Au cours des dernières décennies, des systèmes de taille nanométrique chargés en principes actifs (nanomédicaments) et des nouvelles stratégies thérapeutiques ont été développés afin de surmonter les limitations liées à la chimiothérapie conventionnelle telles qu’une distribution non spécifique, une mauvaise accumulation dans les tissus cibles ainsi qu’une métabolisation rapide. Cependant, le succès des nouveaux médicaments en clinique reste encore limité et seulement un faible nombre de nanomédicaments est actuellement commercialisé.Une divergence entre les résultats précliniques in vitro et les performances obtenues in vivo est souvent observée dans la première étape du développement d'un médicament. Cet écart pourrait être attribué au manque de modèles pertinents, représentatifs de la pathologie observée chez l’Homme et qui soient de bons prédicteurs de la réponse thérapeutique chez les patients. En effet, les modèles utilisés aujourd’hui (généralement culture cellulaire en deux dimensions, 2D) ne reproduisent pas la structure complexe de la tumeur in vivo. Ainsi, ils ne permettent pas une évaluation fiable du potentiel thérapeutique réel des médicaments. Dans cette optique, les méthodologies de culture de cellules en trois dimensions (3D) sont extrêmement avantageuses. Ces méthodologies permettent, en effet, la construction de systèmes cellulaires pertinents qui reproduisent in vitro la relation entre les cellules cancéreuses et leur microenvironnement. Parmi ces modèles, l'assemblage de cellules sous forme de sphéroïdes multicellulaires a été largement exploré. Néanmoins, les sphéroïdes décrits jusqu'à présent correspondent à des nodules formés exclusivement de cellules cancéreuses, ce qui constitue une vraie limitation. En effet, ces sphéroïdes ne reproduisent pas l’organisation de la tumeur et l'hétérogénéité du microenvironnement, et par conséquent ils ne parviennent pas à mimer les multiples barrières biologiques que les médicaments doivent traverser pour atteindre les cellules cibles.Dans cet esprit, l'objectif de cette thèse de doctorat était de surmonter ces limitations et de construire des modèles pertinents qui reproduisent in vitro la relation entre les cellules cancéreuses et leur microenvironnement afin de i) mieux comprendre les mécanismes de passage des nanomédicaments et ii) mieux prédire l’efficacité des nouveaux traitements.Au cours de cette thèse nous nous sommes intéressés au cancer du pancréas qui est caractérisé par la présence d'un abondant stroma formant un bloc fibreux (réaction desmoplastique) qui limite la pénétration des médicaments et réduit ainsi leur efficacité. Cette tumeur représente donc un bon exemple de barrière biologique tumorale.La partie principale de ce travail de recherche repose sur la construction et la caractérisation complète d’un nouveau type de sphéroïde multicellulaire, capable de reproduire in vitro la relation entre les cellules cancéreuses et leur microenvironnement, grâce à la co-culture de cellules cancéreuses pancréatiques, de fibroblastes et de cellules endothéliales. Les études de cytotoxicité in vitro nous ont permis d’investiguer la capacité de ce modèle à reproduire la résistance des cellules cancéreuses aux traitements observés in vivo. Grâce à la Microscopie de Fluorescence à Feuillet de Lumière nous avons pu étudier la pénétration de la doxorubicine, soit en forme libre, soit encapsulée dans des nanoparticules, au sein des sphéroïdes. Ensuite, afin de mieux comprendre comment les médicaments et nanomédicaments interagissent avec la tumeur, nous avons cherché à combiner la culture 3D avec des conditions dynamiques contrôlées dans un dispositif microfluidique. Pour atteindre cet objectif, nous avons conçu et fabriqué une puce sur mesure, adaptée pour loger à la fois le sphéroïde et des canaux dans lesquels les cellules endothéliales pourront s’organiser sous forme de vaisseaux. / In the last decades, various engineered systems for drug delivery (i.e., nanomedicines) have been developed with the aim to overcome the limits associated to conventional chemotherapy, such as non-specific drug distribution, poor delivery to the target tissue and rapid metabolism. However, the success of new therapeutic strategies in the clinic is still suboptimal and only a limited number is currently marketed.A discrepancy between promising preclinical in vitro results and the in vivo