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Shahi, Thakuri Pradip. "MODELING ANTI-CANCER DRUG RESISTANCE USING TUMOR SPHEROIDS." University of Akron / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=akron1574725861735168.
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Karamikamkar, Solmaz. "Development and evaluation of a novel approach to producing uniform 3-D tumor spheroid constructs." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/56296.
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In vitro tumor spheroid models have been developed using microfluidic systems to generate 3-D hydrogel beads containing components of alginate and ECM protein, such as collagen, with high uniformity and throughput. During bead gelation, alginate acts as a fast gelling component helping to maintain the spherical shape of beads and to prevent adjacent or underlying beads from coalescing when working with the slower gelling temperature and pH-sensitivity of collagen components. There are also well-known limitations in using microfluidic systems when working with temperature-sensitive components of collagen type I, and it is determined that to produce uniform hydrogel droplets through a microfluidic system, the mixtures must be homogeneous. However, the issue of collagen’s sensitivity to temperature causes concern for chunks of collagen gel inside of the mixture before bead encapsulation; therefore causing the mixture to become non-uniform and risking chip clogging. In order to overcome this limitation, previous approaches have used a cooling system during bead encapsulation while tumor cells were also present in the mixture, but this procedure assisted in postponing collagen gelation prior to bead production and potentially contributing to a delay in cell proliferation.
Here a novel yet simple method is developed to prepare homogeneous pre-bead-encapsulation-mixtures containing collagen through ultrasonication, while extending cell viability and proliferation. This method allows the cultivation of homogenous TS cultures with high uniformity and compact structure, and not only maintains cell viability but also stimulates the proliferation of cells in alginate/collagen hydrogel bead cultures. Depending on the sonication parameters, time and temperature, gelation of collagen is controlled by small sized fibrils to thick fibers. Human-source-Michigan-Cancer-Foundation-7 (MCF-7) cells isolated from a breast cancer cell line are successfully incorporated into alginate/collagen mixtures, followed by sonication, and then bead production. After bead gelation, the encapsulated MCF-7 cells remained viable and proliferated to form uniform TSs when the beads contained alginate and collagen. Results indicate that ultrasound treatment provides a powerful technique to change the structure of collagen from fiber to fibril, and to disperse collagen fibers in the mixture homogeneously for an application to generate uniform hydrogel beads and spheroids while not disturbing cell proliferation.<br>Applied Science, Faculty of<br>Graduate
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Kim, Kihwan. "MULTICEULLULAR TUMOR HEMI-SPHEROID: A NOVEL IN VITRO 3D MODEL PLATFORM FOR ACCELERATED DRUG DEVELOPMENT." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1481900120946458.
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Inamdar, Sharvari Satish. "THE EFFECT OF CHEMOTHERAPY DRUGS ON GLOBAL OXYGEN UPTAKE IN A MULTICELLULAR TUMOR HEMI-SPHEROID." Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1595958820583196.
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Lama, Rati. "Preclinical evaluation and identification of potent tubulin and Hsp27 inhibitors as anticancer agents." Cleveland State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=csu1430232901.
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Morrone, Luigi. "The Influence of 3D Cell Organization in Tumor Spheroid on Natural Killer Cell Infiltration and Migration." Thesis, KTH, Skolan för kemi, bioteknologi och hälsa (CBH), 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-286605.
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Natural Killer cells are a type of lymphocyte belonging to the innate immune system and they operate cell-mediated cytotoxicity and release of pro-inflammatory cytokines against cancerous cells. However, in vivo testings have shown a reduced activity of NK cells against solid tumors probably due to the negative influence of the immunosuppressive tumor microenvironment. Multicellular tumor spheroids may constitute an advantageous model in cancer biology for studying the mechanisms behind cancer immune editing since it more closely mimics the complexity of the human body compared with the 2D model counterpart. This study investigated the interaction between NK cells isolated from blood and tumor spheroids obtained from A498 renal carcinoma cells, using light-sheet microscopy imaging which allows satisfactory cell tracking in the inner layers of the spheroids. NK cells not only indeed interact with tumor spheroids, but many of them were able to penetrate the spheroids inducing some changes in the structure of the latter. NK cells were also tracked over time, displaying the migration path and calculating the speed. The fluorescence intensity of the NK cells was found reduced as soon as they penetrate the spheroid but, conversely, the speed seems to increase inside the spheroid, a possible sign of the fallibility of the tracking algorithm in this specific case. We propose solutions for more sophisticated future implementations, involving the use of marks during the experimental phase and drift corrections at the data analysis level.
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Maekawa, Hisatsugu. "A Chemosensitivity Study of Colorectal Cancer Using Xenografts of Patient-Derived Tumor Initiating Cells." Kyoto University, 2018. http://hdl.handle.net/2433/235985.
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Mascheroni, Pietro. "Mathematical modeling of avascular tumor growth." Doctoral thesis, Università degli studi di Padova, 2016. http://hdl.handle.net/11577/3425310.
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Cancer is an extremely complex disease, both in terms of its causes and consequences to the body. Cancer cells acquire the ability to proliferate without control, invade the surrounding tissues and eventually form metastases. It is becoming increasingly clear that a description of tumors that is uniquely based on molecular biology is not enough to understand thoroughly this illness. Quantitative sciences, such as physics, mathematics and engineering, can provide a valuable contribution to this field, suggesting new ways to examine the growth of the tumor and to investigate its interaction with the neighboring environment. In this dissertation, we deal with mathematical models for avascular tumor growth. We evaluate the effects of physiological parameters on tumor development, with a particular focus on the mechanical response of the tissue. We start from tumor spheroids, an effective three-dimensional cell culture, to investigate the first stages of tumor growth. These cell aggregates reproduce the nutrient and proliferation gradients found in the early stages of cancer and can be grown with a strict control of their environmental conditions. The equations of the model are derived in the framework of porous media theory, and constitutive relations for the mass transfer terms and the mechanical stress are formulated on the basis of experimental observations. The growth curves of the model are compared to the experimental data, with good agreement for the different experimental settings. A new mathematical law regulating the inhibitory effect of mechanical compression on cancer cell proliferation is also presented. Then, we perform a parametric analysis to identify the key parameters that drive the system response. We conclude this part by introducing governing equations for transport and uptake of a chemotherapeutic agent, designed to target cell proliferation. In particular, we investigate the combined effect of compressive stresses and drug action. Interestingly, we find that variation in tumor spheroid volume, due to the presence of a drug targeting cell proliferation, depends considerably on the compressive stress level of the cell aggregate. In the second part of the dissertation, we study a constitutive law describing the mechanical response of biological tissues. We introduce this relation in a biphasic model for tumor growth based on the mechanics of fluid-saturated porous media. The internal reorganization of the tissue in response to mechanical and chemical stimuli is described by enforcing the multiplicative decomposition of the deformation gradient tensor associated with the solid phase motion. In this way, we are able to distinguish the contributions of growth, rearrangement of cellular bonds, and elastic distortion, occurring during tumor evolution. Results are presented for a benchmark case and for three biological configurations. We analyze the dependence of tumor development on the mechanical environment, with particular focus on cell reorganization and its role in stress relaxation. Finally, we conclude with a summary of the results and with a discussion of possible future extensions.<br>Il cancro è una malattia estremamente complessa, sia per quanto riguarda le sue cause che per i suoi effetti sul corpo. Le cellule del cancro acquisiscono la capacità di proliferare senza controllo, invadere i tessuti vicini e infine sviluppare metastasi. Negli ultimi anni sta diventando sempre più chiaro che una descrizione dei tumori basata unicamente sulla biologia molecolare non può essere sufficiente per comprendere interamente la malattia. A questo riguardo, scienze quantitative come la Fisica, la Matematica e l'Ingegneria, possono fornire un valido contributo, suggerendo nuovi modi per esaminare la crescita di un tumore e studiare la sua interazione con l'ambiente circostante. In questa tesi ci occupiamo di modelli matematici per la crescita avascolare dei tumori. Valutiamo gli effetti dei parametri fisiologici sullo sviluppo del tumore, con un'attenzione particolare alla risposta meccanica del tessuto. Partiamo dagli sferoidi tumorali, una cultura cellulare tridimensionale, per studiare le prime fasi della crescita tumorale. Questi aggregati cellulari sono in grado di riprodurre i gradienti di nutriente e proliferazione che si ritrovano nei tumori avascolari. Inoltre, possono essere fatti crescere con un controllo molto severo delle condizioni ambientali. Le equazioni del modello sono derivate nell'ambito della teoria dei mezzi porosi dove, per chiudere il problema, definiamo opportune relazioni costitutive al fine di descrivere gli scambi di massa tra i diversi componenti del sistema e la risposta meccanica di quest'ultimo. Tali leggi sono formulate sulla base di osservazioni sperimentali. Le curve di crescita del modello sono quindi confrontate con dati sperimentali, con un buon accordo per le diverse condizioni. Presentiamo, inoltre, una nuova espressione matematica per descrivere gli effetti di inibizione della crescita da parte della compressione meccanica sulle cellule cancerose. In seguito, eseguiamo uno studio parametrico per identificare i parametri chiave che guidano la risposta del sistema. Concludiamo infine questa parte introducendo le equazioni di governo per il trasporto e il consumo di un agente chemioterapico, studiato per essere efficace sulle cellule proliferanti. In particolare, consideriamo l'effetto combinato di stress meccanici compressivi e di tale farmaco sullo sviluppo del tumore. A questo proposito, i nostri risultati indicano che una variazione di volume degli sferoidi tumorali, a causa dell'azione del farmaco, dipende sensibilmente dal livello di tensione a cui è sottoposto l'aggregato cellulare. Nella seconda parte di questa trattazione, studiamo una legge costitutiva per descrivere la risposta meccanica di tessuti biologici. Introduciamo questa relazione in un modello bifasico per la crescita tumorale basato sulla meccanica di mezzi porosi saturi. La riorganizzazione interna del tessuto in risposta a stimoli meccanici e chimici è descritta attraverso la decomposizione moltiplicativa del gradiente di deformazione associato con il moto della fase solida del sistema. In questo modo, risulta possibile distinguere i contributi di crescita, riarrangiamento dei legami cellulari e distorsione elastica che prendono luogo durante l'evoluzione del tumore. In seguito, presentiamo risultati per un caso di test e per tre configurazioni di interesse biologico. In particolare, analizziamo la dipendenza dello sviluppo del tumore dal suo ambiente meccanico, con un'attenzione particolare sulla riorganizzazione dei legami tra le cellule e il suo ruolo sul rilassamento degli stress meccanici. Infine, concludiamo la discussione con un breve riassunto dei risultati ottenuti e un resoconto dei possibili sviluppi.
