell cycle. We characterized the localization of fibroblasts within the spheroid by fluorescence microscopy experiments. The expression and localization of the cytoskeletal protein α-SMA was also determined by immunofluorescence. We then established a 24-day co-culture model to maximize communication between cancer cells and fibroblasts. This long-term co-culture model was subsequently analyzed for cytokine, chemokine and metabolite content. Finally, we performed drug screening on day 24 of our long-term co-culture to evaluate a therapeutic response of cancer cells under conditions more similar to the tumor microenvironment.