This research utilizes Electrical Impedance Spectroscopy, a technique classically used for electrochemical analysis and material characterization, as the basis for a non-destructive, label-free assay platform for three dimensional (3D) cellular spheroids. In this work, a linear array of microelectrodes is optimized to rapidly respond to changes located within a 3D multicellular model. In addition, this technique is coupled with an on chip micro-pH sensor for monitoring the environment around the cells. Finally, the responses of both impedance and pH are correlated with physical changes within the cellular model. The impedance analysis system realized through this work provides a foundation for the development of high-throughput drug screening systems that utilize multiple parallel sensing modalities including pH and impedance sensing in order to quickly assess the efficacy of specific drug candidates. The slow development of new drugs is mainly attributed to poor predictability of current chemosensitivity and resistivity assays, as well as genetic differences between the animal models used for tests and humans. In addition, monolayer cultures used in early experimentation are fundamentally different from the complex structure of organs in vivo. This requires the study of smaller 3D models (spheroids) that more efficiently replicate the conditions within the body. The main objective of this research was to develop a microfluidic system on a chip that is capable of deducing viability and morphology of 3D tumor spheroids by monitoring both the impedance of the cellular model and the pH of their local environment. This would provide a fast and reliable method for screening pharmaceutical compounds in a high-throughput system.

No description available.

Natural Killer cells are a type of lymphocyte belonging to the innate immune system and they operate cell-mediated cytotoxicity and release of pro-inflammatory cytokines against cancerous cells. However, in vivo testings have shown a reduced activity of NK cells against solid tumors probably due to the negative influence of the immunosuppressive tumor microenvironment. Multicellular tumor spheroids may constitute an advantageous model in cancer biology for studying the mechanisms behind cancer immune editing since it more closely mimics the complexity of the human body compared with the 2D model counterpart. This study investigated the interaction between NK cells isolated from blood and tumor spheroids obtained from A498 renal carcinoma cells, using light-sheet microscopy imaging which allows satisfactory cell tracking in the inner layers of the spheroids. NK cells not only indeed interact with tumor spheroids, but many of them were able to penetrate the spheroids inducing some changes in the structure of the latter. NK cells were also tracked over time, displaying the migration path and calculating the speed. The fluorescence intensity of the NK cells was found reduced as soon as they penetrate the spheroid but, conversely, the speed seems to increase inside the spheroid, a possible sign of the fallibility of the tracking algorithm in this specific case. We propose solutions for more sophisticated future implementations, involving the use of marks during the experimental phase and drift corrections at the data analysis level.

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required.

No description available.

Le microenvironnement tumoral, l'angiogenèse tumorale et l'hypoxie jouent un rôle crucial dans la progression tumorale et le développement de thérapies de nombreux cancers. Les limites de pénétration des médicaments, les phénomènes de résistance aux anti-cancéreux, la vascularisation de la tumeur et l’hypoxie sont tous des paramètres influençant les effets du médicament. La culture cellulaire 3D permet de créer un microenvironnement qui imite l’architecture et la fonction des tissus in vivo. L’expression de gènes et de protéines modifiée par l’environnement 3D est une autre caractéristique qui impacte l’effet d’une molécule thérapeutique. Dans notre première étude, afin de développer un modèle 3D vascularisé imitant celle des tumeurs in vivo, nous avons mis en culture des cellules endothéliales en 2D avec des cellules tumorales en 3D. Après 2 semaines de culture, un réseau vasculaire s’est organisé avec des structures de type tubulaire présentant une lumière et exprimant différents marqueurs angiogéniques tels que VEGF, CD31 et Collagène IV. Dans notre deuxième étude, nous avons développé un modèle d’hypoxie in vitro intégrant l'environnement 3D et un agent mimétique de l'hypoxie (CoCl2). Le but de ce modèle est de créer un modèle d'hypoxie imitant les tumeurs in vivo et de montrer l'importance de l'hypoxie dans la réponse et la résistance aux médicaments. Ces résultats ont révélé que la meilleure condition était la combinaison 3D+CoCl2, conduisant à la surexpression des gènes relatifs à l’hypoxie (GLUT1/3, VEGF) et à la résistance aux médicaments (ABCG2, MRP1). L'angiogenèse et l'hypoxie sont des facteurs clés pour le microenvironnement tumoral in vivo et ils doivent être adoptés dans la conception de modèles tumoraux in vitro pour mieux sélectionner et cribler les médicaments anticancéreux. / The tumor microenvironment, tumor angiogenesis, and hypoxia play a critical role in the tumor progression and therapy development of many cancers. Limitations in drug penetration, multidrug resistance phenomena, tumor vascularization, and oxygen deficiency are all parameters influencing drug effects. 3D cell culture allows to create a microenvironment that more closely mimics in vivo tissue architecture and function, thus, gene and protein expression modified by the 3D environment are further features that affect treatment outcome. In our first study, in order to develop a vascularized 3D model like in vivo tumors, we co-cultured 2D endothelial cells with 3D tumor cells. After 2 weeks of this combination, a vascular network was formed and organized with tubule-like structures presenting a lumen and expressing different angiogenic markers such as VEGF, CD31 and Collagen IV. In our second study, we developed an in vitro hypoxia model integrating the 3D environment and a hypoxia mimetic agent (CoCl2) to mimic the in vivo tumors and to show the importance of hypoxia in drug response and resistance. Results revealed that the best condition was the combination 3D+CoCl2 model, leading to overexpression oh hypoxia (GLUT1/3, VEGF) and drug resistance (ABCG2, MRP1) related genes. Taken together, angiogenesis and hypoxia are key factors for in vivo tumor microenvironment and they should be adopted in in vitro model design to better select and screen anticancer drugs.