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Marrero, Bernadette. "Evaluation of Immunogene Therapy Using a Plasmid Encoding IL-15 Delivered by Electroporation in a 3D Tumor Model and a Mouse Melanoma Model." Scholar Commons, 2010. http://scholarcommons.usf.edu/etd/3520.
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Melanoma is an aggressive disease with few effective treatment options. Non-toxic, anti-tumor therapies and prophylactic approaches are currently being investigated to identify treatment options that will control and remove late-stage melanoma.
The overall goal of this project was to establish an effective delivery method for a plasmid encoding human interleukin (phIL-15) into mouse melanoma cells (B16.F10) using the gene transfer technique electroporation (EP)1. The EP delivery phIL-15 was optimized using an in vitro 3D tumor model. The purpose was to translate these IL-15 delivery conditions into an in vivo mouse melanoma model to study IL-15 signal transduction and stimulate immune cells to destroy tumor antigens as well as promote an anti-tumor immune memory response.
The in vitro 3D tumor model and the mouse model demonstrated similar expression patterns when delivering phIL-15 with different EP conditions. Intra-tumoral delivery using 500V/cm 20ms enhanced gene delivery and increased IL-15 protein expression compared to 1300V/cm 100μs. There was also a visible increase in transfection efficacy between tumor cells compared to skin cells when delivering pmIL-12 and phIL-15 plasmid constructs in vivo. The plasmid+EP groups 1300V/cm and 500V/cm stimulated increased expression of cytokines IL-1β, IL-6, INFγ, MIP-1β and TNFα. These EP groups also promoted tumor regression by up-regulating CD8+ T cells and CD4+ T cells which targeted melanoma. Regression and survival studies demonstrated that 73.3% of mice cleared B16.F10 cells when treated with phIL-xi15+1300V/cm and pVax+500V/cm. In addition, 53% of the mice responded to the phIL-15+500V/cm treatment group. Furthermore, 75% of the mice from group phIL-15+500V/cm survived secondary inoculation and tumor challenge. In conclusion, plasmid with encoding gene insert phIL-15 delivered by EP has the potential to act as an anti-tumor therapy because it promotes melanoma regression and enhances mouse survival through innate and adaptive cell-mediated immune responses.
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Hoarau, Jessica. "Halfway Between 2D Models and Animal Models : a New Multicellular 3D Spheroid Model Organized to Study Tumor-Endothelium Interactions in Ovarian Cancer." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS111.
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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<br>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
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COSTABILE, FRANCESCA. "Development of an in vitro murine three-dimensional tumor model to study the micro-environment ability to tune cell’s features." Doctoral thesis, Università degli studi di Genova, 2022. http://hdl.handle.net/11567/1079876.
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Background: The tumor microenvironment (TME) is a complex system, shaped by direct interactions among different cell types and by the interactions between cells and extracellular matrix (ECM). Given the emerging importance of the TME in modulating cells’ morphology and function, more sophisticated tumor models incorporating TME features are needed to elucidate cellular, molecular, and immunologic mechanisms of tumor response or resistance. An intensive investigation of in vitro models able to study tumor biology has led to the generation of different three-dimensional (3D) culture methods that better mimic in vivo conditions compared to the usual 2D culture methods. The 3D mono- and co-cultures are able to reproduce some “in vivo features” such as 3D cell morphology, which permits cells to better execute their function and enables them to deposit significant increased amount of ECM.
Aim of this study is the development of an accurate in vitro 3D tumor model to study the impact of tumor ECM on the cells of the microenvironment. The understanding of the specific contributions that ECM proteins make to the tumor microenvironment became crucial due to the emergency of find alternative cures to the many cancer harboring patients resistant to the existing therapies.
Methods: Our experimental approach can be divided in two main experimental plans:
• the development of tumor spheroid model, helpful to understand the beginning stage of the nascent tumor; at day 7, 14 and 21 of spheroids culture we evaluated ECM deposition through immunofluorescence (IF) analysis of collagen type VI; we also analyzed how much the 3D co-culture model we generated was similar compared to the in vivo microenvironment regarding gene expression;
• the development of a scaffold obtained by murine tumor tissue decellularization, utilized for the study of the advanced tumor stage, with a more complex ECM compared to the spheroid model; some scaffolds underwent to collagen type VI IF analysis while other scaffolds were used for recellularization experiments with B16f10 or NIH/3T3 cell lines. Some scaffolds were also used for scanning electron microscopy analysis (SEM).
Results and Discussion: Spheroids generated by the co-culture of B16f10 and NIH/3T3 cells were the only ones lasting for 21 days and the only ones where collagen type VI was detected, compared to spheroids made just by B16f10. These results let us deduce that ECM deposition is fibroblast-dependent. According to the 3D morphology classification of Kenny et al. 2007, B16f10 + NIH/3T3 derived spheroids we generated belong to the Mass class, characterized by cells organized in a regular manner around the center of the colony; B16f10 derived spheroids seems to belong instead to the Grape-like class, characterized by colonies with poor cell-cell contacts and distinguished by their grape-like appearance. B16f10-spheroid’s morphology clearly show a lack of robust cell-cell adhesion, result that has to be overlapped with the absence of collagen type VI. Therefore, fibroblasts are needed for the generation of an early tumor stage 3D model because of their major role as ECM producers and tumor micro-environment organizers. We even investigated spheroid gene expression signature. We focused on the genes that were expressed at the same level between the tumor cells derived from the 3D coculture and the actual in vivo tumor microenvironment. Clusters analysis highlights the similarity between the 2 groups showing 23.1% of the found pathways referring to cancer microenvironment, giving grand importance to the spheroid as in vitro model compared to the 2D systems.
To define an in vitro 3D model suitable to the experimentation on advanced solid tumor phase, murine B16f10 derived tumors were processed for decellularization and studied to verify the efficacy as a natural scaffold for 3D culture system. As observed also from other research groups, cell attachment was limited to the border of the tissue, and it seems cells were not able to pass through the ECM. Control experiment with polyurethane demonstrate the capability of cells to infiltrate a synthetic matrix. To have a better understanding of the ECM structure, frozen sections of decellularized tissue were analyzed by confocal microscope for detection of collagen type VI. Interestingly, both immunofluorescence and histochemistry analysis showed an increase of ECM in decellularized tissue compared to the not decellularized one. This may be due to a slight collapse of the tumor structure since cells have been removed from it. We implemented the ECM observation with the support of SEM: after the decellularization treatment the extracellular fibres are exposed and as hypothesized, the lack of cells makes the fibres twist on themselves, generating a complex net, probably impenetrable from cells. To create a method to measure the collapse of the extracellular matrix that a decellularization treatment may provokes, we compared a fresh and a decellularized specimen of mouse pulmonary parenchyma, where structural modifications are easy to be detected. In this way we were able to quantify the collapse of the ECM through alveoli area reduction.
