The use of nanomedicines for cancer treatment holds a great potential due to their improved efficacy and safety. During the nanomedicine preclinical in vitro evaluation stage, these are mainly tested on cell culture monolayers. However, these 2D models are an unrealistic representation of the in vivo tumors, leading to an inaccurate screening of the candidate formulations. To address this problem, spheroids are emerging as an additional tool to validate the efficacy of new therapeutics due to the ability of these 3D in vitro cancer models to mimic the key features displayed by in vivo solid tumors. In this review, the application of spheroids for the evaluation of nanomedicines' physicochemical properties and therapeutic efficacy is discussed.

Interpretar uma cena, do ponto de vista computacional, geralmente se resume a interpretar e classificar seus elementos, percebendo como eles estão organizados pelo cenário. Essa ação é feita através do reconhecimento dos modelos apresentados como entrada por um sistema que faz a busca por modelos semelhantes em um banco de dados. Esse processo é chamado de recuperação de modelos, sendo que a correspondência e a indexação geralmente fazem parte desse processo. Embora a tecnologia tenha se desenvolvido, a interpretação robusta de cenas do ambiente ainda é um desafio para a computação em geral, dificultando áreas que utilizam da Visão Computacional, como por exemplo a Robótica. Levando em consideração a necessidade de novos algoritmos para a recuperação de formas, este trabalho objetiva aprofundar os estudos na área de Visão Computacional, propondo um novo algoritmo capaz de fazer a recuperação de formas tridimensionais em grandes bancos de dados. Embora os critérios de similaridade sejam importantes, a indexação das características para a busca de objetos semelhantes também tem a sua importância e impacta diretamente em velocidade e qualidade para o problema. Por este motivo, o foco deste trabalho é propor um novo método para a recuperação de modelos tridimensionais, utilizando a estrutura de dados chamada árvore de vocabulários, que apresenta resultados significativos na literatura para consultas por modelos bidimensionais. Neste trabalho, o método é apresentado de forma detalhada e é implementado através de um algoritmo. Os resultados obtidos por este algoritmo são apresentados e analisados utilizando bases de dados e métricas, os quais foram empregados em trabalhos anteriores. O método apresenta resultados promissores, porém com grande espaço para melhorias.

Triple-negative breast cancer (TNBC), the most aggressive and radioresistant subtype of breast cancer, is associated with a high risk of relapse following radiotherapy (RT). The tumor microenvironment (TME) consists not only of tumor cells but of a different type of cell population populations, including infiltrating immune cells, that are also a target and have influence in the efficacy of RT. TNBC tumors are highly infiltrated with macrophages, that are associated with therapy resistance and a worse prognosis. Therefore, uncovering the macrophage-mediated molecular mechanisms behind TNBC radioresistance will reveal new targets for radiosensitizing therapies and improve disease management. At the same time, understanding the underlying mechanisms and alterations driving tumor cell radioresistance is also essential for developing a more precise and effective treatment strategy. Therefore, our work focuses on the development of experimental models to study radioresistance mechanisms in TNBC. Firstly, as previously reported by our team, we have successfully established 3D in vitro tumor and tumor-immune spheroids, using liquid overlay technique, with TNBC MDA-MB-231 cell line and human macrophages, isolated from healthy donors’ buffy coats. These spheroids were subjected to the RT scheme of 5x5.2 Gy, mimicking a week of patients’ treatment. After 24h of the last irradiation, we unveil that macrophages confer protection from RT to tumor cells. The presence of macrophages not only maintained tumor cell viability, but also increased tumor cell proliferation. To better understand if this effect is cell line dependent, we established tumor spheroids and tumor-immune spheroids with TNBC BT-20 cell line. After 8 days, viability, metabolic activity, morphology, and response to RT were assessed. With this we found that the high proliferation ratio of BT-20 cell line impacts the viability of human macrophages in TIS model, not allowing for the same protective effect than observed in MDA-MB-231 TIS. In parallel, we developed radioresiresistant (RR) cell lines of parental radiosensitive (RS) MDA-MB-231 and Hs578T cell lines. For this, cells received intermittent single doses of irradiation of 2 → 4 Gy, in the first week with a 2-day interval, and weekly single doses of 6 → 8 → 10 → 10 → 10Gy, totalizing a radiation dose of 50Gy irradiated, as previously described in other studies. Then, morphology and colony formation capacity of both RR cell lines was assessed and both RR cell lines have higher colony formation capacities than parental cell lines, at some of these doses. Additionally, migration and invasion capacities of the RR cell lines were assessed. On both a collective and single-cell level, RR cell lines demonstrated an increase in migration capacity. In relation to invasion capacity, while no differences were seen in the Hs578T RR cell line, the MDA-MB-231 RR cell line had an increase in invasion when compared to the parental RS cell line. Lastly, the expression of gelatinases (MMP-2 and MMP-9) revealed no significant difference in MDA-MB-231 RR when compared to the parental RS cell line, indicating that other molecular mechanisms may be involved in the increase of invasion of this cell line. With this thesis, we developed new tools, 3D models and RR cell lines, that allow to better understand the role of macrophages on radioresistance, as well as understand the underlaying mechanisms of radioresistance in TNBC cells. In this way, this work paves the way to identify new therapeutic targets, that in the future may allow for a better survival rate and quality of life for these patients.

