Dissertations / Theses on the topic 'Multicellular tumour spheroids'

The work presented in this thesis is concerned with modelling the effects of cell movement on the growth and formation of cell cycle phase specific regions within solid tumours. A model is proposed in the context of multicellular tumour spheroids (MCTS) and includes a simple model of the cell cycle, where cells move between each cell cycle phase depending on the availability of extracellular nutrient, as well as cell movement via chemotaxis, which varies depending upon the respective cell cycle phase of the cell. Numerical and asymptotic solutions show the model re-produces the well known MCTS structure of an internal quiescent cell region surrounded by a rim of proliferating cells. A further, more interesting result, describes a tumour surrounded by a rim of quiescent cells, with an inner quiescent and an interim proliferating cell region. The resultant solutions are a result of the different cell velocity profiles along with the effects of the cell cycle kinetics in different regions of the tumour. The non-linear form of the conservation equations describing the movement of cells means that solutions with spatial discontinuities in the cell concentrations (shocks) are observed for specific parameter values. Analysis of the effects of the chemotactic response and the cell cycle kinetics, both spatial and temporal, provide insight in to the model's behaviour and shows an understanding of cell cycle kinetics, cell movement and the spatial structure of tumours is important in assisting therapeutic strategies. The effectiveness of apoptosis, as an anti-cancer strategy, is shown to be dependent upon the concentration and spatial organisation of proliferating cells within the respective tumour. Comparison with the experimentally verified model of tumour growth developed by Gompertz allows specific model parameters to be expressed in terms of experimentally known variables. Such analysis shows that Gompertz's model is good at predicting the growth of solid tumours with a proliferating rim, but other models are required to understand the growth of non-uniform, heterogeneous tumours. Experimental justification of the model is provided by considering the observed internalisation of H3 Thymidine labelled cells and inert microspheres within MCTS. Here experimental results show that following adherence to the spheroid edge, the microspheres were all advected towards the centre of the spheroids whilst the labelled cells were spread throughout the proliferating and quiescent outer regions. The cell cycle model which is developed is, unlike previous models, able to account for this observed behaviour. Various simulations are discussed in relation to the original experimental results. These results show the importance of cell movement in providing possible ways of assisting with drug delivery to the more therapeutically resistant regions of solid tumours. Finally the importance of necrosis formation is discussed by a simple extension to the model. Necrosis as a result of quiescent cell death leads to the commonly observed formation of a necrotic core in each case. However, using the model to consider the more recent hypothesis that apoptosis leads to the formation of necrotic regions provides interesting theoretical results.

Tumour necrosis has long been associated with poor prognosis and reduced survival in cancer. Hypotheses to explain this include the idea that as aggressive tumours tend to grow rapidly, they outgrow their blood supply leading to areas of hypoxia and subsequently necrosis. However whilst this and similar hypotheses have been put forward to explain the association, the biological significance of the cells which make up necrotic tissue has been largely ignored. This stems from the belief that because a tumour is more aggressive and fast growing it develops areas of necrosis, rather than, the tumour is more aggressive because it contains areas of necrosis. Which came first like the egg and chicken is yet to be determined, however to date most research has only considered the possibility of the former. Viable cells were found in the necrotic core of Multicellular Tumour Spheroids. When examined these cells were found to be different to the original cell line in terms of proliferation, migration, and chemosensitivity. A proteomic analysis showed that these phenotypical changes were accompanied by changes in a large number of proteins within the cells, some of which could be potential therapeutic targets. Furthermore this has led to a new hypothesis for tumour necrosis and its association with poor prognosis. Necrotic tissue provides a microenvironemental niche for cells with increased survival capabilities. Protected from many chemotherapeutics by their non-proliferative status once conditions improve these cells can return to proliferation and repopulate the tumour with an increasingly aggressive population of cells.
Yorkshire Cancer Research

Cellular redox potential is incredibly important for the control and regulation of a vast number of processes occurring in cells. Disruption of the fine redox balance within cells is has been associated with disease. Of particular interest to my research is the redox gradient that develops in cancer tumours, in which the internal regions are further from vascular blood supply and therefore become starved of oxygen and hypoxic. This makes treatment of these areas a lot more challenging, as radiotherapy approaches rely on the presence of oxygen and, with a poor vascular blood supply, drugs delivered through the blood stream will have poor access to these regions. Currently, there is limited knowledge regarding the quantitative nature of this redox gradient in cancerous tumours. To aid the development of drugs and therapies to overcome this problem, a system that enables quantitative mapping of redox potential through a tumour would be a vital tool. In this work redox sensitive molecules attached to gold nanoparticles (NPs) are delivered to cells and give signals using surface enhanced Raman scattering (SERS). Redox potential changes are monitored quantitatively by ratiometric changes in signal intensity of selected signals in the SER spectra acquired. Multicellular tumour spheroids (MTS) are used as a three dimensional (3D) in vitro tumour model, in which the 3D architecture and gradients observed in tumours in vivo develop. As redox potential is pH dependent and pH is another important physiological characteristic in its own right, a SERS pH sensor was developed and ultimately a system that multiplexes intracellular pH and redox measurement by SERS. Initially, simultaneous redox potential and pH measurements were performed in monolayer culture before extending this to MTS. Photothermal optical coherence tomography (OCT) was used to investigate overall 3D NP distribution in the MTS models. It was possible to control NP delivery to MTS to localise NPs to various regions. Redox potential and pH could then be measured using a fibre optic Raman probe, and spatial response to drug treatment monitored. Intracellular NP localisation was investigated using transmission electron microscopy (TEM), scanning electron microscopy (SEM), helium ion microscopy (HIM) and confocal fluorescence microscopy (CFM) and attempts were made to control NP delivery to particular intracellular compartments.

Tumour necrosis has long been associated with poor prognosis and reduced survival in cancer. Hypotheses to explain this include the idea that as aggressive tumours tend to grow rapidly, they outgrow their blood supply leading to areas of hypoxia and subsequently necrosis. However whilst this and similar hypotheses have been put forward to explain the association, the biological significance of the cells which make up necrotic tissue has been largely ignored. This stems from the belief that because a tumour is more aggressive and fast growing it develops areas of necrosis, rather than, the tumour is more aggressive because it contains areas of necrosis. Which came first like the egg and chicken is yet to be determined, however to date most research has only considered the possibility of the former. Viable cells were found in the necrotic core of Multicellular Tumour Spheroids. When examined these cells were found to be different to the original cell line in terms of proliferation, migration, and chemosensitivity. A proteomic analysis showed that these phenotypical changes were accompanied by changes in a large number of proteins within the cells, some of which could be potential therapeutic targets. Furthermore this has led to a new hypothesis for tumour necrosis and its association with poor prognosis. Necrotic tissue provides a microenvironemental niche for cells with increased survival capabilities. Protected from many chemotherapeutics by their non-proliferative status once conditions improve these cells can return to proliferation and repopulate the tumour with an increasingly aggressive population of cells.