Conclusions: The development of the spheroid made by tumor and fibroblast cells is a more realistic environment compared to the usual 2D culture and to the spheroid made simply by tumor cells. Fibroblasts are needed for the generation of a better 3D model because of their major role as ECM producers and tumor micro-environment organizers. The limit of the spheroid model is the time, since, to date, the system does not provide oxygen import, necessary for the tumor bulk growth. Tumor derived 3D scaffolds by decellularization most truthfully represents the real in vivo scenario of a tumor 3D model, but the recellularization lack is very often a big limit. Our data demonstrate that the decellularizzation process provokes the collapse of the matrix that does not allow the tissue to be utilized as scaffold for in vitro cell experimentation. Here we also provide, for the first time, a method to test the damage that the treatment provokes on the samples, avoiding the loss of precious materials.
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Christakou, Athanasia. "Ultrasound-assisted Interactions of Natural Killer Cells with Cancer Cells and Solid Tumors." Doctoral thesis, KTH, Biomedicinsk fysik och röntgenfysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-158522.
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In this Thesis, we have developed a microtechnology-based method for culturing and visualizing high numbers of individual cells and cell-cell interactions over extended periods of time. The foundation of the device is a silicon-glass multiwell microplate (also referred as microchip) directly compatible with fluorescence microscopy. The initial microchip design involved thousands of square wells of sizes up to 80 µm, for screening large numbers of cell-cell interactions at the single cell level. Biocompatibility and confinement tests proved the feasibility of the idea, and further investigation showed the conservation of immune cellular processes within the wells. Although the system is very reliable for screening, limitations related to synchronization of the interaction events, and the inability to maintain conjugations for long time periods, led to the development of a novel ultrasonic manipulation multiwell microdevice. The main components of the ultrasonic device is a 100-well silicon-glass microchip and an ultrasonic transducer. The transducer is used for ultrasonic actuation on the chip with a frequency causing half-wave resonances in each of the wells (2.0-2.5 MHz for wells with sizes 300-350 µm). Therefore, cells in suspension are directed by acoustic radiation forces towards a pressure node formed in the center of each well. This method allows simultaneous aggregation of cells in all wells and sustains cells confined within a small area for long time periods (even up to several days). The biological target of investigation in this Thesis is the natural killer (NK) cells and their functional properties. NK cells belong to the lymphatic group and they are important factors for host defense and immune regulation. They are characterized by the ability to interact with virus infected cells and cancer cells upon contact, and under suitable conditions they can induce target cell death. We have utilized the ultrasonic microdevice to induce NK-target cell interactions at the single cell level. Our results confirm a heterogeneity within IL-2 activated NK cell populations, with some cells being inactive, while others are capable to kill quickly and in a consecutive manner. Furthermore, we have integrated the ultrasonic microdevice in a temperature regulation system that allows to actuate with high-voltage ultrasound, but still sustain the cell physiological temperature. Using this system we have been able to induce formation of up to 100 solid tumors (HepG2 cells) in parallel without using surface modification or hydrogels. Finally, we used the tumors as targets for investigating NK cells ability to infiltrate and kill solid tumors. To summarize, a method is presented for investigating individual NK cell behavior against target cells and solid tumors. Although we have utilized our technique to investigate NK cells, there is no limitation of the target of investigation. In the future, the device could be used for any type of cells where interactions at the single cell level can reveal critical information, but also to form solid tumors of primary cancer cells for toxicology studies.<br><p>QC 20150113</p>
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Alessandri, Kévin. "The cellular capsules technology and its applications to investigate model tumor progression and to engineer tissues in vitro." Thesis, Paris 5, 2013. http://www.theses.fr/2013PA05T062/document.
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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<br>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
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Jardet, Claire. "Développement d’un modèle humain de mélanome ex vivo basé sur l’implantation de sphéroïdes dans des explants de peau." Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30282.
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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<br>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
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Huang, Yingbo. "Intrapulmonary Inoculation of Multicellular Tumor Spheroids to Construct an Orthotopic Lung Cancer Xenograft Model that Mimics Four Clinical Stages of Non-small Cell Lung Cancer." Scholarly Commons, 2019. https://scholarlycommons.pacific.edu/uop_etds/3596.
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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.
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Reza, Poor Fatemeh. "?IGH3 promotes apoptosis in osteosarcoma tumor spheroids." Thesis, The University of Texas at San Antonio, 2015. http://pqdtopen.proquest.com/#viewpdf?dispub=1598532.
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<p> Transforming Growth Factor Beta Induced Gene Human Clone 3, BIGH3, is an extra cellular matrix protein expressed by different cell types. BIGH3 promotes cell adhesion and has been recognized as a tumor suppressor protein in many studies, a function consistent with the finding that the expression of BIGH3 is reduced in various tumors and transformed cells when compared to healthy counterparts. </p><p> In the present study, we found that BIGH3 induces MG63 multi tumor spheroid (MTS) cells apoptosis and antagonized the development of tumor cells into large aggregate, supporting BIGH3 tumor suppressor role. MG63 spheroids were cultured in recombinant BIGH3 and vascular smooth muscle cells (VSMCs) conditioned medium . We have shown BIGH3 to be abundantly expressed by VSMCs. In addition, stimulation of BIGH3 gene by TGF-β1 in MG63 cells resulted in overexpression of BIGH3 and subsequent increase in apoptosis by almost 3 fold. TUNEL assay was performed to detect apoptotic cells. Smaller and scattered tumor spheroids were observed in TGF-β1 treated cells. Importantly, in-house developed anti- BIGH3 antibody reduced apoptosis percentage by almost one-half and antagonized the development of osteosarcoma cells into large aggregate spheroids. Within the formed spheroids, BIGH3 was immunologically detected in in the stroma and at cell bodies, suggesting a possible binding of BIGH3 to the cell surface. Collectively, these data suggest that BIGH3 plays a suppressive role in development of osteosarcoma tumor spheroids. </p><p> MG63 were cultured in agar-coated wells, where they developed into 3D aggregates. Unlike classical monolayer-based models (2D), multicellular tumor spheroid (MTS) cell culture system mimics the in vivo 3D structure of a solid tumor.</p>
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Chaddad, Hassan. "Development of vascularized tumor spheroids mimicking the tumor environment : angiogenesis and hypoxia." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ001.
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Le microenvironnement tumoral, l'angiogenèse tumorale et l'hypoxie jouent un rôle crucial dans la progression tumorale et le développement de thérapies de nombreux cancers. Les limites de pénétration des médicaments, les phénomènes de résistance aux anti-cancéreux, la vascularisation de la tumeur et l’hypoxie sont tous des paramètres influençant les effets du médicament. La culture cellulaire 3D permet de créer un microenvironnement qui imite l’architecture et la fonction des tissus in vivo. L’expression de gènes et de protéines modifiée par l’environnement 3D est une autre caractéristique qui impacte l’effet d’une molécule thérapeutique. Dans notre première étude, afin de développer un modèle 3D vascularisé imitant celle des tumeurs in vivo, nous avons mis en culture des cellules endothéliales en 2D avec des cellules tumorales en 3D. Après 2 semaines de culture, un réseau vasculaire s’est organisé avec des structures de type tubulaire présentant une lumière et exprimant différents marqueurs angiogéniques tels que VEGF, CD31 et Collagène IV. Dans notre deuxième étude, nous avons développé un modèle d’hypoxie in vitro intégrant l'environnement 3D et un agent mimétique de l'hypoxie (CoCl2). Le but de ce modèle est de créer un modèle d'hypoxie imitant les tumeurs in vivo et de montrer l'importance de l'hypoxie dans la réponse et la résistance aux médicaments. Ces résultats ont révélé que la meilleure condition était la combinaison 3D+CoCl2, conduisant à la surexpression des gènes relatifs à l’hypoxie (GLUT1/3, VEGF) et à la résistance aux médicaments (ABCG2, MRP1). L'angiogenèse et l'hypoxie sont des facteurs clés pour le microenvironnement tumoral in vivo et ils doivent être adoptés dans la conception de modèles tumoraux in vitro pour mieux sélectionner et cribler les médicaments anticancéreux<br>The tumor microenvironment, tumor angiogenesis, and hypoxia play a critical role in the tumor progression and therapy development of many cancers. Limitations in drug penetration, multidrug resistance phenomena, tumor vascularization, and oxygen deficiency are all parameters influencing drug effects. 3D cell culture allows to create a microenvironment that more closely mimics in vivo tissue architecture and function, thus, gene and protein expression modified by the 3D environment are further features that affect treatment outcome. In our first study, in order to develop a vascularized 3D model like in vivo tumors, we co-cultured 2D endothelial cells with 3D tumor cells. After 2 weeks of this combination, a vascular network was formed and organized with tubule-like structures presenting a lumen and expressing different angiogenic markers such as VEGF, CD31 and Collagen IV. In our second study, we developed an in vitro hypoxia model integrating the 3D environment and a hypoxia mimetic agent (CoCl2) to mimic the in vivo tumors and to show the importance of hypoxia in drug response and resistance. Results revealed that the best condition was the combination 3D+CoCl2 model, leading to overexpression oh hypoxia (GLUT1/3, VEGF) and drug resistance (ABCG2, MRP1) related genes. Taken together, angiogenesis and hypoxia are key factors for in vivo tumor microenvironment and they should be adopted in in vitro model design to better select and screen anticancer drugs
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RIZZUTI, ILARIA FRANCESCA. "STRENGTHEN OF DPNS FEATURES FOR THERANOSTIC APPLICATIONS AND MECHANICAL-CONTROL OF CHEMOTHERAPEUTIC EFFICACY THROUGH MODULATION OF CELL PROLIFERATION." Doctoral thesis, Università degli studi di Genova, 2020. http://hdl.handle.net/11567/1000310.