performances is often observed in the early stage of drug development and might be ascribed to the lack of capacity of the models commonly used for in vitro studies to faithfully reproduce the pathophysiology of solid tumors. These models mainly consist of cancer cells cultured as flat (two dimensional, 2D) monolayers or assembled to form three dimensional (3D) multicellular tumor spheroids (MCTS).However, being composed exclusively of one cell type, these models are too simplistic. They do not allow to reproduce the heterogeneous cellular composition, as well as, the complex architecture of the tumor and its surrounding microenvironment. Thus, they fail to replicate the multiple biological barriers that drugs and nanomecidines have to cross in order to reach the target cells.The aim of this PhD thesis was to overcome these limitations and construct a reliable tool for an appropriate in vitro evaluation of the therapeutic potential of nanomedicines and other chemotherapies. Attention has been focused on the pancreatic ductal adenocarcinoma (PDAC) whose strong fibrotic reaction represents a well-known example of a tumor biological barrier responsible of the limited efficacy of the treatments. The main part of this research work relies on the construction and complete characterization of novel hetero-type MCTS based on a triple co-culture of pancreatic cancer cells, fibroblasts and endothelial cells, and thus capable to integrate the cancerous component and the microenvironment of the tumor. The constructed 3D model has demonstrated the capacity to reproduce in vitro the influence of the microenvironment on the sensitivity of cancer cells to chemotherapy. In addition, by combining the 3D model and the innovative Light Sheet Fluorescence Microscopy (LSFM), we have been able to investigate the penetration of the anticancer drug doxorubicin (in a free form and loaded into nanoparticles (NPs)) in a high informative manner. Then, in order to acquire a better understanding on how nanomedicines and other anticancer chemotherapies interact with the tumor, we sought to combine the hetero-type 3D culture with controlled flow conditions in a microfluidic device. To reach this goal we have designed and fabricated a tailor-made chip suitable to host both a MCTS and a perfusable microvascular network (i.e., MCTS-on-a-chip).

Diffuse Gliome bei Erwachsenen sind aggressive, unheilbare Hirntumore. Humanisierte Mausmodelle helfen, molekulare Mechanismen zu verstehen und therapeutische Ziele zu identifizieren, aber der Vergleich mit Proben von Patienten gestaltet sich schwierig. Ich habe eine computergestützte Plattform namens CAPE entwickelt, um Tumormodelle und Patienten-Expressionsprofile mit Hilfe der nicht-negativen Matrixfaktorisierung zu vergleichen. Die Anwendung von CAPE auf humanisierte Maus-Gliom-Avatar-Modelle (GSA) und diffuse Glioma-Patienten zeigte eine starke Übereinstimmung zwischen den Modellen und dem proneuralen Glioblastom-Subtyp. CAPE hat gezeigt, dass durch die Transplantation der Erwerb neuer Tumorzustände in den Modellen verbessert wurde. Durch die Kombination von reporterbasiertem genetischem Tracing und CAPE zeigte sich, dass eine Untergruppe der in vivo GSA-Populationen mit Patienten zusammenfällt, die astrozytische Merkmale aufweisen. Die Behandlung von GSA-Modellen in vitro mit menschlichem Serum, TNFα oder ionisierender Strahlung führte zu einer Verschiebung in den mesenchymalen Zustand. Einzelzell-Transkriptomik annotierte GSA-Populationen unter verschiedenen Bedingungen und zeigte alle Glioblastomzustände in vivo und bei Aktivierung durch externe Faktoren. Der Vergleich von GSA-Einzelzellpopulationen und Patienten bestätigte diese Identitäten. Die Studie etablierte einen umfassenden Rahmen für die Erprobung und Validierung von Verbesserungen der Tumormodelle, um Patienten besser abzubilden, und erweiterte das Verständnis der Tumorbiologie und Ansprechen auf Therapie. / Adult-type diffuse gliomas are aggressive, incurable adult brain cancers. Humanized mouse models help understand molecular mechanisms and identify therapeutic targets, but comparing them with patient samples is difficult. I developed a computational framework, CAPE, for comparing tumor models and patient expression profiles using non-negative matrix factorization. Applying CAPE to humanized mouse glioma subtype avatar models (GSA) and adult-type