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases capable of digesting the extracellular matrix (ECM), which is essential for tissue structure and transmitting messages between cells. MMPs play an important role in cancer, controlling cell migration, proliferation, apoptosis, regulation of tumour expansion, angiogenesis and invasion. Previous research has indicated high expression of MT1-MMP in breast cancers suggesting a potential role in tumour progression. Our results confirm that 3D multicellular tumour spheroids (MCTS) using phenotype-specific breast cancer cell lines are a valuable experimental model of the tumour microenvironment. Optimisation of MCTS culture growth conditions using different breast cancer cell lines (MCF-7, T47D, MDA-MB-468 and MDA-MB-231) was performed. Unexpected detection of MT1-MMP in MCF-7 MCTS warranted further investigation. MT1-MMP expression in different micro-environmental conditions, including hypoxia and nutrient deprivation (serum-free induced autophagy) were measured in MCF-7 monolayer cultures and MCTS models using immunofluorescence (IF), immunohistochemistry (IHC) and western blot (WB). MT1-MMP expression was rapidly and irreversibly up-regulated in MCF-7 breast cancer cells under conditions of stress (hypoxia and autophagy) compared to normal conditions suggesting an important role of the culture environment on cells behaviour and protein expression. We employed isobaric tags for relative and absolute quantitation (iTRAQ) technology to correlate MT1-MMP increase with proteomic profiles in MCF-7 breast cancer cell grown under hypoxic, serum-free and 3D MCTS conditions. More than 3500 proteins were identified, which were clustered into groups based on response to unique or shared microenvironment changes. Hypoxic monolayer and spheroid cells exhibited changes in anaerobic metabolism and lipid synthesis, respectively, whereas autophagy resulted in up-regulation of cellular component disassembly. The result indicated multiple drivers of MT1-MMP expression in MCF-7 cells.
Al-Mstansiriya University, Iraq

The toxicity of platinum anticancer drugs presents a major obstacle in the effective treatment of tumours. Much of the toxicity stems from a lack of specificity of the drugs for the sites at which they are able to exert maximum anticancer activity. An improved understanding of the behaviour of the drugs in the tumour environment may assist in the rational design of future platinum anticancer agents with enhanced specificity and reduced toxicity. In the work presented herein, the specificity of two classes of platinum anticancer agents was assessed (platinum(IV) cisplatin analogues and platinum(II) anthraquinone complexes). The interaction of the platinum(IV) agents with DNA, believed to be their main cellular target, was examined using XANES spectroscopy. This experiment was designed to assess the ability of the drugs to interact with DNA and thus exert their anticancer activity. It was shown that the platinum(IV) complexes were not reduced by DNA during 48 hr incubation. It was not possible to conclusively determine whether the interaction of the complexes with DNA was direct or platinum(II) catalysed, or whether interaction had occurred at all. The distribution of platinum(II) anthraquinone complexes and their corresponding anthraquinone ligands in tumour cells (A2780 ovarian and DLD-1 colon cancer cell lines) was investigated. The cytotoxicity of the compounds in DLD-1 cells was also assessed. It was found that the compounds were efficiently taken up into the cells and entered the lysosomal compartments almost exclusively. This suggested that the cytotoxicity of the drugs was caused by lysosomal disruption, or that the platinum complexes were degraded, leaving a platinum species to enter the cell nuclei and interact with DNA. Alternatively, the complexes may bind to proteins and transport into the nuclei of the cells, though with their fluorescence quenched by the protein. The penetration and distribution of platinum(IV) complexes was assessed in DLD-1 multicellular tumour spheroids (established models of solid tumours) using a number of synchrotron techniques, including micro-tomography, micro-SRIXE, and micro-XANES. The complexes were found to be capable of penetrating throughout the entire volume of the spheroids. Micro-XANES indicated that in central and peripheral spheroidal regions, bound platinum species were present largely as platinum(II).

Doctor of Philosophy (PhD)
The toxicity of platinum anticancer drugs presents a major obstacle in the effective treatment of tumours. Much of the toxicity stems from a lack of specificity of the drugs for the sites at which they are able to exert maximum anticancer activity. An improved understanding of the behaviour of the drugs in the tumour environment may assist in the rational design of future platinum anticancer agents with enhanced specificity and reduced toxicity. In the work presented herein, the specificity of two classes of platinum anticancer agents was assessed (platinum(IV) cisplatin analogues and platinum(II) anthraquinone complexes). The interaction of the platinum(IV) agents with DNA, believed to be their main cellular target, was examined using XANES spectroscopy. This experiment was designed to assess the ability of the drugs to interact with DNA and thus exert their anticancer activity. It was shown that the platinum(IV) complexes were not reduced by DNA during 48 hr incubation. It was not possible to conclusively determine whether the interaction of the complexes with DNA was direct or platinum(II) catalysed, or whether interaction had occurred at all. The distribution of platinum(II) anthraquinone complexes and their corresponding anthraquinone ligands in tumour cells (A2780 ovarian and DLD-1 colon cancer cell lines) was investigated. The cytotoxicity of the compounds in DLD-1 cells was also assessed. It was found that the compounds were efficiently taken up into the cells and entered the lysosomal compartments almost exclusively. This suggested that the cytotoxicity of the drugs was caused by lysosomal disruption, or that the platinum complexes were degraded, leaving a platinum species to enter the cell nuclei and interact with DNA. Alternatively, the complexes may bind to proteins and transport into the nuclei of the cells, though with their fluorescence quenched by the protein. The penetration and distribution of platinum(IV) complexes was assessed in DLD-1 multicellular tumour spheroids (established models of solid tumours) using a number of synchrotron techniques, including micro-tomography, micro-SRIXE, and micro-XANES. The complexes were found to be capable of penetrating throughout the entire volume of the spheroids. Micro-XANES indicated that in central and peripheral spheroidal regions, bound platinum species were present largely as platinum(II).