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Solid tumors are complex biological structures which are composed of cellular and matrix components, everything being perfused by blood vessels. During tumor development, modifications of both biochemical and mechanical parameters are observed and can feedback on one another. Cancer cells constantly interact with their mechanical environment and the whole tissue is mostly confined by its surrounding. Compressive mechanical stress develops in part from cell proliferation and could eventually result in the clamping of blood vessels leading to increased interstitial fluid pressure (hydrostatic pressure). The consequent hypoperfusion poses important obstacles to drug delivery and nanomedicines. In fact, the tortuous tumor microvasculature has blood velocities up to one order of magnitude lower compared to healthy capillary networks. Moreover, the fast angiogenesis during tumor progression leads to high vascular density in solid tumors, large gaps exist between endothelial cells in tumor blood vessels, and tumor tissues show selective extravasation and retention of macromolecular drugs (Enhanced Permeation Retention – EPR – effect). These effects have served as a basis for the development of drug delivery systems which are aimed at enhancing tumor tissue targeting and drug therapeutic effectiveness. Over the last 15 years, a plethora of materials and different formulations have been proposed for the realization of nanomedicines. Yet, drug-loading efficiency, sequestration by phagocytic cells, and tumor accumulation of nanoparticle-loaded agents - nanomedicines - are sub-optimal.
Starting from these considerations, during my PhD, I studied two complementary approaches: in the first two years my work was focused on implementing the characteristics of Discoidal Polymeric Nanoconstructs designed with controlled geometries and mechanical properties. In the last year, I investigated the role of mechanical stress on chemotherapeutic efficacy.
More precisely, this work first reviews the use of deformable discoidal nanoconstructs (DPNs) as a novel delivery strategy for therapeutic and imaging agents. Inspired by blood cell behavior, these nanoconstructs are designed to efficiently navigate the circulatory system, minimize sequestration by phagocytic cells, and recognize the tortuous angiogenic microvasculature of neoplastic masses. In this work, the synthesis, drug loading and release, and physico-chemical characterization of DPNs were enhanced with particular emphasis on the ability to independently control size, shape, surface properties, and mechanical stiffness. Two different loading strategies were tested, namely the straightforward “direct loading” and the “absorbance loading”. In the former case, the agent was directly mixed with the polymeric paste to realize DPNs whereas, in the latter case, DPNs were first lyophilized and then rehydrated upon exposure to a concentrated aqueous solution of the agent. Under these two loading conditions, the encapsulation efficiencies and release profiles of three different molecules and their corresponding prodrugs were systematically assessed (1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine lipid chains or 1 kDa PEG chains were directly conjugated with Cy5.5 or methotrexate and Doxorubicin). Moderately hydrophobic compounds with low molecular weight showed encapsulation efficiencies of 80%, with absorption loading (direct loading has efficiencies around 1%). The DOX-DPN showed on triple negative breast cancer cells a toxicity comparable to free DOX. Preliminary in vivo preliminary studies conducted with directly loaded Cy5-DPN demonstrated a fairly solid integration of the imaging compound with the polymer matrix of the particles.
The second part of the work dissect what happens to free drugs or to drugs carried by nanovectors once they reach the tumor site. As we mention above, the elevated mechanical stress derived from tumor progression could result in blood vessels clamping with consequent reduction of drug efficacy. It is quite obvious to imagine that if the drug fails to reach the tumor it cannot act on it. Indeed, mechanical stress within the tumor site is present from the early stages of the disease. Our goal was to understand what happens when mechanical stress is not yet so large enough to fully collapse the blood vessels. Are there mechanical alterations that can affect the efficacy of a chemotherapeutic? We studied how mechanical perturbations of the tumor microenvironment could contribute to the mechanical-form of Gemcitabine drug resistance.
Specifically, we developed a new in vitro strategy to mimic the mechanical compression stress induced by the stroma during tumor progression. We embedded pancreatic tumor spheroids into agarose polymeric matrix in order to demonstrate the effect of mechanical compressive stress on tumor proliferation. Then, we validated our results with other types of mechanical stresses. Finally, we investigated the therapeutic efficacy of a proliferation-based chemotherapy: Gemcitabine.
Collectively, having the physical cues of cancer in mind, it can be important to cross-fertilize the fields of physical oncology and nanomedicine.
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Senkowski, Wojciech. "High-throughput screening using multicellular tumor spheroids to reveal and exploit tumor-specific vulnerabilities." Doctoral thesis, Uppsala universitet, Cancerfarmakologi och beräkningsmedicin, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-320598.
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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.
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Sheth, Disha B. "Multielectrode platform for measuring oxygenation status in multicellular tumor spheroids." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1301516012.
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Riffle, Stephen. "Multicellular Tumor Spheroids as a Model to Study Tumor Cell Adaptations within a Hypoxic Environment." University of Cincinnati / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ucin151188562556805.
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McMillan, Kay Seonaid. "Development of a microfluidic platform for multicellular tumour spheroid assays." Thesis, University of Strathclyde, 2016. http://digitool.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=27926.
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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.
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Ohlin, Mathias. "Ultrasonic Fluid and Cell Manipulation." Doctoral thesis, KTH, Biomedicinsk fysik och röntgenfysik, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-166779.
