Das maligne Melanom ist die aggressivste Form von Hautkrebs und verschiedene Familien von Rezeptortyrosinkinasen sind an der Entwicklung und der verstärkten Malignität beteiligt. Eph-Rezeptoren stellen die größte Klasse der Rezeptortyrosinkinasen dar und spielen eine wichtige Rolle bei der Tumorangiogenese und -progression. Die Genexpression und Proteinsynthese verschiedener Eph-Rezeptoren und Ephrin-Liganden ist bei vielen Tumorentitäten erhöht. Aus diesem Grund sollten sie sich als Zielproteine für die Entwicklung neuer Radiopharmaka eignen. Zudem zeigen Literaturbefunde einen Einfluss der hypoxischen Zellumgebung auf die Genexpression und die Proteinsynthese verschiedener Eph-Rezeptoren und Ephrin-Liganden. Das Ziel dieser Arbeit war es, Regulationsmechanismen bei verschiedenen Eph-Rezeptoren und Ephrin-Liganden aufzuklären, welche durch ein hypoxisches Umfeld hervorgerufen werden. Dazu wurde neben einem extrinsischen Hypoxiemodell an Monolayerzellkulturen auch ein intrinsisches Hypoxiemodell in Form von Tumorsphäroiden untersucht. Da die Genexpression und die Proteinsynthese von EphA2, EphB4, EphrinA1 und EphrinB2 laut Literatur vom Malignitätsgrad abhängig sind, wurden die metastatischen Melanomzelllinien A375, A2058 und MeWo und die prämetastatische Melanomzelllinie MEL-JUSO verwendet. Die Verifizierung der experimentellen Hypoxie erfolgte durch den etablierten Hypoxiemarker [18F]Fluormisonidazol, sowie dem Nachweis der VEGF-Genexpression unter den verwendeten Kulturbedingungen. Damit konnte die Eignung der hypoxischen Systeme gezeigt werden. Unabhängig vom Hypoxiemodell war in keiner der untersuchten Zelllinien ein Einfluss der Hypoxie auf die Genexpression und Proteinsynthese von EphA2, EphB4, EphrinA1 und EphrinB2 nachweisbar. Ein gesteigerter EphA2-Gehalt in Melanomzellen ist laut Literatur mit einer Erhöhung des Metastasierungspotentials verbunden. Um diesen Einfluss innerhalb einer Zelllinie zu untersuchen, wurden transgene A375-Zellen generiert. Mit dieser Zelllinie fanden Untersuchungen zu verschiedenen Metastasierungseigenschaften statt. Dabei wurde festgestellt, dass sich die Migration der Zellen durch den erhöhten EphA2-Gehalt verringerte, dabei war die hypoxische Umgebung ohne Einfluss. Weiterhin wurde festgestellt, dass der EphA2-Rezeptor das Adhäsionsverhalten von A375-Zellen nicht beeinflusst. Auch ein Einfluss auf das invasive Verhalten konnte nicht festgestellt werden. Eine hypoxische Umgebung war in beiden Fällen nicht von Bedeutung. Aus den Ergebnissen der vorliegenden Arbeit kann geschlussfolgert werden, dass bei den untersuchten Melanomzelllinien keine Regulation der Eph-Rezeptoren und Ephrin-Liganden durch ein hypoxisches Umfeld erfolgt. Durch die ausführliche Charakterisierung des EphA2-Rezeptors in der Arbeit kann jedoch geschlussfolgert werden, dass sich der Rezeptor als potentielles Zielmolekül für die Entwicklung neuer Radiotherapeutika und Radiodiagnostika eignet, nicht jedoch für die Detektion hypoxischer Bereiche in Tumoren. Durch die nunmehr etablierte Generierung von Sphäroiden und einer Zelllinie, welche den Rezeptor verstärkt exprimiert und synthetisiert, stehen nun Zellmodelle für die weiterführende Charakterisierung und Analyse neuer Radiodiagnostika und Radiotherapeutika auf der Basis von Inhibitoren und Antikörper gegen EphA2 zur Verfügung.

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.