Intracellular redox potential (IRP) is a measure of how oxidising or reducing the environment is within a cell. It is a function of numerous factors including redox couples, antioxidant enzymes and reactive oxygen species. Disruption of the tightly regulated redox status has been linked to the initiation and progression of cancer. However, there is very limited knowledge about the quantitative nature of the redox potential and pH gradients that exist in cancer tumour models. Multicellular tumour spheroids (MTS) are three-dimensional cell cultures that possess their own microenvironments, similar to those found in tumours. From the necrotic core to the outer proliferating layer there exist gradients of oxygen, lactate, pH and drug penetration. Tumours also have inadequate vasculature resulting in a state of hypoxia. Hypoxia is a key player in metabolic dysregulation but can also provide cells with resistance against cancer treatments, particularly chemotherapy and radiation therapy. The primary hypoxia regulators are HIFs (Hypoxia Inducible Factors) which under low O2 conditions bind a hypoxia response element, inhibiting oxidative phosphorylation and upregulating glycolysis which has two significant implications: the first is an increase in levels of NADPH/NADH, the main electron donors found in cells which impacts the redox state, whilst the second is a decrease in intracellular pH (pHi) because of increased lactate production. Thus, redox state and intracellular pHi can be used as indicators of metabolic changes within 3D cultures and provide insight into cellular response to therapy. Surface-Enhanced Raman Spectroscopy (SERS) provides a real-time, high resolution method of measuring pHi and IRP in cell culture. It allows for quick and potentially portable analysis of MTS, providing a new platform for monitoring response to drugs and therapy in an unobtrusive manner. Redox and pH-active probes functionalised to Au nanoshells were readily taken up by prostate cancer cell lines and predominantly found to localise in the cytosol. These probes were characterised by density functional theory and spectroelectrochemistry, and their in vitro behaviour modelled by the chemical induction of oxidative and reductive stress. Next, targeting nanosensors to different zones of the MTS allowed for spatial quantification of redox state and pHi throughout the structure and the ability to map the effects of drug treatments on MTS redox biology. The magnitude of the potential gradient can be quantified as free energy (ΔG) and used as a measurement of MTS viability. Treatment of PC3 MTS with staurosporine, an apoptosis inducer, was accompanied by a decrease in free energy gradients over time, whereas treatment of MTS with cisplatin, a drug to which they are resistant, showed an increase in viability indicating a compensatory mechanism and hence resistance. Finally, using this technique the effects of ionising radiation on IRP and pHi in the tumour model was explored. Following exposure to a range of doses of x-ray radiation, as well as single and multi-fractionated regimes, IRP and pHi were measured and MTS viability assessed. Increased radiation dosage diminished the potential gradient across the MTS and decreased viability. Similarly, fractionation of a single large dose was found to enhance MTS death. This novel SERS approach therefore has the potential to not only be used as a mode of drug screening and tool for drug development, but also for pre-clinical characterisation of tumours enabling clinicians to optimise radiation regimes in a patient-specific manner.

Microfluidics is a valuable technology for a variety of different biomedical applications. In particular, within cancer research, it can be used to improve upon currently used in vitro screening assays by facilitating the use of 3D cell culture models. One of these models is the multicellular tumour spheroid (MCTS), which provides a more accurate reflection of the tumour microenvironment in vivo by reproducing the cell to cell contact, the development of a nutritional gradient and the formation of a heterogeneous population of cells. Therefore, the MCTS provides a more physiologically relevant in vitro model for testing the efficacy of treatments at the preclinical level. Currently, methods for the formation and culture of spheroids have several limitations, including being labour intensive, being low throughput, producing shear stress towards cells and the hanging drop system being unstable to physical shocks. Recently, microfluidics (especially droplet microfluidics) has been employed for the culture and screening of spheroids, providing a high-throughput methodology which only requires small volumes of fluids and small numbers of cells. However, current issues with droplet microfluidics include complicated droplet gelation procedures and short cell culture times. In this thesis, the use of microfluidic technologies as an approach for spheroid formation and culture are investigated with the aim to create a platform for radiotherapeutic and chemotherapeutic treatment of spheroids using cell lines. Initially, the use of emulsion technology at the macro scale was evaluated to determine the best conditions for spheroid culture. Once this was achieved the spheroids were compared to spheroids using a traditional method and radiotherapeutic treatment was conducted. Subsequently, avenues for miniaturising the developed emulsion-based methods were studied to provide a microfluidic technology. Finally, along with identifying the optimal culture conditions using hydrogels, a microfluidic system that integrated both droplet and single phase microfluidics features was developed for the formation and culture of spheroids. Using the latter, proof of principle experiments were conducted to demonstrate the suitability of the platform for both chemotherapeutic and radiotherapeutic assays within the same device.

High-throughput drug screening (HTS) in live cells is often a vital part of the preclinical anticancer drug discovery process. So far, two-dimensional (2D) monolayer cell cultures have been the most prevalent model in HTS endeavors. However, 2D cell cultures often fail to recapitulate the complex microenvironments of in vivo tumors. Monolayer cultures are highly proliferative and generally do not contain quiescent cells, thought to be one of the main reasons for the anticancer therapy failure in clinic. Thus, there is a need for in vitro cellular models that would increase predictive value of preclinical research results. The utilization of more complex three-dimensional (3D) cell cultures, such as multicellular tumor spheroids (MCTS), which contain both proliferating and quiescent cells, has therefore been proposed. However, difficult handling and high costs still pose significant hurdles for application of MCTS for HTS. In this work, we aimed to develop novel assays to apply MCTS for HTS and drug evaluation. We also set out to identify cellular processes that could be targeted to selectively eradicate quiescent cancer cells. In Paper I, we developed a novel MCTS-based HTS assay and found that nutrient-deprived and hypoxic cancer cells are selectively vulnerable to treatment with inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). We also identified nitazoxanide, an FDA-approved anthelmintic agent, to act as an OXPHOS inhibitor and to potentiate the effects of standard chemotherapy in vivo. Subsequently, in Paper II we applied the high-throughput gene-expression profiling method for MCTS-based drug screening. This led to discovery that quiescent cells up-regulate the mevalonate pathway upon OXPHOS inhibition and that the combination of OXPHOS inhibitors and mevalonate pathway inhibitors (statins) results in synergistic toxicity in this cell population. In Paper III, we developed a novel spheroid-based drug combination-screening platform and identified a set of molecules that synergize with nitazoxanide to eradicate quiescent cancer cells. Finally, in Paper IV, we applied our MCTS-based methods to evaluate the effects of phosphodiesterase (PDE) inhibitors in PDE3A-expressing cell lines. In summary, this work illustrates how MCTS-based HTS yields potential to reveal and exploit previously unrecognized tumor-specific vulnerabilities. It also underscores the importance of cell culture conditions in preclinical drug discovery endeavors.