La thérapie photodynamique (PDT) est un traitement alternatif du cancer plus ciblé et moins invasif que les modalités traditionnelles. La Temoporfine (mTHPC, nom sous forme médicamenteuse : Foscan®), est l'un des PS les plus puissants cliniquement approuvés. Cependant, sa faible solubilité en milieu aqueux a provoqué plusieurs complications lors de son administration. La présente étude vise à mettre au point des nanoparticules constituées d’une molécule anticancéreuse couplée à la cyclodextrine intégré dans un liposome (drug-in-cyclodextrin-in-liposome, DCL) en couplant deux systèmes d'administration indépendants : les complexes d'inclusion cyclodextrine-mTHPC et les vésicules liposomales pour améliorer le transport et la pénétration de la mTHPC dans le tissu cible. La formation de complexes d'inclusion entre les cyclodextrines et la mTHPC a été étudiée en détail. Sur la base de ces données, des mTHPC-DCL à simple et double charge ont été préparées, optimisées et caractérisées. Il a été démontré que les mTHPC-DCL sont stables et que presque tous les mTHPC-DCL sont liés à β-CDs dans la lumière aqueuse interne des liposomes. L'influence des DCLs sur l'accumulation, la distribution et l'efficacité photodynamique de la mTHPC a été étudiée dans des modèles cellulaire en monocouche et sphéroïde multicellulaires 3D d’adénocarcinome de pharynx humain (HT29). En utilisant des sphéroïdes, nous avons démontré que le DCL à base de triméthyl-β-CD fournissait une accumulation homogène de la mTHPC dans tout le volume des sphéroïdes tumoraux, suggérant ainsi une distribution optimale de la mTHPC. / Photodynamic therapy (PDT) is an alternative cancer treatment which offers a more targeted and less invasive treatment regimen compared to traditional modalities. Temoporfin (mTHPC, medicinal product name: Foscan®), is one of the most potent clinically approved PS. However, its poor solubility in aqueous medium caused several complications of its administration. The present study is aimed at the development of drug-in-cyclodextrin-in-liposome (DCL) nanoparticles by coupling two independent delivery systems: cyclodextrin/mTHPC inclusion complexes and liposomal vesicles to improve the transport and penetration of mTHPC to the target tissue. The formation of inclusion complexes between cyclodextrins and mTHPC was studied in detail. Based on these data, single and double loaded mTHPC-DCLs have been prepared, optimized and characterized. It was demonstrated that mTHPC-DCLs are stable and almost all mTHPC is bound to β-CDs in the inner aqueous liposome lumen. The influence of DCLs on mTHPC accumulation, distribution and photodynamic efficiency was studied in human adenocarcinoma HT29 cellular monolayer and spheroid models. Using 3D multicellular HT29 tumor spheroids we demonstrated that trimethyl-β-CD-based DCL provides homogenous accumulation of mTHPC across tumor spheroid volume thus supposing optimal mTHPC distribution.