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During the last decade, ultrasonic manipulation has matured into an important tool with a wide range of applications, from fundamental cell biological research to clinical and industrial implementations. The contactless nature of ultrasound makes it possible to manipulate living cells in a gentle way, e.g., for positioning, sorting, and aggregation. However, when manipulating cells using ultrasound, especially using high acoustic amplitudes, a great deal of heat can be generated. This constitutes a challenge, since the viability of cells is dependent on a stable physiological temperature around 37°C. In this Thesis we present applications of ultrasonic manipulation of fluids, particles, and cells in temperature-controlled micrometer-sized devices fabricated using well established etching techniques, directly compatible with high-resolution fluorescence microscopy. Furthermore, we present ultrasonic manipulation in larger up to centimeter-sized devices optimized for fluid mixing and cell lysis. In the present work, two new ultrasonic manipulation platforms have been developed implementing temperature control. These platforms are much improved with increased performance and usability compared to previous platforms. Also, two new ultrasonic platforms utilizing low-frequency ultrasound for solubilization and cell lysis of microliter-volumed and milliliter-volumed samples have been designed and implemented. We have applied ultrasound to synchronize the interaction between large numbers of immune, natural killer cells, and cancer cells to study the cytotoxic response, on a single cell level. A heterogeneity was found among the natural killer cell population, i.e., some cells displayed high cytotoxic response while others were dormant. Furthermore, we have used temperature-controlled ultrasound to form up to 100, in parallel, solid cancer HepG2 tumors in a glass-silicon multi-well microplate. Next, we investigated the immune cells cytotoxic response against the solid tumors. We found a correlation between the number of immune cells compared to the size of the tumor and the cytotoxic outcome, i.e., if the tumor could be defeated. Finally, the effect of high acoustic pressure amplitudes in the MPa-range on cell viability has been studied in a newly developed platform optimized for long-term stable temperature control, independent on the applied ultrasound power. Lastly, we present two applications of ultrasonic fluid mixing and lysis of cells. One platform is optimized for small microliter-sized volumes in plastic disposable chips and another is optimized for large milliliter-sized volumes in plastic test tubes. The latter platform has been implemented for clinical sputum sample solubilization and cell lysis for genomic DNA extraction for subsequent pathogen detection<br>Ultraljudsmanipulering har under de senaste tio åren mognat och utvecklats till ett verktyg med ett brett användningsområde. Idag kan man finna applikationer inom allt från cellbiologisk grundforskning till industri samt sjukvård. Ultraljudsmanipuleringens kontaktlösa natur gör det till en varsam metod för att manipulera celler, till exempel inom positionering, sortering och aggregering. När ultraljud med hög amplitud används kan värmeutvecklingen, som är oundviklig, bli ett problem. För att kunna säkerställa hög cellviabilitet krävs temperaturkontroll som kan hålla en fysiologisk, stabil temperatur på 37°C. I denna avhandling presenterar vi tillämpningar av temperaturkontrollerad ultraljudsmanipulering i mikrometerstora anordningar fabricerade med väletablerade etsningstekniker. Dessa anordningar är optimerade för att vara fullt kompatibla med högupplöst fluorescensmikroskopi. Vi demonstrerar även ultraljudsmanipulering i centimeterstora anordningar optimerade för omrörning och blandning av vätskor samt lysering av celler. Två nya plattformar för ultraljudsmanipulering med inbyggd temperaturkontroll har utvecklats. Dessa två plattformar erbjuder ökad prestanda, flexibilitet samt även användarvänlighet. Utöver dessa plattformar har ytterligare två anordningar för lågfrekvent ultraljudssolubilisering och cellysering av mikroliter- och milliliterstora prover konstruerats. I denna avhandling har vi tillämpat ultraljud för att synkronisera interaktionen mellan populationer utav immunceller (natural killer-celler) och cancerceller för att på cellnivå studera det cytotoxiska gensvaret. Vi fann en heterogenitet hos immuncellspopulationen. Det manifesterade sig i en fördelning av immuncellerna, från celler med stort cytotoxiskt gensvar till inaktiva immunceller. Vi har dessutom använt temperaturkontrollerad ultrasljudsmanipulering för att skapa solida cancertumörer utav HepG2-cancerceller, upp till 100 stycken parallellt, i en multihåls-mikrotiterplatta bestående av glas och kisel. Med hjälp av dessa tumörer har vi studerat det cytotoxiska gensvaret från immuncellerna. Vi fann att förhållandet mellan antalet immunceller och storleken på tumören bestämde utfallet, det vill säga om tumören kunde bekämpas. Vi presenterar dessutom effekten utav högamplitudsultraljudsexponering av cancerceller i en plattform speciellt designad för höga tryckamplituder med implementerad ultraljudseffektsoberoende temperaturkontroll. Slutligen presenterar vi två tillämpningar av ultraljud för vätskeblandning och cellysering. Den första tillämpningen är anpassad för små volymer i plastchip för engångsbruk och den andra är optimerad för större volymer i plastprovrör. Den senare tillämpningen är speciellt framtagen för ultraljudssolubilisering och cellysering utav kliniska sputumprover för att möjliggöra DNA-extrahering för detektion av smittämnen.<br><p>QC 20150522</p>
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Ward, John P. "Mathematical modelling of avascular tumour growth." Thesis, University of Nottingham, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.339566.
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Monazzam, Azita. "Multicellular Tumour Spheroids in a Translational PET Imaging Strategy." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8196.
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Bloch, Katarzyna. "Structural and bioenergetic changes in tumour spheroids during growth." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:1d7b8669-b62a-4554-bb66-157f54e3ded2.
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Multicellular tumour spheroids (TS) are an in vitro model of avascular tumours, and have been widely used to investigate tumour growth, metabolism and hypoxia. The geometry of the TS lends itself to mathematical representation, and theoretical models of TS growth and the development of hypoxia are abundant. With some notable exceptions however, these models have been developed independently of the biological data collection process and are overwhelmingly based upon data from multiple sources. Thus, whilst mathematical modeling has the potential to help explain and guide biological experiments, without reliable data it is unlikely to live up to this expectation. In this thesis, a combination of experimental and theoretical approaches was used to characterize the relationship between proliferation, hypoxia and metabolism during the growth of TS derived from the DLD-1 human colon adenocarcinoma cell line. Experimental data were collected over the entire period of TS growth, generating a high volume of predominantly imaging data. To facilitate the extraction of quantitative information from this, a suite of image analysis software, which is readily applicable to other data sets, was developed. During growth, the DLD-1 TS maintained a macroscopic spherical geometry but at the microscale level the TS boundary was increasingly irregular, with TS disintegrating rapidly after 20 days. Immunofluorescence (IF) studies showed that hypoxia developed soon after TS initiation, followed by the characteristic onset of necrosis. Reduced proliferation was found to be concomitant with the development of hypoxia, although some cells retained proliferative capacity even under severely hypoxic conditions. Towards the end of culture, TS were primarily comprised of severely hypoxic and necrotic cells, a probable cause of disintegration. Mathematical simulation of oxygen gradients in TS using literature-based values for the maximal rate of oxygen consumption was used to estimate the partial oxygen pressure (pO<sub>2</sub>) at which the IF marker of hypoxia was bound. Assuming a spatially-invariant rate of oxygen consumption, the model predicted that the onset of hypoxic binding occurs at pO<sub>2</sub> levels similar to those reported in the literature, however the onset of necrosis was overestimated. Mathematical simulations predicted that oxygen consumption decreases as TSs increase in size, supporting previous observations. The Warburg Effect, where glucose metabolism is favoured even under aerobic conditions, is a hallmark of tumours. Although development of the glycolytic phenotype during TS growth was observed in the form of an elevated activity of the lactate dehydrogenase V (LDHV) enzyme, the activity and expression of other glycolytic enzymes, such as hexokinase II (HKII), was unaltered. Whilst the spatial distribution of HKII was unrestricted throughout the TS's viable fraction, LDHV expression was elevated in regions of hypoxia, suggesting constant adaptation of tumour cells to their microenvironment. In addition to the above findings, the data generated have been collected and analysed in the context of the requirements of theoretical modelling at each step; thus, they can be used to parameterise and inform more sophisticated models of tumour metabolism.
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Smith, Hannah. "Metabolic adaptations to micro-environmental stress in tumour spheroids." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:3651d265-ddc0-4258-b3f7-2a0242697d21.
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Alterations in energy metabolism due to factors including cellular stress from the hostile tumour micro-environment are a emerging cancer hallmark. Distinct hypoxic and quiescent cell populations develop, which are resistant to chemotherapy due to lack of proliferation, drug inactivity in the altered redox status of the cell and enhanced drug biotransformation. The present study characterises the metabolic strategies employed by these distinct populations of cancer cells. The in vitro 3-dimensional tumour spheroid model, which reflects tumour architecture and behaviour, cultured under different micro-environmental conditions was utilized in this study. Metabolic enzyme activity and expression, overall metabolic flux rates for nutrients, metabolomics profiles of specific pathways and tissue status were assessed. Metabolic adaptations consistent with the Warburg effect were observed in fully oxygenated, proliferative tumour spheroids, with glucose being metabolised to produce lactate. Additionally, metabolomics investigations determined glucose was metabolised by the pentose phosphate pathway, demonstrated by high enrichment of glucose-derived carbon in 6-phophogluconate. The extraction of 39.7 ± 7.6 μ moles (mg protein) <sup>-1</sup> glutamine from the medium over 24 hours was observed in these spheroids, consistent with glutaminolysis pathway activity. A 2-fold higher rate of glycolytic flux (measured by production of 3h2O from 5-<sup>3</sup>H-glucose) was measured in hypoxic tumour spheroids, despite reduced levels of glycolytic enzymes being determined. Surprisingly, although lower rates of glycolysis (2.6-fold) were measured in quiescent spheroids, increased glycolytic enzyme activities (HK 1.9 fold, PK 2 fold and LDH 1.8 fold), glucose (1.9 fold over 24 hours) and glutamine uptake (5.5 fold over 12 hours) as well as lactate production (1.8 fold) were measured, relative to their proliferating counterparts. This study demonstrates that metabolic strategies employed by tumour spheroids differ upon exposure to distinct micro-environmental stresses, additionally identifying hexokinase as a potential therapeutic target for the inhibition of glycolysis under all micro-environmental stress conditions analysed.
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Paczkowski, Marcin. "Biological interactions between cell populations in heterogeneous tumour spheroids." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:91853f0a-9bb5-4190-a731-7bbe2d68bfff.