Le cancer de l'ovaire (CO) est la cinquième cause de décès chez les femmes qui se caractérise par son diagnostic tardif (stades FIGO III et IV) et par l’importance de ses métastases abdominales souvent observées au moment du diagnostic. Le traitement repose sur une chirurgie cytoréductive complète associée à une chimiothérapie. Malheureusement, parmi les patientes ayant une rémission clinique complète après la fin du traitement initial, 60% des personnes atteintes d'un cancer épithélial de l'ovaire (CEO) à un stade avancé rechuteront dans les cinq ans.L'importance de la néo-angiogenèse dans le développement des tumeurs a poussé les chercheurs à étudier d'autres stratégies. Les thérapies anti-angiogéniques ciblant le système vasculaire tumoral sont désormais utilisées en association avec la thérapie cytotoxique standard dans le traitement des CEO. Malheureusement, les progrès réalisés grâce à cette approche offrent encore un succès limité, qui peut s'expliquer en partie par l'interaction hétérotypique entre les cellules endothéliales et la tumeur. Plusieurs études suggèrent un dialogue complexe entre les cellules cancéreuses de l'ovaire (OCC) et les cellules endothéliales (EC) pouvant entraîner une sensibilité différente à la chimiothérapie et aux traitements anti-angiogéniques conduisant à la progression tumorale.L’objectif de la présente étude est d’étudier le rôle des interactions entre EC et OCC dans la prolifération et la chimiorésistance des CEO. Pour modéliser l'endothélium de la tumeur, nous avons utilisé notre modèle de cellules endothéliales AKT activées (E4+EC). Nous avons démontré en utilisant un modèle de coculture 2D que l’endothélium activé induit une prolifération et une chimiorésistance accrues dans les CEO par l’activation de la signalisation de Notch. Nous avons montré que l’expression et l’activation des récepteurs Notch étaient augmentées dans les cultures en coculture et dans les OCC résistantes à la chimiothérapie.L’accumulation d’ascite dans l’abdomen des patientes atteintes de CO semble jouer un rôle clé dans le mécanisme de propagation des OCC. Les OCC isolées flottent généralement dans l'ascite et forment des sphéroïdes multicellulaires. Dans ce contexte, nous avons développé un nouveau modèle 3D de sphéroïde pour étudier les interactions tumeur-endothélium dans un modèle plus proche des conditions in vivo. Nous avons démontré que les E4+EC et OCC formaient des angiosphères organisées avec un noyau de cellules endothéliales entourées par des OCC qui prolifèrent rapidement. Nous avons établi que l'activation de l'AKT dans les EC était nécessaire pour la formation d'angiosphères organisées. Fait intéressant, dans les ascites de patientes CEO, nous avons pu trouver des structures très similaires à nos angiosphères. De plus, dans une cohorte rétrospective de 59 patientes, nous avons montré que les EC étaient AKT activé chez des patientes atteintes de CEO, ce qui confirme l'importance de l'activation d’AKT dans la CEO. De plus, nous avons démontré l'importance du FGF2, de la Pentraxine 3 (PTX3), du PD-ECGF et du TIMP-1 dans l'organisation de l'angiosphères. Enfin, nous avons confirmé le rôle de Notch3/Jag1 dans le cross-talk des OCC-EC dans la prolifération et l'invasion des OCC au péritoine.En conclusion, notre étude illustre l’importance des EC AKT activé dans les CEO. Au vu des résultats mitigés des traitements anti-angiogéniques, se concentrer sur la normalisation vasculaire dans l'angiogenèse pathologique pourrait être plus efficace. Bien que l'AKT soit difficilement ciblable, la caractérisation génétique des tumeurs pourrait potentiellement identifier un sous-ensemble de tumeurs avec une signalisation NOTCH aberrante qui constituerait une cible idéale pour des inhibiteurs spécifiques. Alors que nous nous dirigeons vers la médecine personnalisée et de précision, il pourrait y avoir une place pour l'inhibition de NOTCH dans les CO en combinaison avec d'autres stratégies thérapeutiques
Ovarian cancer (OC) is the most lethal gynecologic malignancy in developed countries and the fifth cause of death among women. OC is a heterogeneous disease, which is characterized by its late diagnosis (FIGO III and IV stages) and the importance of abdominal metastases often observed at the time of diagnosis. The mainstay of treatment involves complete cytoreductive surgery associated with platinum and taxane-based chemotherapy. Unfortunately, among patients achieving complete clinical remission after completion of initial treatment, 60% with advanced epithelial ovarian cancer (EOC) will relapse within five years.The importance of neo-angiogenesis in tumor formation, growth and dissemination has driven researchers to investigate into alternative strategies. Anti-angiogenic therapies targeting tumor vasculature are now used in combination with standard cytotoxic therapy in the treatment of EOC. Unfortunately, the progress achieved by this approach still offers limited success which can partly be explained by the heterotypic interaction between the tumor and endothelial cells. Evidence suggests a complex cross-talk between ovarian cancer cells (OCCs) and endothelial cells (ECs) that can result in the emergence of a heterogeneous tumoral and endothelial population with different sensitivity to chemotherapy and anti-angiogenic therapies leading to an increase of OCC proliferation and dissemination.The objective of the present study is to investigate the role of ECs and OCCs interactions in the proliferation and chemoresistance of EOC. To model tumor endothelium, we used our model of Akt-activated endothelial cells (E4+ECs). We demonstrated using a 2D co-culture model that activated endothelium induces increased proliferation and chemoresistance in EOC through the activation of Notch signaling. We showed that Notch receptor expression and activation are increased in co-culture and in OCCs resistant to chemotherapy.The accumulation of ascites in the abdomen of an OC patient seems to play a key role in the mechanism of OCC spreading. Detached cancer cells usually float in ascites and form multicellular spheroids. In this context, we developed a new model of organized multicellular 3D spheroid to study tumor-endothelium interactions in a model closer to in vivo conditions. We demonstrated that when cocultured in 3D condition, E4+ECs and OCCs formed organized tumor angiospheres with a core of endothelial cells surrounded by highly proliferating OCCs. We established that AKT activation in ECs was mandatory for the formation of organized angiospheres. Interestingly, in EOC patient ascites, we were able to find structures that were very similar to our angiospheres. In addition, in a retrospective cohort of 59 patients, we showed that ECs were AKT activated in EOC patients which support the importance of AKT activation in EC in EOC. Besides, we demonstrated the importance of FGF2, Pentraxin 3 (PTX3), PD-ECGF and TIMP-1 in angiosphere organization. Finally, we confirmed the role of Notch3/Jagged1 in OCCs-ECs crosstalk for OCC proliferation but also during peritoneum invasion.Altogether, our study illustrates the importance of AKT activated ECs in EOC. In a context of poor results of anti-angiogenic therapies in clinical settings, focusing on vascular normalization in pathological angiogenesis could be more efficient. While AKT is hardly targetable, the genetic characterization of tumors could potentially identify a subset of tumors with aberrant NOTCH signaling that would constitute an ideal target for specific inhibitors. As we move toward personalized and precision medicine, there might be a place for notch inhibition in advanced ovarian cancer in combination with other therapeutic strategies