Cancer is a dreadful illness with ever-increasing rates, and it is one of the world's top causes of death. Most treatment options, such as systemic chemotherapy, are painfully slow and have many associated side effects due to their lack of specificity to cancer cells and their intravenous nature. Besides, chemotherapy alone may be susceptible to the occurrence of new metastasis after the treatment stops. Therefore, a more specialized treatment may benefit the patient. Among the therapeutic approaches currently used in clinical cancer immunotherapy offers a considerable advantage over existing conventional therapies in terms of avoiding metastasis and recurrence due to the phenomenon of immunological memory. Several modalities have been evaluated and the most promising one seems to be cancer vaccines. Effector cells can target tumor-associated antigens produced by cancer cells with the help of nanoparticles, which are a potent antigen presentation and delivery mechanism that can maximize the immune response. As such colloidal gels present themselves as a new approach to this problem due to their in situ applicability, and moldability conjugated with their capacity to transport both hydrophobic and hydrophilic molecules depending on the nanoparticles present on their structure. Considering this, the work presented on this thesis focuses on the formulation of a double network colloidal gel assembled from two polymer particles with opposite charges. Nanoparticles of Zein and methacrylated hyaluronic acid (234.0 ± 7.4 nm), for the encapsulation of doxorubicin, were combined with the plastic particles of PLGA and metacrylated poly(ethyleneimine) (264.8 ± 4.0 nm), for the encapsulation of indocyanine green. They both showed high encapsulation efficiency, for DOX (96.78%) and ICG (98.86%) respectively, through the nanoprecipitation method. The obtained colloidal gels were administered to a monolayered culture of Panc-1 cell line, irradiated with a laser to stimulate the chemo-photothermal therapy and the cytotoxicity was evaluated. The release profile of the colloidal gel was assessed with the assistance of laser irradiation which proved to boost the release of doxorubicin. The effect of an immunostimulant drug (GDC-0152) was also evaluated in 3D scaffold comprising Panc-1 cells. Different concentrations of this compound were administered to both 3D and 2D cultures and their cell viability was evaluated.

Triple-negative breast cancer is one of the most aggressive forms of breast cancer, characterized by high heterogeneity, lack of specific therapeutic targets and poor response to conventional treatments. Despite the widespread use of 2D cell cultures, they do not faithfully reproduce the structural and functional complexity of tumors in vivo, making it difficult to translate the results obtained into clinical practice. In this context, we sought to evaluate the usefulness of 3D models as an intermediate tool that is more representative of tumor physiology, comparing them with 2D cultures. For this purpose, spheroids of E0771 mouse and MDA- MB-231 human cells were formed on very low adhesion plates with variable cell densities, and the spheroids were compared for cell viability by AlamarBlue, and structural integrity with Calcein-AM and propidium iodide dyes. Furthermore, E0771 spheroids were tested with doxorubicin chemotherapy at different stages of formation. E0771 and MDA-MB-231 spheroids showed growth and structural differences. Furthermore, using E0771 cells, doxorubicin treatment had a greater impact when cells were grown in 2D compared to spheroids. Additionally, the development time of the spheroids led to a decreased impact of doxorubicin, even at higher concentrations. The spheroids proved to be an adaptable model that could answer more complex questions, through the manipulation of variables such as extracellular matrix or the inclusion of other cell populations, among others, to this originally simpler model.