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When radiotherapy is prescribed in the clinic, the tumour is assumed to be homogeneous. However, tumours are composed of many distinct cell populations that interact with each other and their environment. It is unclear how this heterogeneity affects tumour growth and response to treatment. Additionally, much of cancer research considers tumour cells in isolation, neglecting the context in which the disease develops. Experimental approaches alone are not suficient to understand the complexity of the interactions occurring in the cancer ecosystem. In this thesis we use a multidisciplinary approach combining in vitro experiments, mathematical modelling and parameter inference methods to explore the impact of cellular heterogeneity on tumour growth and radiation response. Our objectives are: i) to determine if intratumour heterogeneity affects bulk radiation response; ii) to characterise the interactions between co-cultured cell populations; iii) to determine how interactions between different cell populations are affected by radiation. In the first part of the thesis we design a 3D experimental model in which we co-culture pairs of prostate cancer cell lines with distinct phenotypes and derived from the same tumours. We use the experimental model to study the growth and radiation response of heterogeneous tumours (Chapter 2). We then use nonlinear regression and approximate Bayesian computation algorithm to fit the Verhulst logistic and Lotka-Volterra mathematical models to our data to characterise how the cell populations interact when co-cultured in tumour spheroids before and after exposure to ionising radiation (Chapters 3 and 4). Our third piece of work involves the development of a cellular automaton model of avascular tumour growth to allow for spatial variation within the tumour. The cellular automaton model is simulated for a range of parameter values and fitted to the logistic model to determine the relationship between microscale and macroscale parameters describing tumour growth and to study the dynamics between co-cultured cell populations. The work presented in this thesis highlights the benefits of multidisciplinary research for understanding cancer heterogeneity. We show that intratumour heterogeneity affects bulk tumour growth and radiation response. We demonstrate how complex biological interactions can be identified and quantified via mathematical modelling and inference. We also show how experimental design studies can streamline biological experiments. Taken together, this work presents a framework for uncovering the effect of cellular heterogeneity on tumour growth with a particular emphasis on interclonal interactions.
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Mallet, Daniel Gordon. "Mathematical Modelling of the Role of Haptotaxis in Tumour Growth and Invasion." Queensland University of Technology, 2004. http://eprints.qut.edu.au/15941/.
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In this thesis, a number of mathematical models of haptotactic cell migration are developed. The modelling of haptotaxis is presented in two distinct parts - the first comprises an investigation of haptotaxis in pre-necrotic avascular tumours, while the second consists of the modelling of adhesion-mediated haptotactic cell migration within tissue, with particular attention paid to the biological appropriateness of the description of cell-extracellular matrix adhesion. A model is developed that describes the effects of passive and haptotactic migration on the cellular dynamics and growth of pre-necrotic avascular tumours. The model includes a description of the extracellular matrix and its effect on cell migration. Questions are posed as to which cell types act as a source of the extracellular matrix, and the model is used to simulate the possible effects of different matrix sources. Simulations in one-dimensional and spherically symmetric geometry are presented, displaying familiar results such as three-phase tumour growth and tumours comprising a rim of proliferating cells surrounding a non-proliferating region. Novel effects are also described such as cell population splitting and tumour shrinkage due to haptotaxis and appropriate extracellular matrix construction. The avascular tumour model is then extended to describe the internalisation of labelled cells and inert microspheres within multicell tumour spheroids. A novel model of adhesion-receptor mediated haptotactic cell migration is presented and specific applications of the model to tumour invasion processes are discussed. This model includes a more biologically realistic description of cell adhesion than has been considered in previous models of cell population haptotaxis. Through assumptions of fast kinetics, the model is simplified with the identification of relationships between the simplified model and previous models of haptotaxis. Further simpli.cations to the model are made and travelling wave solutions of the original model are then investigated. It is noted that the generic numerical solution routine NAG D03PCF is not always appropriate for the solution of the model, and can produce oscillatory and inaccurate solutions. For this reason, a control volume numerical solver with .ux limiting is developed to provide a better method of solving the cell migration models.
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Mallet, Daniel Gordon. "Mathematical Modelling of the Role of Haptotaxis in Tumour Growth and Invasion." Thesis, Queensland University of Technology, 2004. https://eprints.qut.edu.au/15941/1/Daniel_Mallet_Thesis.pdf.
Full textAbstract:
In this thesis, a number of mathematical models of haptotactic cell migration are developed. The modelling of haptotaxis is presented in two distinct parts - the first comprises an investigation of haptotaxis in pre-necrotic avascular tumours, while the second consists of the modelling of adhesion-mediated haptotactic cell migration within tissue, with particular attention paid to the biological appropriateness of the description of cell-extracellular matrix adhesion.
A model is developed that describes the effects of passive and haptotactic migration on the cellular dynamics and growth of pre-necrotic avascular tumours. The model includes a description of the extracellular matrix and its effect on cell migration. Questions are posed as to which cell types act as a source of the extracellular matrix, and the model is used to simulate the possible effects of different matrix sources. Simulations in one-dimensional and spherically symmetric geometry are presented, displaying familiar results such as three-phase tumour growth and tumours comprising a rim of proliferating cells surrounding a non-proliferating region. Novel effects are also described such as cell population splitting and tumour shrinkage due to haptotaxis and appropriate extracellular matrix construction. The avascular tumour model is then extended to describe the internalisation of labelled cells and inert microspheres within multicell tumour spheroids.
A novel model of adhesion-receptor mediated haptotactic cell migration is presented and specific applications of the model to tumour invasion processes are discussed. This model includes a more biologically realistic description of cell adhesion than has been considered in previous models of cell population haptotaxis. Through assumptions of fast kinetics, the model is simplified with the identification of relationships between the simplified model and previous models of haptotaxis.
Further simpli.cations to the model are made and travelling wave solutions of the original model are then investigated. It is noted that the generic numerical solution routine NAG D03PCF is not always appropriate for the solution of the model, and can produce oscillatory and inaccurate solutions. For this reason, a control volume numerical solver with .ux limiting is developed to provide a better method of solving the cell migration models.
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Grist, Samantha Marie. "A microfluidic platform to study real-time tumour cell and tumour spheroid response to chronic and transient hypoxia." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/58212.
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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.<br>Applied Science, Faculty of<br>Electrical and Computer Engineering, Department of<br>Graduate
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Norman, Julia. "Investigating Peptide Conjugation for the Selective Activation of Metal Complexes in Tumours." Thesis, The University of Sydney, 2013. http://hdl.handle.net/2123/9506.