Les sphéroïdes multicellulaires tumoraux (SMT) constituent un outil prometteur dans le domaine de l’étude biologique des tumeurs. Le but de la thèse était de développer une technique de la formation de SMT et de démontrer la disponibilité de ces sphéroïdes comme modèle in vitro 3D pour tester l’efficacité de principes actifs anticancéreux ainsi que celle de formulations de délivrance de médicaments. L'effet d’auto-assemblage de cellules induit par une addition des peptides RGD cycliques a été étudié pour 16 lignées cellulaires de différentes origines. Le peptide cyclique RGDfK et sa modification avec le cation triphenylphosphonium (TPP) ont permis de mettre en évidence l’induction de formation de sphéroïdes. Les sphéroïdes ont été employés comme modèles pour évaluer la cytotoxicité de principes actifs antitumoraux (doxorubicine, curcumine, temozolomide) et un certain nombre de formulations nano- et micrométriques (microréservoirs, nano-émulsions et micelles)
Multicellular tumor spheroids (MTS) are a promising tool in tumor biology. The aim of the Thesis was to develop a novel highly reproducible technique for MTS formation, and to demonstrate the availability of these spheroids as 3D in vitro model to test anticancer drugs and drug delivery vehicles. Cell self-assembly effect induced by an addition of cyclic RGD-peptides directly to monolayer cultures was studied for 16 cell lines of various origin. Cyclo-RGDfK peptide and its modification with triphenylphosphonium cation (TPP) were found to induce spheroid formation. The spheroids were used as a model to evaluate the cytotoxicity of antitumor drugs (doxorubicin, curcumin, temozolomide) and a number of nano- and micro- formulations (microcontainers, nano-emulsions and micelles)

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

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.

Le mélanome métastatique est le cancer de la peau le plus agressif. Bien que son taux d’incidence soit inférieur à 1%, plus de 75% des décès associés à un cancer de la peau lui sont attribués. Au cours des dernières années, de nouvelles stratégies thérapeutiques ont permis d’améliorer la survie des patients. Cependant, des mécanismes de résistance à ces traitements se développent dans la majorité des cas, conduisant à une phase de rechute, et une survie à 5 ans inférieure à 20%. Des modèles d’étude expérimentaux sont nécessaires afin de comprendre les mécanismes impliqués dans l’apparition de ces résistances et développer de nouvelles stratégies thérapeutiques. Différents modèles in vitro sont actuellement utilisés pour le développement de drogues anti-tumorales, tels que celui du sphéroïde. Bien qu’il permette de reproduire l’organisation tridimensionnelle d’une tumeur, l’absence de microenvironnement tumoral empêche l’étude des interactions entre les cellules tumorales et celui-ci alors que ces facteurs jouent un rôle primordial dans la croissance tumorale et le développement de métastases. Dans ce contexte, mes travaux ont porté sur le développement et la caractérisation d’un modèle ex vivo de mélanome humain complet permettant l’étude de l’évolution d’une tumeur dans le tissu sain et l’évaluation de composés pharmacologiques. Les travaux réalisés ont tout d’abord conduit au développement d’un modèle de cancer cutané basé sur la combinaison d’un modèle de sphéroïde de lignée cellulaire de mélanome humain et du modèle de peau humaine ex vivo NativeSkin®, développé par la société Genoskin. Une procédure a été développée et validée pour permettre l’implantation reproductible d’un sphéroïde dans le derme des explants de peau. Parallèlement, j’ai développé une approche d’imagerie in situ par microscopie à feuille de lumière après transparisation des modèles. J’ai également développé une stratégie d’analyse d’images permettant la caractérisation quantitative de l'évolution du sphéroïde implanté en 3 dimensions et de suivre la dispersion des cellules du tumorales au sein de l’explant de peau. La caractérisation histologique du modèle implanté a révélé de façon très inattendue une perte progressive de l’intégrité du sphéroïde après implantation, associée à une diminution rapide de la prolifération des cellules le constituant et l’apoptose massive des cellules situées à sa périphérie. Ce phénomène a été observé de façon similaire lors de l’implantation de sphéroïdes produits à partir de différents types cellulaires. Afin de comprendre ces résultats, j’ai étudié l’implication potentielle de différents paramètres dans l’induction de la mortalité cellulaire observée tels que les conditions d’implantation, les facteurs synthétisés par le modèle et la contrainte mécanique exercée par le derme. Les résultats obtenus suggèrent que les facteurs sécrétés par les modèles après implantation du sphéroïde ont un effet antiprolifératif sur les sphéroïdes de mélanome et qu’ils induisent la mortalité des cellules situées à sa périphérie. Par ailleurs, l’application d’une contrainte mécanique extérieure sur les sphéroïdes de mélanome entraîne la perte de la cohésion de leur structure. Enfin, l’implantation de sphéroïdes dans le derme de biopsies de peau préalablement desséchées, induisant une perte de la viabilité cellulaire, a conduit à des résultats opposés à ceux observés avec de la peau normale : la structure des sphéroïdes reste cohésive et la prolifération des cellules est maintenue en périphérie du sphéroïde sans qu’aucune apoptose massive ne soit observée. L'ensemble de ces travaux semble suggérer que la mortalité du sphéroïde pourrait être, en partie, la conséquence d’une contrainte mécanique exercée par la peau sur le sphéroïde et/ou de facteurs produits par la peau durant sa culture. Ces données ouvrent des perspectives intéressantes dans le domaine de l’ingénierie tissulaire pour l’évaluation pharmacologique de composés thérapeutiques
Malignant melanoma is the most aggressive form of skin cancer. Although it only occurs in less than 1%, it is responsible for more than 75% of skin cancer-related deaths. Furthermore, melanoma incidence has constantly increased during the last decades. New therapies such as targeted therapy and immunotherapy have emerged over the past years, significantly improving the overall survival rates of patients with advanced melanoma stages. However, resistance to those treatments develops in most cases, leading to relapse with a 5-years survival of those patients under 20%. Experimental models are needed in order to better understand the molecular events underlying these resistance mechanisms, and to develop new therapeutic strategies. MultiCellular Tumor spheroid is an increasingly recognized 3D in vitro model for pharmacological evaluation. Although this model accurately reproduces the 3D architecture, cell-cell interaction and cell heterogeneity found in microtumor in vivo, spheroids lack tumor-microenvironment interactions, which play a key role in tumor growth and metastasis development. In this context, the aim of my project was to develop and characterize a fully ex vivo human melanoma model for the study of tumor growth within the skin and the evaluation of antitumor drugs. Our approach relies on the combination of human melanoma cell lines grown in Multicellular Tumor Spheroids and the NativeSkin® model, an ex vivo human skin model produced by the biotechnology company Genoskin. Hence, I developed and validated a method to reproducibly implant one spheroid into the dermal compartment of skin explants cultured ex vivo. In parallel I have developed in situ imaging strategies based on light-sheet microscopy (SPIM, “Selective Plane Illumination Microscopy”) after optical clearing of the implanted skin biopsies. I also developed analytic methods to allow for the quantitative characterization of the spheroids evolution in 3 dimensions as well as tumor cells dispersal within the dermis of skin explants. Histological characterization of the implanted models over time revealed a progressive loss of the spheroids integrity after implantation associated with a rapid decrease in cell proliferation and massive apoptosis of the cells located in the peripheral layers. These results were shared by implanted spheroids made from different cell types. Further experiments were conducted in order to better understand these results and evaluate the impact of different parameters on the implanted microtumors viability such as the implantation procedure conditions, factors synthesized by the model after spheroid implantation and external mechanical stress. Results suggest that factors produced by the implanted models have an antiproliferative effect on melanoma spheroids and induce mortality in the peripheral layers of the spheroids. Moreover, results show that mechanical stress applied on melanoma spheroids induces loss of their cohesion. Finally, implantation of spheroids within the dermis of previously dessicated biopsies for 7 days, causing loss of skin cells viability, led to opposite results than in normal skin: spheroids maintain both a cohesive structure and proliferation in the peripheral cells without any massive apoptosis. Overall, this work led to the validation of a methodology to reproducibly implant spheroids into an ex vivo skin explant and the setup of an optical clearing technique necessary for in situ imaging of the implanted spheroid. Histological characterization unexpectedly revealed spheroids cells death following their implantation. Results suggest that this mortality could be partly related to mechanical stress exerted on the spheroids by the skin and/or by factors produced by the skin during culture. These data open new perspectives in the research field of tissue engineering for antitumoral pharmacology