Cancer is the second leading cause of death in the US and worldwide, accounting for 16% of deaths worldwide in 2015. Of more than 100 types of cancers affecting humans, breast cancer is the most common cancer among women and is the second leading cause of death in women. Triple negative breast cancer (TNBC) is a subtype of breast carcinomas defined by the lack of the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2 /neu). The prognosis and survival of TNBC patients remains the poor due to the lack of effective targeted therapy. Nanotechnology-based drug delivery systems, such as liposomes, are widely investigated to enhance anticancer efficacy by concentrating the drug molecules in the tissues of interest and by altering the pharmacokinetic profile. Taking advantage of the pH gradient in the tumor microenvironment, pH-triggered release is a promising strategy to enhance the anticancer efficacy of drug delivery systems against TNBC. Previously, a strategy in our lab has been developed to render saturated and pegylated liposomes pH-sensitive: protonation-induced conformational switch of lipid tails, using trans-2-aminocyclohexanol lipids (TACH, flipids) as a molecular trigger. Based on previous work in our lab, pH-sensitive liposomes (fliposomes) composed of C-16 flipids with amine group of morpholine (MOR) and azetidine (AZE) demonstrated optimized triggered release in response to the tumor’s low pH microenvironment. In this study, different preparation methods were developed and optimized to produce viable fliposomes with high doxorubicin (DOX) encapsulation efficiency. In vitro release assays were established and validated to accurately reflect pH-triggered release of fliposomes. The physicochemical properties of DOX-loaded fliposomes were characterized and their pH-dependent release were investigated. Factors influencing the desirable attributes of liposomes, such as size, pH-sensitivity, stability and drug-loading capacity were explored. Based on these characterizations, central composite design (CCD) was utilized to optimize the formulation of fliposome with two critical factors, flipids and cholesterol. Cell viability assays on traditional monolayer and innovative three-dimensional multicellular spheroids (3D MCS) of TNBC cell lines were conducted to evaluate the anticancer efficacy of the resultant fliposomes in vitro. The constructed 3D MCS carried heterogeneously distributed live and apoptotic cells, as well as acidity inside the 3D MCS based on confocal microscopic imaging studies. The distribution and penetration of DOX-loaded fliposomes into 3D MCS was imaged by confocal microscopy in comparison to DOX-loaded non pH-sensitive liposomes and free DOX. As a result, fliposome manifested superior anticancer activity against TNBC 3D MCS by efficient penetration into 3D MCS, followed by tuning up the release rate of the anticancer agent DOX. A TNBC orthotopic xenograft model was established by transplanting TNBC into the murine mammalian fat pad, which maintains the organ-specific tumor microenvironment of the original organ . A pilot pharmacokinetic study was conducted in order to correlate the pH response and stability properties with the in vivo stability of the optimized AZE-C16 fliposome. The antitumor efficacy was comparable between free DOX and DOX-loaded stealth liposome with tumor volumes of ~ 80-90% of the control treatment 32 days post first dose. In contrast, the DOX-loaded fliposome, especially MOR-C16 fliposome, exhibited a significantly higher antitumor efficacy and delayed progression compared to free DOX and stealth liposome treatments. Taken together, DOX-loaded fliposomes were successfully prepared and optimized for in vivo application. They were able to achieve superior activity against TNBC in vitro and in vivo, facilitated by enhanced release of the anticancer drug DOX after penetration inside TNBC tumor.

