Browsing by Author "Costa, Elisabete Cristina da Rocha"
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- Modelos de co-culturas de células in vitro para desenvolvimento de novos sistemas de entrega de fármacosPublication . Costa, Elisabete Cristina da Rocha; Correia, Ilídio Joaquim Sobreira; Gaspar, Vítor Manuel AbreuThe demanding applications of nanocarriers in cancer biology require the existence of testing platforms that mimic the in vivo tumor microenvironment and its unique biological features. For this, highly informative methodologies such as animal experimentation are the current gold-standard. However, very recent reports issued by regulatory agencies appeal for the reduction of the used animal research models due to economical and ethical issues, thus evidencing the urgent necessity for novel alternatives. Co-culture cell models have the potential to bridge the gap between the required reduction of animal use and the existence of suitable models that closely reproduce in vivo tumors. This is a novel type of in vitro cell culture that is mainly characterized by the culture of cancer cells in contact with stromal cells, mimicking the tumor microenvironment in vitro trough the establishment of cancer-stroma synergic interactions. However, this evaluation was until now limited to co-culture systems established with precise cell ratios, not addressing the natural heterogeneity commonly found in tumors of different patients. The research work presented in this thesis describes the development and optimization of novel 2D co-culture models of breast and cervical cancers with various cell-to-cell ratios, in order to unravel the influence of heterogeneous conditions on the evaluation of nanocarrier biological performance and ultimately in the therapeutic outcome. As a proof of concept these novel platforms were used to evaluate a multifunctional gene delivery system designed for cancer therapy and revealed that in fact different co-culture ratios may influence the overall assessment of nanocarrier targeting specificity. In addition, since recent reports demonstrate the high influence of the 3D architecture of tumor masses in the response to anti-cancer drugs or delivery systems, the engineering and optimization of suitable substrates for generation of organotypic 3D co-culture models with various cancer-fibroblast cell ratios was also investigated. The 3D multicelular spheroid models of breast and cervix cancer produced at various time points, possess all the major characteristics of in vivo tumors including the structural rearrangement, the diffusional limit of oxygen or nutrients and most importantly, the distinctive necrotic core of solid tumors. Overall, these newly developed co-culture and 3D models assume crucial importance for the future design and optimization of new drug delivery providing a new level of in vitro reproducibility of in vivo tumors.
- Study of 3D tumor spheroids for biomedical applicationsPublication . Costa, Elisabete Cristina da Rocha; Correia, Ilídio Joaquim SobreiraCancer is a major health issue for the worldwide population. It is the second most common cause of death in developed countries. According to the World Health Organization (WHO; 2018), 1 in 6 deaths occurs as a result of this disease. This scenario occurs due to the limited effectiveness of the currently available treatments, namely chemotherapy, radiotherapy and surgery. To overcome such drawbacks, the medical/scientific community as well as the pharmaceutical companies have been effortfully pursuing the development of new cancer treatments exhibiting a higher efficacy. Before the new drugs can be used in the clinic, they must fulfill all the requirements established by the regulatory agencies, namely by the Food and Drug Administration (FDA) and the European Medicine Agency (EMA). The initial assessment of anticancer agents’ efficacy is performed through in vitro and in vivo assays (preclinical studies). This preclinical stage includes physicochemical and biological characterization of the compounds as well as their safety and toxic profile, by using cells and animals. The 2D cell cultures, i.e. cells grown as a monolayer on artificial flat plastic surfaces, are the most common in vitro model used for drug development purposes. These cell culture models are easy to handle, cost-effective and display a good reproducibility. However, they are unable to fully predict the effect of therapeutic agents in in vivo tumors. Due to that, a huge number of compounds showing effectiveness in 2D cell culture models fail when tested in in vivo models. This drawback leads to an over-use of animals in experimentation, and also contributes to increase the time and the overall cost of the drug development process. The limitations presented by 2D cell cultures result from their inability to mimic the characteristics of the in vivo tumor tissues, specially their cellular organization and function. Therefore, it is crucial to