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

Les thérapies anticancéreuses basées sur des principes physiques (radiofréquences, ultrasons, laser, électroporation...) ont considérablement augmenté lors de la dernière décennie. Leurs objectifs sont de détruire directement les cellules cancéreuses, de favoriser l'entrée ciblée de molécules thérapeutiques ou encore de stimuler le système immunitaire du patient afin d'éliminer la tumeur. Le plasma froid suscite l'intérêt dans le domaine de l'oncologie grâce à sa capacité à générer des espèces réactives oxygénées (ROS) et azotées (RNS) qui peuvent être génotoxiques et cytotoxiques pour les cellules cancéreuses. Deux approches d'utilisation du plasma sont étudiées : soit l'exposition directe de cellules au jet plasma, soit l'exposition indirecte via l'utilisation d'un Milieu Activé par Plasma (PAM). Le PAM étant plus facile à délivrer par injection dans la tumeur, c'est cette approche qui est choisie lors de ces travaux. Le travail de thèse présenté consiste à étudier l'effet génotoxique et cytotoxique du PAM, obtenu après exposition du milieu au jet de plasma d'hélium, sur des tumeurs in vitro et in vivo. Pour les études in vitro, nous avons choisi d'utiliser un modèle 3D : le sphéroïde (MCTS - MultiCellular Tumor Spheroid). Ce modèle présente des caractéristiques proches du modèle in vivo grâce à son organisation en sphéroïde. Les MCTS présentent en effet des gradients de pénétration d'oxygène, de nutriments et de prolifération cellulaire. La première partie de la thèse concerne l'identification et la quantification des espèces générées dans le PAM. Les méthodes d'analyses utilisées sont la résonance paramagnétique électronique, la fluorimétrie, la colorimétrie, la chromatographie en phase liquide et la spectrométrie de masse. Ces analyses ont mis en évidence que la toxicité du PAM était due à plusieurs facteurs : d'un côté la génération de ROS et RNS mais aussi à la dégradation des nutriments pour les cellules contenues dans le milieu via par exemple l'oxydation et la nitrosylation des acides aminés. La deuxième partie est dédiée à l'étude des effets du PAM sur les MCTS HCT-116 (cancer du côlon).[...]
Cancer therapies based on physical principles (radiofrequency, ultrasound, laser, electroporation...) have considerably increased in the last decade. Their objectives are to directly destroy cancer cells, to favor the targeted entry of therapeutic molecules or to stimulate the patient's immune system in order to eliminate the tumor. Cold plasma still arouses interest in the field of oncology through its ability to generate reactive oxygen species (ROS) and nitrogen species (RNS) which can be genotoxic and cytotoxic for cancer cells. Two approaches to the use of plasma are studied: either direct exposure of cells to the plasma jet, or indirect exposure via the use of a Plasma Activated Medium (PAM). The PAM being easier to deliver by injection into the tumor, this approach was chosen in this work. The work presented consists in studying the genotoxic and cytotoxic effects of PAM resulting from exposure of the medium to the helium plasma jet on in vitro and in vivo tumors. For in vitro studies, we chose to use a 3D model: the spheroid (MCTS - MultiCellular Tumor Spheroid). This model has similar characteristics to the in vivo model thanks to its spheroidal organization. The spheroids have indeed gradients of oxygen penetration, nutrients and cell proliferation. The first part of the thesis concerns the identification and quantification of the species generated in PAM. The analytical methods used are paramagnetic electronic resonance, fluorimetry, colorimetry, liquid chromatography and mass spectrometry. These analyses revealed that the toxicity of PAM was due to several factors: on the one hand to the generation of ROS and RNS and on the other hand to the degradation of cell nutrients contained in the medium via, for example, the oxidation and nitrosylation of the amino acids. The second part is dedicated to the study of the effects of PAM on HCT-116 (colon cancer) spheroids[...]