diffuse glioma patients revealed a strong resemblance between models and proneural glioblastoma subtype. CAPE showed that transplantation improved new tumor state acquisition in models. Combining genetic tracing reporter phenotypic selection with CAPE showed a subset of in vivo GSA populations clustering with patients having astrocytic-like identities. In vitro treatment of GSA models with human serum, TNFα, or ionizing radiation led to a mesenchymal state shift upon reporter selection. Single-cell transcriptomics annotated GSA populations in different conditions, revealing all glioblastoma states in vivo and upon external factor activation. Comparing GSA single-cell populations and patients confirmed these identities. The study established a comprehensive framework for testing and validating tumor model improvements to resemble patients, advancing tumor biology and treatment response understanding.

Cancer is a complex and dynamic disease that is difficult to treat due to its heterogeneous nature at multiple scales. Standard therapies such as surgery, radiation, and chemotherapy often fail, therefore superior therapies must be developed. Electroporation-based therapies offer an alternative to standard treatments, utilizing pulsed electric fields to permeabilize cell membranes to either enhance drug delivery (electrochemotherapy) or induce cancer cell death (irreversible electroporation). Electroporation treatments show promise in the clinic, however, are limited in the size of tumors that they can safely treat without increasing the applied voltage to an extent that induces thermal damage or muscle contractions in patients. A method to increase ablation size safely is needed. To make this advancement and to advance other cancer treatments as well, better in vitro tumor models are needed. Heterogeneity not only makes cancer difficult to treat, but also difficult to recapitulate in vitro. This dissertation addresses the complementary need to develop both better cancer therapies and more physiologically relevant in vitro tumor models. My results demonstrate that by using a calcium adjuvant with irreversible electroporation treatment, ablation size can be increased without using a higher applied voltage. Additional mechanistic studies identified signaling pathways that were differentially dysregulated under calcium and no calcium conditions, impacting cell death. Finally, I have successfully encapsulated cells in fibrin microgels which may enable the creation of more physiologically relevant and complex 3D in vitro and ex-vivo platforms to investigate IRE as well as other tumor therapies. / Doctor of Philosophy / Cancer is a complex and dynamic disease. Heterogeneity exists at the single cell, tumor, and patient levels making it difficult to establish a unified target for therapy. Standard therapies such as surgery, radiation, and chemotherapy often fail for this reason, therefore superior therapies must be developed. Electroporation-based therapies offer an alternative to standard treatments, utilizing pulsed electric fields to permeabilize cell membranes to either enhance drug delivery (electrochemotherapy) or induce cancer cell death (irreversible electroporation). Electroporation treatments show promise in the clinic, however, are limited in the size of tumors that they can safely treat without increasing the applied voltage to an extent that induces thermal damage or muscle contractions in patients. A method to increase ablation size safely is needed. To make this advancement and to advance other cancer treatments as well, better tumor models are needed. Many of the same challenges in treating cancer serve as challenges in creating physiologically relevant tumor models. In this dissertation, I have developed a simplified platform to test whether using a calcium additive with irreversible electroporation therapies enhances ablation size. My results demonstrate that by using a calcium additive with irreversible electroporation treatment, ablation size can be increased without using a higher applied voltage. In addition, the biological pathways responsible for cell death in irreversible electroporation treatment with and without calcium were studied. Finally, I have successfully encapsulated cells in fibrin microgels that can be used to create better tumor models that encompass the heterogeneity of tumors found in the body.