Abstract  Lack of clinical relevance is assumed to be the main reason behind the high failure rate of medical drugs in the very initial phases of clinical trials. Clinical relevance is difficult to achieve with current tools as they lack the biological and physiological cues found in vivo. Microfluidics, the knowledge of fluid manipulation in small channels, has proven to be a promising science to bridge the gap between the current in vitro and the real in vivo features. In this thesis, a scaffold for the growth of spheroids inside a microfluidic device for potential drug screening was developed. Firstly, the surface of a microfluidic device was coated with the polymers cellulose nanofibrils, polyallylamine hydrochloride, and polyethyleneimine using the Layer-by-Layer technique to achieve an even surface coverage. Here, different chip designs, polymer concentrations, and pressure directions were tested. It was decided that using a negative pressure direction with a polymer concentration of 50 mg/L in a chip design with micropillars was optimal and these conditions were then used for testing the spheroid formation. Secondly, spheroids were grown inside the microfluidic channels using different coatings: the previously mentioned polymer buildup, one non-coated channel, and one coated with attachment factor proteins. These three surface conditions were compared and it was shown that the polymer-based surface cover was indeed superior as a scaffold as it encouraged and promoted cell growth in the spheroid formation of liver cancer cells from the HepG2 cell line. Further development of this cellulose nanofibrils-coated microfluidic device displays a promising future for functioning as an in vitro 3D cell culture model that better mimics the close-to-cell microenvironments by imitating cell proliferation, cell-to-cell, and cell-to-extracellular matrix interactions. / Sammanfattning Den främsta orsaken bakom den höga antal misslyckade kliniska läkemedelsprövningar i de initiala faserna antas bero på brist på klinisk relevans. Klinisk relevans är mycket svår att uppnå med dagens verktyg då de saknar de biologiska och fysiologiska förhållandena som återfinns in vivo. Mikrofluidik, kunskapen om vätskemanipulation i små kanaler har visat sig vara lovande vetenskap för att överbrygga klyftan mellan de nuvarande in vitro och de faktiska in vivo funktionerna. I detta arbete utvecklades en matris för sfäroider att växa på inuti en mikrofluidisk kanal för att potentiellt användas till läkemedelsscreening. Först användes Layer-by-Layer teknologi för att jämnt betäckta ytan inuti en mikrofluidisk kanal med polymererna cellulosananofibriller, polyallylamin hydroklorid samt polyetylenimin. Här testades olika designer på mikrofluidiska chip, polymerkoncentrationer samt tryckriktningar. Utifrån detta gick det att fastställa att negativt tryck med en polymerkoncentration på 50 mg/L i en chippdesign med mikropelare var optimal för en jämn ytbetäckning och dessa förhållanden användes sedan för att pröva sfäroidernas tillväxt. Härnäst testades därmed sfäroidernas tillväxt inuti mikrofluidiska kanaler under tre olika förhållanden: ett med polymerbetäckningen, ett utan betäckning och ett då ytan var täckt med proteiner med fästfaktorer. Dessa tre förhållanden jämfördes sedan med varandra och således gick det att konstatera att den polymerbaseradebetäckningen fungerade överlägset som matris för tillväxt av HepG2 lever cancer cell sfäroider eftersom den tycks främja dess tillväxt och bildning. Det pekar mot att ytterligare utveckling av denna cellulostäckta yta skulle innebära en lovande modell för in vitro 3D cellodling som bättre efterliknar den cellulära mikromiljön genom att imitera cellproliferation, interaktioner celler emellan samt mellan cell och extracellulär matrisen.

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.

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

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.

Les glioblastomes sont des tumeurs primaires du cerveau les plus malignes. L’identification des cellules souches cancéreuses de glioblastome (CSGs) a transformé notre vision globale des glioblastomes en révélant une hiérarchie cellulaire au sein de ces tumeurs. Les CSGs sont douées de propriétés d’auto-renouvellement, de différenciation et peuvent entrer en quiescence. Elles sont considérées comme les cellules entretenant les tumeurs, responsables de leur dissémination et des rechutes après traitement. La découverte des CSGs a conduit à un changement de paradigme dans le développement des thérapies anticancéreuses, avec la nécessité de cibler dans le traitement non seulement les cellules de la masse tumorale, mais aussi les CSGs. Un criblage différentiel de la chimiothèque Prestwick réalisé au laboratoire a permis d’identifier le bisacodyl comme une molécule présentant une cytotoxicité spécifique sur les CSGs en quiescence.Cette thèse présente un travail sur la caractérisation des CSGs, la compréhension du mode d’action du bisacodyl, ainsi que l’évaluation de son potentiel thérapeutique sur un modèle 3D in intro et des modèles in vivo. / Glioblastomas are the most malignant primary brain tumors. The identification of glioblastoma stemcells (GSCs) has transformed our comprehension of those tumors by revealing a hierarchical organization. GSCs can self-renew, differentiate and enter into a quiescent state. They are considered as cells which fuel and as the main culprits of tumor relapse. The discovery of GSCs triggered a change in paradigm for cancer therapy. Indeed to gain in efficacy, therapies need to target, not only the cells forming the bulk of the tumor, but also GSCs particularly resistant and endowed with a high tumorigenic potential. Chemical screening of the Prestwick chemical library in our laboratory, unveiled bisacodyl with a specific activity on quiescent GSCs.This thesis presents work on the characterization of GSCs, study of the mode of action of bisacodyl on GSCs, as well as a preclinical evaluation of bisacodyl on a 3D model in vitro and animal models in vivo.