D.Tech. (Biomedical Technology)
Photodynamic therapy (PDT) is an alternative treatment modality for malignant tumours based on the photodamage to tumour cells through a photochemical reaction (Ahn et al., 2013). PDT utilizes a light sensitive photosensitizer (PS) that selectively localizes in tumour cells and is excited by light of a specific wavelength in the presence of molecular oxygen. The excited PS leads to the generation of singlet oxygen or other reactive oxygen species(ROS) which induces cytotoxic damage to cellular organelles and eventually cell death. Singlet oxygen has a very short life and its generation is controlled by the presence of the PS and the laser light (Senge and Radomski, 2013).The subcellular localization site of the PS plays a vital role in determining the effectiveness and the extent of cellular damage as well as the mechanism involved in cell death. Lung cancer is the leading cause of cancer death worldwide in both males and females, with an estimated 1.4 million deaths each year (American Cancer Society, 2011). Therapeutic modalities used in the treatment of lung cancer such as chemotherapy, radiotherapy and immunotherapy have rarely yielded a good prognosis and effective treatment remains a challenging problem to date. An alternative treatment modality with minimal complications such as PDT needs to be explored. Most in vitro PDT experiments are conducted on monolayer cultures and the cellular environment of these cultures does not correspond to that of in vivo studies. Multicellular tumour spheroids (MCTSs) serves as an important model in cancer research for the evaluation of therapeutic interventions since they mimic different aspects of the human tumour tissue environment.

The cell cycle is a tightly regulated process that is functioning optimally when all factors contribute at the precise time and level that is required. However, in tumours some of these pathways are malfunctioning and to study this, we are utilizing spheroids as our in vitro tumour model system. In this thesis, we hypothesize that spheroids once adequately characterized will provide an invaluable means to assess the potential for using cytostatic agents to minimize accelerated repopulation and target radioresistant cells during radiation therapy of multicellular systems. The general methods involved for the majority of the work included immunoblotting, flow cytometry, as well as clonogenic assays. We have studied differences in the cell cycle time of subpopulations within each spheroid, where cells near the necrotic center showed a prolonged cell cycle time. Cycle time required by the peripheral cell layers was more rapid suggesting that cell kinetic variations may likely be a result of the local microenvironmental conditions such as hypoxia. Cyclin B1 and D levels were measured in spheroid populations that were subjected to a range of anoxic to aerobic conditions where cyclin levels decreased with decreasing concentrations of oxygen. Interestingly, these observations illustrate that measuring cyclin levels can provide a quick and convenient index for proliferation rate. The cytostatic agent, rapamycin, was analyzed for its influence on cell cycle progression and its affect on cyclin levels. Using human WiDr tumour xenografts, we found that the combination of rapamycin treatment with radiation had additive therapeutic benefits. Accelerated repopulation has been established as a limitation in radiation therapy of some cancers. Therapeutic advances have led to using accelerated fractionation regimens in an attempt to counteract the tumours’ inherent ability to grow in the face of continued therapy. Since accelerated repopulation of irradiated tumours may be associated with the recruitment of quiescent cells into the cell cycle, rapamycin can potentially be used to control those cells. By incorporating rapamycin and concurrently administering radiation, we found a synergistic effect where the cell cycle time of the solid xenograft tumours were even slower than with either treatment alone.
Medicine, Faculty of
Medicine, Department of
Experimental Medicine, Division of
Graduate

Multicellular tumor spheroid cultures (MCTS), have a wide variety of uses in the field of cancer treatment. Current methods, however, do not provide spheroids adequate large for therapy testing. In order to circumvent this problem, a bioreactor made using Polydimethylsiloxane (PDMS) was constructed to allow the adequate growth of spheroid clusters. Liver Hepatic Carcinoma cells (Hep G2) and Anaplastic Thyroid Carcinoma (SW 1736) were cultured and isolated. They were then further matured using the “hanging drop” method, involving spheroid formation using gravity. The optimal growth time for hanging drop cultures was determined to be 72 hours. PDMS wells of different diameter were then constructed using a 24-well plate as a base, and clusters of cells were transferred into them for MCTS formation. The wells were fabricated using PDMS as a mold, then carving out wells for cell growth. Development of the spheroids in the bioreactor was monitored using microscopy paired with various staining techniques, and measurements and media changes were done daily. Cell cluster volume and height were analyzed as a function of PDMS well diameter. Optimum spheroid formation occurred in 5mm diameter wells, with peaking growth around 10 days. Further research was done regarding cancer cells and HIFU (High Intensity Focused Ultrasound) and Ethanol treatments, as tests of combination cancer therapies. Cells were first treated as cell pellets, then using the spheroid method above. The use of combination therapies proved more effective than either therapy alone. Chemo-ablation is also an upcoming therapy method to be used in combination therapies. This research can be used as a starting point for larger spheroid formation, and eventually in vivo testing of cancer therapies. Further analysis can be done to compare these models for comparison of shape, viability, and the effects of anti-cancer treatments.
acase@tulane.edu