screen anticancer agents in in vitro models that can better represent the features of solid tumors affecting humans. Sphere-like tightly bound cellular aggregates, known as spheroids, are in vitro 3D cell culture models that reproduce closely the complexity of cancerous human tissues. Such is owed to the fact that spheroids display inherent metabolic (oxygen, carbon dioxide, nutrients and waste) gradients like those found in poorly vascularized tumors. Such gradients endow spheroids a cellular similar organization to that exhibited by solid tumors, i.e. three main cellular layers composed of an external layer of proliferative cells, an intermediate layer formed by quiescence cells and an inner acidic and hypoxic layer comprised of necrotic cells. On the other hand, compared to 2D cell cultures, spheroids demonstrate increased cell-cell interactions, as well as ECM proteins deposition and cell-ECM interactions. Despite of the potential of 3D tumor spheroids for drug screening purposes, their use is still scarce. In one hand, spheroids’ production techniques that can be used to form spheroids (e.g. Liquid Overlay Technique (LOT) and Hanging Drop Technique) demand optimization for allowing the production of spheroids under highly reproducible conditions. On the other hand, the analysis of the therapeutics’ effect on spheroids is still challenging, due to the limited availability of protocols, techniques and equipment required to perform such assays. Particularly, although the fluorescence microscopy is one of the most used techniques to assess the therapeutics distribution within the spheroids or the cellular death in response to a potential therapeutic, some fluorescence microscopes are not suitable for the imaging of spheroids. The imaging of spheroids by confocal fluorescence microscopy modalities is challenging due to spheroids’ thickness and to the light scattering phenomenon. When light cross a biological sample, it is scattered due to the refractive index (RI) mismatches between acellular and cellular constituents. Due to this fact, the excitation light is dispersed through the sample, thus limiting its penetration into the deepest regions of spheroids. Therefore, it is challenging to obtain high-resolution images of intact spheroids, specially from their interior. Having this in mind, the main objective of this thesis’ work plan was to address the limitations of spheroids imaging by confocal laser scanning microscopy (CLSM). To achieve this goal, optical clearing methods, namely ClearT and ClearT2, that were previously applied for the imaging of animals’ biological samples, were optimized for spheroids analysis. These methods were used to reduce the light scattering phenomenon in the spheroids by performing the homogenization of the spheroids overall RI, to accomplish an improved spheroids’ transparency and a light penetration depth. In the first experimental study that was performed within the scope of this thesis, the ClearT optical clear method was, from the best of my knowledge, used for the first time for spheroids’ analysis. To accomplish that, spheroids were cleared by immersing them on solutions with increasing concentrations of formamide. The obtained results demonstrated that the ClearT method did not affect the structure of the spheroids and improved the PI signal penetration depth by about 43%. Additionally, ClearT also enhanced the cells imaging within the spheroid by increasing the imaging cross-section depth by 47% (at 100 μm of depth in the Z-axis). In the second study, the ClearT2 optical clearing method was investigated and optimized for the analysis of spheroids. The ClearT2 protocol involves the immersion of the samples in aqueous solutions with increasing concentrations of formamide and polyethylene glycol (PEG). Herein, it was investigated for the first time the influence of PEG molecular weight on ClearT2 method ability to clear spheroids. For this purpose, several PEG molecular weights were investigated, namely 4000, 8000 and 10000 Da. The obtained results demonstrated that the ClearT2 method enhances spheroids’ transparency and contributes to preserve the PI fluorescence intensity for all the PEG MW used. Further, the ClearT2 method performed using PEG 4000 Da allowed a better PI signal penetration depth (up to 212 μm in the Z-axis) and an enhanced of the cross-section depth (about 26% more than that of the non-cleared spheroids at a penetration depth of 100 μm). Overall, the obtained results demonstrate that the ClearT and ClearT2 methods were able to improve the CLSM imaging of PI stained spheroids. Furthermore, these methods may also contribute for the development of new anticancer treatments, since they may be used for the evaluation of the cellular death within spheroids prompted by therapeutics and even for the analysis of drugs distribution and dispersion within spheroids as well as to perform the analysis of the expression of specific fluorescent proteins in spheroids.