No description available.

Le neuroblastome pédiatrique à haut risque est incurable malgré l’intensification des traitements. Chez le patient, les cellules de neuroblastome échappent à l’activité anticancéreuse des cellules immunitaires Natural Killer (NK). Or, lorsque cultivées in vitro en monocouche (2D), les cellules de neuroblastomes redeviennent sensibles à l’activité cytotoxique des cellules NK ce qui ne reflètent pas leur résistance dans les tumeurs in situ. Nous faisons l'hypothèse que lorsque cultivées en 3D sous forme de sphéroïdes, les cellules de neuroblastome pourraient retrouver certaines caractéristiques qui les rendraient plus représentatives des tumeurs in situ au niveau immunologique. Ainsi, un tel modèle préclinique pourrait mieux refléter la résistance aux cellules NK et servir de modèle de criblage pour la découverte de médicaments potentialisant la cytotoxicité des cellules NK. Pour répondre à cette question, nous avons développé un système de culture cellulaire 3D utilisant plusieurs lignées cellulaires de neuroblastome. À ce système, une co-culture en 3D avec une lignée de cellules Natural Killer (NK92) a été mise en place. Nous avons mis en évidence que les sphéroïdes de neuroblastome présentent des changements d’expression de certains gènes qui sont retrouvées chez les patients ainsi qu’une plus grande résistance à l’activité cytotoxique des cellules NK92 en comparaison avec les lignées en monocouche. Les co cultures de sphéroïdes ont été exposées à des inhibiteurs de protéines impliquées à différents niveaux de l’épigénome afin de découvrir des médicaments qui sensibiliseraient les cellules de neuroblastome à l’activité cytotoxique des NK92. Une différence dans la sensibilité aux médicaments entre les sphéroïdes et les cellules en 2D ainsi qu’en monoculture ou en co-culture a été observée et certains composés ont été identifiés en vue de potentialiser l’activité des cellules NK92. Ainsi, nos études ont permis de mieux comprendre les mécanismes impliqués dans la résistance des cellules du neuroblastome à l’activité cytotoxique des cellules NK dans un modèle plus représentatif de la tumeur in situ. / High-risk pediatric neuroblastoma remains incurable despite intensified treatments. In patients, neuroblastoma cells evade the anti-cancer activity of Natural Killer (NK) immune cells. However, when cultured in vitro in a monolayer (2D), neuroblastoma cells become sensitive to the cytotoxic activity of NK cells, which does not reflect their resistance in tumors in situ. We hypothesize that when cultured in 3D in the form of spheroids, neuroblastoma cells could regain certain characteristics that would make them representative of tumors in situ at the immunological level. Thus, such a preclinical model could better reflect NK cell resistance and serve as a screening model for drug discovery to discover a treatment that can potentiate NK cell cytotoxicity. To answer this question, we developed a 3D cell culture system using several neuroblastoma cell lines. To this system, a 3D coculture model with a Natural Killer (NK92) cell line was set up. We have shown that neuroblastoma spheroids develop changes in the expression of certain genes that are found in patients as well as greater resistance to NK92 cells compared to monolayer cell lines. Spheroid cocultures were exposed to inhibitors of proteins involved at different levels of the epigenome to discover drugs that would sensitize neuroblastoma cells to the cytotoxic activity of NK92. A difference in drug sensitivity between spheroids and cells in 2D as well as in monoculture or coculture was observed and some compounds were identified to potentiate the activity of NK92 cells. Thus, our studies have provided a better understanding of the mechanisms involved in the resistance of neuroblastoma cells to the cytotoxic activity of NK cells in a more representative model of the tumor in situ.