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There are various strategies in the treatment of cancer including surgical excision, radiotherapy, chemotherapy and immunotherapy. Chemotherapy offers the distinct advantage of systemic distribution, with the potential to reach primary tumours as well as metastases. A significant drawback of the vast majority of chemotherapeutic agents is their lack of specificity for cancer cells as well as poor tumour penetration, meaning that cells distal to blood vessels receive an inadequate dose and remain viable. There is an imperative need for the development of anti-tumour drugs which exhibit good tumour penetration and distribution with minimal systemic toxicity. Tumour activated prodrugs (TAPs) are one of the main strategies now employed in antitumour drug development, and involve administering an inert, less toxic form of a particular drug which is then selectively transformed into its toxic version by the body in the vicinity of the tumour.1 In this study, TAPs which contain a matrix-metalloproteinase II (MMP-2) specific peptide cleavage sequence were synthesised. There is significant evidence that there is increased expression of MMP-1, -2, -3, -7, -9, -13, -14 in both primary tumours and metastases, and that there is a positive correlation between high levels of MMP expression and poor patient prognosis.2 In this work, recombinant DNA techniques were used to visualise the expression of MMP-2 and MMP-9 in vitro. Plasmid constructs in which MMP-2 and MMP-9 were fused to the AmCyan fluorescent protein gene were generated for insertion into a mammalian transfection vector. Transfection into three cell lines that exhibited different levels of MMP-2 and MMP-9 expression was evaluated by measuring the fluorescence emission in the 480 – 550 nm range. The results demonstrated potential for monitoring MMP expression and secretion in monolayer and 3D cell culture by transfection with MMP-fluorescent protein plasmid vectors. However, employing AmCyan as the fluorescent protein resulted in an unexpected interaction with the riboflavin component of cell culture media whereby fluorescence emission could not be observed in the 500 - 550 nm range as expected. The use of other fluorescent proteins should also be pursued, as well as the use of cell-culture media which is riboflavin-free. In order to evaluate the potential of targeting MMP-2 overexpression in solid tumours for selective activation of a TAP, a series of model fluorophore-peptide substrates were investigated in cell monolayers and 3D multicellular tumour spheroids. The target compound DDDDK(FITC)DIPVSLRSK(RhB) (4.8) contained a tetra-aspartate (DDDD) uptake-blocking group designed to prevent influx of the compound into the cell prior to activation by MMP-2 cleavage. Additionally the fluorescein isothiocyanate and rhodamine B fluorophores we attached to the peptide on opposing sites of the MMP-2 cleavage site, facilitating visualisation of the intact peptide as well as the post-cleavage fragments. LC-MS studies showed that in the absence of the uptake-blocking group K(FITC)DIPVSLRSK(RhB) (4.5) is almost entirely cleaved into its respective fragments K(FITC)DIPVS (4.6) and LRKS(RhB) (4.7) after 24 hours. LC-MS results also showed that the uptake-blocking group slows cleavage of the peptide, a trait which was also observed in fluorescence confocal microscopy. K(FITC)DIPVSLRSK(RhB) (4.5) and DDDDK(FITC)DIPVSLRSK(RhB) (4.8) were tested in DLD-1 cell monolayers in the absence of MMP-2 activity. After 4 hours K(FITC)DIPVSLRSK(RhB) (4.5) was found to have entered the cells intact and undergone a small degree of cleavage, while DDDDK(FITC)DIPVSLRSK(RhB) (4.8) did not enter cells intact, and instead only a very minor amount of intracellular fluorescence in the red channel was visible, indicative of non-specific cleavage. In the presence of MMP-2, less uptake of the intact K(FITC)DIPVSLRSK(RhB) (4.5) peptide was observed and instead discreet localised fluorescence was observed in the red and green channels, suggesting that cleavage was occurring in the extracellular space. This result was also observed, but to a lesser degree, for DDDDK(FITC)DIPVSLRSK(RhB) (4.8). These compounds were then also tested in multicellular tumour spheroid models, where the intact peptide K(FITC)DIPVSLRSK(RhB) (4.5) underwent cleavage in the surrounding media, resulting in sequestration of the LRSK(RhB) (4.7) fragment by the outermost cells, preventing it from penetrating further into the spheroid. Contrastingly, the slower cleavage of DDDDK(FITC)DIPVSLRSK(RhB) (4.8) improved the distribution of fluorescence in the red channel, having allowed the intact peptide to diffuse further into the spheroid before cleavage and cellular uptake of the LRSK(RhB) (4.7) fragment. These results confirmed the importance of the uptake-blocking group for ensuring delivery of the payload to the less accessible regions of solid tumour. Overall, this work demonstrated the potential of using an MMP-2 specific cleavage sequence for the selective delivery of chemotherapeutic agents to solid tumours, and as such a series of cytotoxin-peptide substrates containing the MMP-2 specific cleavage sequence were synthesised. The cytotoxic compounds investigated were the platinum(IV) complex cis, cis, trans-acetato[(1R,2R)-cyclohexane-1,2-diamine-N,N’]succinatooxalatoplatinum(IV) and ruthenium(II) complex [(4-methyl-4'-carboxy-2,2'-bipyridine)bis(4,4'-di-tert-butyl-2,2'-bipyridine)]ruthenium(II) hexafluorophosphate. The cytotoxic properties of library of platinum(IV)-peptide conjugates were tested, with none of the peptide-Pt(IV) conjugates possessing IC50 values similar to the free platinum(II) precursor. The intact DIPVSLRSK(Pt) (5.8) peptide is slightly less toxic than its post-cleavage fragment LRSK(Pt) (5.7) in the absence of MMP-2, but the IC50 of DIPVSLRSK(Pt) (5.8) is almost identical to LRSK(Pt) (5.7) in the presence of MMP-2, suggesting that MMP-2 is contributing to the activation and subsequent cytotoxicity of the compound. In both cell lines, the conjugate which contains the uptake-blocking group DDDDGDIPVSLRSK(Pt) (5.9) is not cytotoxic, proving that incorporation of the tetra-aspartate moiety can modify the activity of these compounds. Platinum accumulation studies in cell monolayers did not show a significant difference between the levels of intracellular platinum following incubation with LRSK(Pt) (5.7), DIPVSLRSK(Pt) (5.8), and DDDDGDIPVSLRSK(Pt) (5.9). However, no platinum was detected in the hypoxic/necrotic regions or periphery of spheroids treated with LRSK(Pt) (5.7) and DDDDGDIPVSLRSK(Pt) (5.9) when analysed by SRIXE mapping. After observing the poor spheroid penetration of the hydrophilic platinum(IV)-peptide compounds, the substitution of the cytotoxic platinum(IV) complex for a ruthenium(II)-bipyridyl complex saw an improvement in spheroid penetration. Peptide conjugation of the ruthenium(II) complex improved the cellular uptake of the ruthenium(II) and fluorescence confocal microscopy of LRSK(Ru) (5.10) showed that the compound was localised in the cytoplasm, while little of the free complex [Ru(tBu2bpy)2(HOOC-4’-CH3bpy)]2PF6 (5.6) was observed inside cells. This increase in uptake is likely to have contributed to the increased cytotoxicity of the compound, reducing the IC50 value by a factor of 2.
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Tindall, Marcus John. "Modelling cell movement and the cell cycle in multicellular tumour spheroids." Thesis, University of Southampton, 2002. https://eprints.soton.ac.uk/50618/.
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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.
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Evans, Charlotte L. "The biological and therapeutic significance of tumour necrosis. Identification and characterisation of viable cells from the necrotic core of multicellular tumour spheroids provides evidence of a new micro-environmental niche that has biological and therapeutic significance." Thesis, University of Bradford, 2014. http://hdl.handle.net/10454/13961.
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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.<br>Yorkshire Cancer Research
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Aoun, Laurene. "Mechanical properties of growing microtumors : engineering microdevices for the mechanical investigation of tumor spheroids models." Toulouse 3, 2014. http://www.theses.fr/2014TOU30137.
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Le développement tumoral est influencé par des signaux biochimiques mais également biomécaniques. Plusieurs études ont montré que la modification de l'environnement mécanique peut moduler la croissance tumorale des cellules, la migration, l'invasion ainsi que la prolifération et l'apoptose. Cependant, les propriétés mécaniques intrinsèques des tumeurs sont elles peu connues. Le sphéroïde est un modèle multicellulaire reproduisant l'organisation des cellules dans une tumeur par les interactions cellule-cellule et cellule-microenvironnement. Afin d'étudier les propriétés mécaniques des sphéroïdes, mon projet était de déterminer les forces exercées par des sphéroïdes tumoraux en cours de croissance. Dans cet objectif, j'ai développé des micropilliers en PDMS, de rapport de forme élevé et démontré qu'ils se comportent comme des capteurs de force. Nous avons mesuré les forces générées par des sphéroïdes de cancer mammaire et nous avons constaté que la croissance des sphéroïdes induit un déplacement croissant des piliers avec le temps et que les forces correspondantes sont de l'ordre de quelques centaines de nanoNewtons. Nous avons trouvé que les forces générées dépendent de la rigidité de l'obstacle environnant, ce qui s'est traduit par la génération des forces plus grandes envers des micropiliers plus rigides. L'ensemble de ces résultats montre que les sphéroïdes en croissance sont capables de détecter leur micro-environnement et générer des forces en fonction de la résistance du dispositif. L'utilisation de ces microdispositifs comme des microcapteurs de force ouvre de nouvelles perspectives dans les domaines de la mécanique des tissus et en pharmacologie anti-tumorale<br>Tumor growth and progression is influenced not only by biochemical cues but also biomechanical ones. Several studies have shown that the modification of the mechanical environment can modulate tumour cell growth, migration and invasion as well as proliferation and apoptosis. However, little is known about the intrinsic tumour mechanical properties. Spheroids provide a unique 3 dimensional model to study the mechanical properties of tumors since they reproduce the organization of a microtumour in cell-cell and cell-microenvironment interactions. In order to investigate the mechanical properties of spheroids, my project was to determine the forces exerted by tumor spheroids during growth. This was done by engineering biocompatible high aspect ratio PDMS microdevices, using microfabrication technics. We showed that these micropillars serve as force sensors. We have measured the forces generated by mammary cancer cells spheroids and we demonstrated that spheroids under growth induce increasing pillars displacement with time and that the corresponding forces are of the order of a few hundred nanoNewtons. We found as well that the generated forces depend on the rigidity of the surrounding obstacle, which was reflected by the generation of increased forces towards more rigid micropillars. The ensemble of these results shows that growing spheroids are able to sense their microenvironment and generate forces according to the resistance of the device. The use of these microdevices arrays as force microsensors opens new prospects in the fields of tissue mechanics and pharmacological drug screening
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Corrêa, de Sampaio Pedro Vaz de Almada. "Using a novel 3-dimensional in vitro spheroid model to investigate new roles for stromal metalloproteinases in tumour angiogenesis." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610640.