The use of Augmented Reality (AR) technology has recently improved education and training methods. This technology has provided alternatives to the way people learn and relate to their environment. This work-in-progress study presents an approach to develop a smart learning city environment using AR games to change citizens’ sustainable development competences, the EduCITY. The EduCITY – “Smart and Sustainable Cities with Augmented Reality mobile educational games made by and for the citizens” is a research and training project that aims to improve sustainable cities through the creation of a disruptive smart learning environment, supported by a mobile app with multimedia educational resources, such as AR and 3D models. AR is defined as a direct or indirect real-time view of a real physical environment, improved/enhanced by the addition of computer-generated virtual information. It is interactive and combines real and virtual objects. A 3D model is a virtual three-dimensional object that can be viewed from different angles. Unlike traditional 2D images, which are flat and have only length and width, 3D models add depth to the image, making it more lifelike. The EduCITY app developed two types of AR (Augmented Marker and ARBook) and different 3D models, mainly leaves, flowers and fruits of specific trees in the city. To create the 3D models of the leaves, each leaf was photographed from both sides without zooming and on a white background. The model was then assembled using Blender. In addition, the 3D models of the flowers and fruits were created using the Polycam Pro app, a 3D capture app. This paper focuses on analysing the perceptions of university students (most of whom will become teachers in the future) about the use of AR and 3D models in education. This study adopts a qualitative interpretive approach through a case study methodology. The participants are 55 undergraduate students selected through convenience sampling. The research is part of the course 'Nature Integrated Sciences II' at the University of Aveiro (Portugal), during the academic year 2023/24. To answer the research question "What are students' initial perceptions of Augmented Reality and 3D models integrated into the EduCITY app?", the research team organised an activity for these students, which included playing a game with the EduCITY app while walking around the city. In this activity, students explored the different types of AR and 3D models while answering the proposed questions. At the end of the activity, participating students completed an individual online questionnaire, which consisted mainly of closed questions with multiple choice, item selection, and a five-point Likert scale, although there were also open-ended questions. The data is analysed using descriptive analysis. The results show that the students consider AR and the 3D models integrated in the EduCITY app as innovative and believe that they facilitate the understanding of certain content, mainly related to biology and plant species. Future work includes further testing of the EduCITY app in order to finalise it and make it available for free in the stores (PlayStore and AppStore). Once the app is concluded, further user studies will be carried out to evaluate the features and usability of the EduCITY app.

O cancro da mama é um dos tipos de cancro mais frequentemente detetados em mulheres. Os cancros luminais são os cancros de mama mais comuns, onde o luminal B apresenta alta agressividade e genes relacionados à proliferação tumoral. O desenvolvimento do cancro da mama está associado aos fatores celulares e solúveis da matriz extracelular (ECM). A ECM promove a migração celular conectando o citoesqueleto das células com aderências focais (FA). A presença de FA está relacionada com a fosforilação da tirosina, sendo a quinase de adesão focal (FAK) uma das mais prontamente fosforiladas no cancro. A superexpressão de FAK está associada à proliferação de células tumorais, contribuindo também para a ativação das células endoteliais, que expressam a P-selectina na sua superfície. A ação da P-selectina é desencadeada pela sua interação com o ligando-1 da glicoproteína P-selectina (PSGL-1) expresso nas células tumorais, promovendo a circulação de células cancerígenas e metástase. Como a interação entre FAK e P-selectina é pouco compreendida, este projeto tem como objetivo avaliar o efeito antitumoral sinérgico da interação entre os inibidores da FAK e da P-selectina e a nanovacina direcionadas a DC que entregam antígenos associados a tumores e adjuvantes, em modelos esferóides 3D. A expressão de FAK, FAK fosforilada (pFAK), P-selectina e PSGL-1 foram avaliadas em células EO771 e do microambiente tumoral (TME) recolhidas de ratinhos inoculados com EO771. Verificou-se que FAK, pFAK e P-selectina estavam superexpressas em células EO771 TME (> 97%). Para modular o TME em direção a um microambiente efetor imunológico, antes de avaliar o papel do defactinib (inibidor da FAK) e do KF-38789 (inibidor da P-selectina) na função e frequência das células imunes dentro do TME, avaliámos o impacto do defactinib e do KF-38789 na viabilidade das células EO771 e TME EO771. Consequentemente, o defactinib e o KF-38789 induziram um efeito citotóxico significativo nas células EO771 para concentrações acima de 1 e 0,1 μM, respetivamente. Além disso, foi obtida uma diminuição significativa na viabilidade das células EO771 TME para concentrações de defactinib e KF-38789 acima de 0,5 e 0,05 µM, respetivamente. Finalmente, para avaliar o impacto do efeito sinérgico entre a inibição da FAK e da P-selectina combinada com a nanovacina, foi realizada a co-cultura dos esferóides 3D de tumores EO771 (EO771 TME) e esplenócitos. De acordo com as diferentes condições, o tratamento de esferóides 3D do cancro da mama com KF-38789 e a nanovacina isoladamente ou em combinação com o tratamento com defactinib mostrou-se promissor na modulação da invasão tumoral. Finalmente, uma inibição completa do sprouting resultando na morte de células esferóides 3D foi alcançada com a combinação de defactinib e esplenócitos estimulados seguido de KF-38789, representando o efeito sinérgico mais promissor entre os tratamentos testados. Considerando a estratégia terapêutica mais promissora e eficaz, levanta-se a hipótese de que o defactinib primeiro modula o TME para permitir uma melhor infiltração imunológica desencadeada pela vacinação, enquanto a P-selectina previne a proliferação de células cancerígenas.