Inadequate metabolic conditions in solid tumors lead to the formation of quiescent cancer cells that are suspended in a transient cell cycle arrest. When conditions change, quiescent cancer cells can re-enter the cell cycle and cause recurrence. Drug screening efforts have revealed mitochondrial oxidative phosphorylation as a unique metabolic dependency in quiescent cancer cells. The anthelmintic drug nitazoxanide is an inhibitor of oxidative phosphorylation and preferentially active against quiescent cancer cells in multicellular tumor spheroids.  In this thesis, we employed current and developed new models of quiescent cancer cells and applied live cell imaging for improved preclinical evaluation of cancer drugs in hepatocellular and colorectal carcinoma cell lines. As part of this work, a new assay to measure mitochondrial membrane potential in three-dimensional cell models was developed, an application of the JC-1 assay, and we demonstrated that the preferential activity against quiescent cancer cells of nitazoxanide is shared by two kinase inhibitors: sorafenib and regorafenib. The sensitivity of quiescent cancer cells to nitazoxanide, sorafenib, and regorafenib correlated with the disruption of the mitochondrial membrane potential. Nitazoxanide and sorafenib, in combination, caused an additive decrease in viability, mitochondrial membrane potential, and colony regrowth capacity.  Furthermore, we developed a quiescent hollow fiber assay and implemented an improved analysis using live cell imaging and adenosine triphosphate analysis. Hypoxia and cancer cell quiescence were enriched in hollow fiber macrocapsules over time, and the culture conditions affected nitazoxanide sensitivity. Additionally, we used basement membrane extract gel to support cell growth in hollow fiber macrocapsules and implanted macrocapsules in mice. We observed that the in vivo environment was favorable to cell growth. Through this characterization of the quiescent hollow fiber assay, we were able to outline important paths for future research.

Cette thèse présente des travaux sur l’étude et la calibration de modèles d’équations aux dérivées partielles pour la croissance tumorale. La première partie porte sur l’analyse d’un modèle de croissance tumorale pour le cas de métastases au foie de tumeurs gastro-intestinales (GIST). Le modèle est un système d’équations aux dérivées partielles couplées et prend en compte plusieurs traitements dont un traitement anti-angiogénique. Le modèle permet de reproduire des données cliniques. La première partie de ce travail concerne la preuve d’existence/unicité de la solution du modèle. La seconde partie du travail porte sur l’étude du comportement asymptotique de la solution du modèle lorsqu’un paramètre du modèle, décrivant la capacité de la tumeur à évacuer la nécrose, converge vers 0. La seconde partie de la thèse concerne le développement d’un modèle de croissance pour des sphéroïdes tumoraux ainsi que sur la calibration de ce modèle à partir de données expérimentales in vitro. L’objectif est de développer un modèle permettant de reproduire quantitativement la distribution des cellules proliférantes à l’intérieur d’un sphéroïde en fonction de la concentration en nutriments. Le travail de modélisation et de calibration du modèle a été effectué à partir de données expérimentales permettant d’obtenir la répartition spatiale de cellules proliférantes dans un sphéroïde tumoral. / In this thesis, we present several works on the study and the calibration of partial differential equations models for tumor growth. The first part is devoted to the mathematical study of a model for tumor drug resistance in the case of gastro-intestinal tumor (GIST) metastases to the liver. The model we study consists in a coupled partial differential equations system and takes several treatments into account, such as a anti-angiogenic treatment. This model is able to reproduce clinical data. In a first part, we present the proof of the existence/uniqueness of the solution to this model. Then, in a second part, we study the asymptotic behavior of the solution when a parameter of this model, describing the capacity of the tumor to evacuate the necrosis, goes to 0. In the second part of this thesis, we present the development of model for tumor spheroids growth. We also present the model calibration thanks to in vitro experimental data. The main objective of this work is to reproduce quantitatively the proliferative cell distribution in a spheroid, as a function of the concentration of nutrients. The modeling and calibration of this model have been done thanks to experimental data consisting of proliferative cells distribution in a spheroid.