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Evans, Charlotte Louise. "The biological and therapeutic significance of tumour necrosis : identification and characterisation of viable cells from the necrotic core of multicellular tumour spheroids provides evidence of a new micro-environmental niche that has biological and therapeutic significance." Thesis, University of Bradford, 2014. http://hdl.handle.net/10454/13961.
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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.
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Jamieson, Lauren Elizabeth. "Measuring redox potential in 3D breast cancer tumour models using SERS nanosensors." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25964.
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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.
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Ham, Stephanie Lemmo. "Engineering Tumor Models Using Aqueous Biphasic 3D Culture Microtechnology." University of Akron / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=akron150470381711759.
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Akasov, Roman. "Novel 3D in vitro models based on multicellular tumor spheroids to test anticancer drugs and drug delivery vehicles." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAF013/document.
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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)<br>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)
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Kashtl, Ghasaq J. "Differential membrane-type matrix metalloproteinase expression in phenotypically defined breast cancer cell lines: Comparison of MT-MMP expression in environmentally-challenged 2D monolayer cultures and 3D multicellular tumour spheroids." Thesis, University of Bradford, 2018. http://hdl.handle.net/10454/17346.
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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.<br>Al-Mstansiriya University, Iraq
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Alexander, Frank. "RTEMIS: Real-Time Tumoroid and Environment Monitoring Using Impedance Spectroscopy and pH Sensing." Scholar Commons, 2014. https://scholarcommons.usf.edu/etd/5168.
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This research utilizes Electrical Impedance Spectroscopy, a technique classically used for electrochemical analysis and material characterization, as the basis for a non-destructive, label-free assay platform for three dimensional (3D) cellular spheroids. In this work, a linear array of microelectrodes is optimized to rapidly respond to changes located within a 3D multicellular model. In addition, this technique is coupled with an on chip micro-pH sensor for monitoring the environment around the cells. Finally, the responses of both impedance and pH are correlated with physical changes within the cellular model. The impedance analysis system realized through this work provides a foundation for the development of high-throughput drug screening systems that utilize multiple parallel sensing modalities including pH and impedance sensing in order to quickly assess the efficacy of specific drug candidates.
The slow development of new drugs is mainly attributed to poor predictability of current chemosensitivity and resistivity assays, as well as genetic differences between the animal models used for tests and humans. In addition, monolayer cultures used in early experimentation are fundamentally different from the complex structure of organs in vivo. This requires the study of smaller 3D models (spheroids) that more efficiently replicate the conditions within the body.
The main objective of this research was to develop a microfluidic system on a chip that is capable of deducing viability and morphology of 3D tumor spheroids by monitoring both the impedance of the cellular model and the pH of their local environment. This would provide a fast and reliable method for screening pharmaceutical compounds in a high-throughput system.
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Kim, Byung Juen. "Development of Multiple Fluorescent Tumour Spheroid Models To Investigate the use of Transition Metal Complexes as Hypoxia-Activated Prodrugs." Thesis, The University of Sydney, 2014. http://hdl.handle.net/2123/12494.
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The use of transition metal based tumour-activated prodrugs is a promising strategy to improve the selectivity of anticancer agents towards cancer over healthy tissues. The rational design of these agents is difficult, as knowledge and tools to understand their biological activities have been lacking to date. In this study, multiple fluorescent cellular models have been developed and characterised to study metal complexes [M(acac)3] and [M(dbm)3], as models of hypoxia-activated prodrugs, where M = Co, Fe, Ru. The biological activities of these metal complexes were evaluated in both the monolayer cultures as well as spheroids, an exemplary in vitro tumour model. As most anticancer agents have been developed with the purpose of damaging the nucleus and the DNA, the changes in the cell cycle progression caused by the prodrugs was investigated using the fluorescent ubiquitination cell cycle indicator (FUCCI) system. When the metal complexes chaperone the cytotoxin to the hypoxic region of the tumour, the metal complexes that have undergone ligand exchange are also found in the same region. To monitor the changes in spatial and temporal regulation of the intracellular labile iron pool, one copy of the iron responsive element from the human ferritin light gene was utilised to drive the translation of EosFP. Finally, the promoter region of hypoxia-related genes was manipulated to visualise hypoxia in a tumour microenvironment. Also, a robust method to select hypoxia responsive cells has been developed. The outcomes of this study provided insights into how metal complexes are taken up by cells, their cytotoxic effects, their impact on the cell cycle, their indirect involvement in iron metabolism and hypoxia selectivity in spheroids. These new findings will contribute to the design and development of effective prodrugs for treating cancer.
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Hardelauf, Heike, Jean-Philippe Frimat, Joanna D. Stewart, et al. "Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-138739.
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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<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Hardelauf, Heike, Jean-Philippe Frimat, Joanna D. Stewart, et al. "Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits." Royal Society of Chemistry, 2011. https://tud.qucosa.de/id/qucosa%3A27776.
Full textAbstract:
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.<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Zraikat, Manar Saleh Ali. "Development of in vitro models of invasion for the pharmacological investigation of small molecule inhibitors of tumour progression : development and validation of a 3-dimensional tumour spheroid invasion model to evaluate the pharmacological effects of novel small molecule β3 integrin antagonists". Thesis, University of Bradford, 2015. http://hdl.handle.net/10454/7511.
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Tumour dissemination is a major reason for failure of therapy for many tumour types therefore there is a requirement for novel targets & therapies. The αIIbβ3 and αvβ3 integrins have been demonstrated to have significant involvement at many stages of the tumour dissemination process including, tumour cell adhesion, migration, metastasis and angiogenesis, and thus the β3 integrins are a potential target for therapeutic antagonism with small molecules. Because of the clear interaction between the different integrin types, targeting integrins as a therapeutic strategy requires targeting more than one integrin type. Consequently, the ICT is developing a group of novel new αIIbβ3 and αvβ3 integrin dual antagonists. One of the main challenges is having a relevant, validated experimental model that expresses these integrins. The aim of the work presented here is to develop and validate an in vitro αIIbβ3 and αvβ3 integrin expressing assay of tumour cell invasion. The spheroid invasion assay has the advantage over standard monolayer transwell chamber invasion assays of being a 3-dimensional assay, and thus mimics better the cell-cell interactions and architecture that are present in a tumour compared to the monolayer-based assay. A panel of human cancer cell lines known to express one of the molecular targets of interest, αvβ3 integrin was evaluated for the ability to form spheroids and to invade through collagen matrices. One glioma cell line, U87-MG, demonstrated consistent spheroid formation and invasion and was thus selected for further studies. Optimum conditions were established for use of U87-MG in the invasion assay, and the assay was validated using a known inhibitor of invasion, LiCl and known β3 antagonist, cRGDfV. Subsequently a group of novel small molecule β3 antagonists were evaluated at nontoxic concentrations using the assay. Both LiCl and cRGDfV inhibited spheroid invasion through the gel in a dose-dependent manner, thus validating the assay. Furthermore, when the novel small molecule β3 antagonists were evaluated using the model, a dose and time dependent reduction in U87-MG spheroids invasion in collagen was observed. In further work initial steps were taken to construct a cell line which expresses both αIIbβ3 and αvβ3 integrin to use in the model to assess for dual integrin antagonism. In conclusion, this work has established a validated assay which has been utilised for some compounds to evaluate a group of novel small molecule β3 integrin antagonists with encouraging results.
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Rühland, Svenja [Verfasser], and Peter [Akademischer Betreuer] Nelson. "3D imaging and analysis of mesenchymal stem cells and cytotoxic T lymphocytes invading tumor spheroids / Svenja Rühland ; Betreuer: Peter Nelson." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1176409735/34.
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Stöhr, Daniela [Verfasser], and Peter [Akademischer Betreuer] Scheurich. "Characterising heterogeneous TRAIL responsiveness and overcoming TRAIL resistance in multicellular tumour spheroids / Daniela Stöhr ; Betreuer: Peter Scheurich." Stuttgart : Universitätsbibliothek der Universität Stuttgart, 2018. http://d-nb.info/1181099277/34.
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Alderden, Rebecca. "The Distribution of Platinum Complexes in Biological Systems." Thesis, The University of Sydney, 2006. http://hdl.handle.net/2123/1419.
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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).
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Alderden, Rebecca. "The Distribution of Platinum Complexes in Biological Systems." University of Sydney, 2006. http://hdl.handle.net/2123/1419.
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Doctor of Philosophy (PhD)<br>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).