Pancreatic Ductal Adenocarcinoma (PDAC) remains one of the most aggressive and lethal malignancies nowadays. Current treatments are often ineffective, primarily due to the immunosuppressive, densely desmoplastic tumor microenvironment (TME), which current preclinical models hardly reproduce. The PDAC TME results from a unique spatial organization and interaction among different cell types and stromal components. To emulate this arrangement, a stratified, sandwich-like 3D in vitro model was developed that includes tumor, stromal, and immune elements. This multicellular model consists of a basal stromal-immune layer that combines pancreas-derived decellularized extracellular matrix (dECM) with hyaluronic acid (HA) to form a hydrogel hosting cancer-associated fibroblasts (CAFs) and monocyte-derived macrophages. On this base, a spherical dECM-based hydrogel encapsulating PANC-1 cells was placed as a central tumor core and overlaid with a second stromal-immune layer that matches the basal layer in composition. The resulting constructs were mechanically stable, remained viable for at least 14 days, and could be reproducibly handled and analyzed over time. Notably, the platform supported quantitative and qualitative readouts relevant to PDAC biology, including spatial organization of tumor, stromal, and immune populations, secreted cytokine profiling, and tumor invasive behavior. Functionally, the presence of CAFs led to the emergence of a contractile stromal compartment and visible matrix remodeling, while the combined presence of CAFs and macrophage-like cells enhanced collective invasive outgrowth, particularly in pancreatic dECM surrounding hydrogel. In parallel, the system exhibited an inflammatory and pro-tumoral secretome characterized by increased IL-6 and IL-8. Overall, this work establishes a reproducible, immunocompetent 3D PDAC platform that recapitulates stromal activity, macrophage-associated signaling, and cooperative invasive behavior, providing a foundation for microenvironment-targeted therapeutic screening in a preclinical setting.

In vitro 3D tumor microenvironment mimicking models are gathering momentum as alternatives to traditional 2D flat monolayer cultures due to their potential for recapitulating major cancer hallmarks. To fulfill such potential, it is crucial that 3D tumor testing platforms completely emulate in vitro the complex in vivo tumor niche and its cellular constituents. Mesenchymal stem cells (MSCs) are recognized to play a pivotal multi-modulatory role in cancer, generating interest as biological targets and as key tumor suppressing, or tumor promoting effectors. This review discusses the biological influence of different types of MSCs in the tumor microenvironment and showcases recent studies that engineer 3D MSCs-cancer cells co-cultures as advanced in vitro therapy testing platforms. A special focus is given to MSCs-cancer 3D co-culture set-up parameters, challenges, and future opportunities. Understanding cancer-MSCs crosstalk and their underlying effects is envisioned to support the development of advanced 3D in vitro disease models for discovery of forefront cancer treatments.