Cancer is the second-leading cause of death worldwide and regarding the bone-related cancers, osteosarcoma (OS) is the most common primary malignant tumor which is characterized by its high potential to metastasize, predominantly affecting children and adolescents. Despite the innumerable efforts towards the development of new anti-cancer therapies, several efficacious drug candidates identified in preclinical tests fail during clinical trials. Three-dimensional (3D) cell culture systems have been proposed as reliable in vitro platforms for tumor modelling in an attempt to closely reproduce the tumor pathophysiology and properly identify effective therapies. Human methacryloyl platelet lysates (PLMA)-based hydrogels were recently proposed as cost-effective and biologically relevant 3D in vitro platforms for human cell growth and proliferation. Therefore, the aim of this work was, in a first approach, to validate the PLMA hydrogels as reliable 3D platforms to support in vivo-like cell spheroid invasion mechanisms and subsequently explore that potential to establish humanized 3D mono- and co-culture OS invasion models for drug screening and validation. Spheroids of three tumor cell lines (MG-63, SaOS-2 and A549) and human bone marrow-derived mesenchymal stem cells (hBM-MSC) were embedded into PLMA hydrogels (three different concentrations), Matrigel and poly(ethylene glycol) diacrylate. PLMA hydrogels demonstrated to support the phenotypic heterogeneity of solid tumors and the acquisition of an in vivo-like cell polarity. Furthermore, these hydrogels perfectly recapitulated the cell invasiveness ability, demonstrating that invasion speed can be easily controlled through PLMA hydrogel stiffness. The co-culture of MG-63 spheroids with human osteoblasts and hBM-MSCs demonstrated that the crosstalk between tumor invading cells and stromal cells was truly recapitulated into PLMA hydrogels. A doxorubicin treatment in mono- and co-culture OS models clearly reflected the protective role of stromal cells in OS chemoresistance, exhibiting a drug response closest to in vivo. Overall, the results validated the humanized PLMA-based hydrogels as reliable 3D in vitro platforms to support an in vivo-like tumor morphology and invasiveness. Moreover, the complexity of the established co-culture OS model provided a more pathophysiological in vitro environment for screening and validation of anti-metastatic agents in order to predict patients’ response and expedite the availability of effective therapies.
O cancro é a segunda principal causa de morte a nível mundial e, relativamente ao cancro do osso, o osteossarcoma (OS) é o tumor maligno primário mais comum caracterizado pelo seu elevado potencial metastático, afetando predominantemente crianças e adolescentes. Apesar dos inúmeros esforços que visam o desenvolvimento de novas terapias anti-cancerígenas, vários candidatos a fármacos identificados como eficazes nos testes pré-clínicos falham durante os ensaios clínicos. Os sistemas de cultura de células tridimensionais (3D) têm sido propostos como plataformas in vitro fidedignas para o desenvolvimento de modelos tumorais na tentativa de reproduzir a patofisiologia tumoral e identificar terapias eficazes. Hidrogéis baseados em lisados de plaquetas humanos metacrilatados (PLMA) foram recentemente propostos como plataformas in vitro 3D economicamente viáveis e biologicamente relevantes para o crescimento e proliferação de células humanas. Assim, o objetivo deste trabalho foi, numa primeira abordagem, validar os hidrogéis de PLMA como plataformas 3D para suportar mecanismos de invasão de esferóides e, subsequentemente, explorar esse potencial para estabelecer modelos humanizados 3D de OS em mono- e co-cultura para teste e validação de fármacos. Esferóides de três linhas celulares tumorais (MG-63, SaOS-2 e A549) e células estaminais mesenquimais humanas derivadas da medula óssea (hBM-MSC) foram embebidos em hidrogéis de PLMA (três concentrações diferentes), Matrigel e poli(etileno glicol) diacrilatado. Os hidrogéis de PLMA demonstraram suportar a heterogeneidade fenotípica dos tumores sólidos e a aquisição de uma polaridade celular semelhante à in vivo. Além disso, estes hidrogéis recapitularam perfeitamente a capacidade de invasão celular, demonstrando que a velocidade de invasão pode ser facilmente controlada através da rigidez dos hidrogéis de PLMA. A co-cultura de esferóides de MG-63 com osteoblastos humanos e hBM-MSCs demonstrou que a comunicação entre as células tumorais invasivas e as células estromais foi fielmente recapitulada nos hidrogéis de PLMA. Um tratamento com doxorubicina nos modelos de OS em mono- e co-cultura claramente refletiu o papel protetivo das células estromais na quimioresistência em OS, exibindo uma resposta ao fármaco mais próxima da obtida in vivo. Globalmente, os resultados validaram os hidrogéis humanizados baseados em PLMA como plataformas in vitro 3D fidedignas para suportar uma morfologia e invasão tumoral semelhante à in vivo. Além disso, a complexidade do modelo de OS de co-cultura estabelecido forneceu um ambiente in vitro mais patofisiológico para o teste e validação de agentes anti-metastáticos de modo a prever a resposta do paciente e acelerar a disponibilidade de terapias efetivas.
Mestrado em Biotecnologia

碩士
國立成功大學
生物醫學工程學系
106
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. It has a poor prognosis because it is often diagnosed in the advanced stage when treatments are limited. Three-dimensional (3D) cell culture models have become powerful tools in cancer research, as they better simulate the in vivo physiological microenvironment than traditional 2D cell cultures. Tumor cells cultured in a 3D system as multicellular tumor spheroids (MTS) recapitulate several critical in vivo characteristics that allow the study of biological functions and drug discovery. Microwell technology is best platform for generating MTS as it provides geometrically defined microstructures for culturing size-controlled MTS amenable for various downstream functional assays. This thesis presents a simple and economical microwell fabrication methodology that be conveniently incorporated into the conventional workflow used to generate MTS. This study had three main objectives: (1) To perform rapid prototyping of size-controlled microwells using a conventional CO2 laser engraver, and to control the variable sizes by fine-tuning the parameters of microwell prototyping to generate hepatic MTS; (2) To combine microwell technology with conventional multi-well plate-based cell culture methods for proof-of-concept of high-throughput drug screening; (3) To explore novel therapeutic interventions through photothermal treatment of concanavalin A (ConA)-modified silica–carbon hollow spheres (SCHSs). The microwells were 400–700 µm in diameter, and hepatic MTS cultured in them for up to 5 days grew to 250–520 µm with good viability and shape. To demonstrate the ability to integrate the microwell fabrication with a high-throughput workflow using the conventional multi-well plate system, a conventional 96-well plate was employed for proof-of-concept drug screening. The half maximal inhibitory concentrations of doxorubicin were determined to be 9.3 µM in 2D conditions and 42.8 and 52.3 µM in both 3D conditions, namely microwells fabricated at focal lengths and laser powers of 0 mm and 10 W, and -3 mm and 15 W, respectively. The optimal concentration for ConA binding to SCHSs was 500:200 µg/mL after a 2 h incubation to best bind with MTS. Based on this concentration for further photothermal treatment, the live/dead cell viability assay assessed the relative cell viability through exposure to 3 W/cm2 near-infrared laser for 20 min. The relative fluorescence intensity showed an eight-fold reduction in cell viability, confirming the feasibility of photothermal treatment as a potential therapeutic intervention. In conclusion, using the microwell platform to generate MTS may be an effective tool for discovering therapeutic modalities for cancer treatment.