This work investigates the use of irreversible electroporation (IRE) for tissue engineering applications and as a cancer ablation therapy. IRE uses short, high-intensity electric pulses to create pores in a cell's membrane and disrupt its stability. At a certain energy level, damage to the cell becomes too great and it leads to cell death. The particular mechanisms that drive this response are still not completely understood. Thus, further characterization of this behavior for cell death induced by pulsed electric fields (PEFs) will advance the understanding of these types of therapies and encourage their use to treat unresectable tumors that can benefit from the non-thermal mechanism of action which spares critical blood vessels and nerves in the surrounding area. We evaluate the response to PEFs by different cell types through experimental testing combined with computer simulations of these treatments. We show that IRE can be used to kill a specific type of bacteria that produce cellulose which can be used as an implantable material to repair damaged tissues. By killing these bacteria at particular times and locations during their cellulose production, we can create conduits in the overall structure of this material for the transport of oxygen and nutrients to the cells within the area after implantation. The use of tissue models also plays a key role in the investigation of various cancer treatments by providing a controlled environment which can mimic the state of cells within a tumor. We use tumor models comprised of a mix of collagen and cancer cells to evaluate their response to IRE based on the parameters that induce cell death and the time it takes for this process to occur. The treatment of prostate and pancreatic cancer cells with standard monopolar (only positive polarity) IRE pulses resulted in different time points for a full lesion (area of cell death) to develop for each cell type. These results indicate the presence of secondary processes within a cell that induce further cell death in the border of the lesion and cause the lesion to increase in size several hours after treatment. The use of high-frequency irreversible electroporation (H-FIRE)--comprised of short bursts of high-intensity, bipolar (both positive and negative polarity) pulses--can selectively treat cancer cells while keeping healthy cells in the neighboring areas alive. We show that H-FIRE pulses can target tumor-initiating cells (TICs) and late-stage, malignant cancer cells over non-malignant cells using a mouse ovarian cancer model representative of different stages of disease progression. To further explore the mechanisms that drive this difference in response to IRE and H-FIRE, we used more complex tumor models. Spheroids are a type of 3D cell culture model characterized by the aggregation of one or more types of cells within a single compact structure; when embedded in collagen gels, these provide cell-to-cell contact and cell-to-matrix adhesion by interactions of cells with the collagen fibers (closely mimicking the tumor microenvironment). The parameters for successful ablation with IRE and H-FIRE can be further optimized with the use of these models and the underlying mechanisms driving the response to PEFs at the cellular level can be revealed. / Ph. D. / This work investigates the use of irreversible electroporation (IRE) for tissue engineering applications and as a cancer ablation therapy. IRE uses short, high-intensity electric pulses to create pores in a cell’s membrane and disrupt its stability. At a certain energy level, damage to the cell becomes too great and it leads to cell death. The particular mechanisms that drive this response are still not completely understood. Thus, further characterization of this behavior for cell death induced by pulsed electric fields (PEFs) will advance the understanding of these types of therapies and encourage their use to treat unresectable tumors that can benefit from the non-thermal mechanism of action which spares critical blood vessels and nerves in the surrounding area. We evaluate the response to PEFs by different cell types through experimental testing combined with computer simulations of these treatments. We show that IRE can be used to kill a specific type of bacteria that produce cellulose which can be used as an implantable material to repair damaged tissues. By killing these bacteria at particular times and locations during their cellulose production, we can create conduits in the overall structure of this material for the transport of oxygen and nutrients to the cells within the area after implantation. The use of tissue models also plays a key role in the investigation of various cancer treatments by providing a controlled environment which can mimic the state of cells within a tumor. We use tumor models comprised of a mix of collagen and cancer cells to evaluate their response to IRE based on the parameters that induce cell death and the time it takes for this process to occur. The treatment of prostate and pancreatic cancer cells with standard monopolar (only positive polarity) IRE pulses resulted in different time points for a full lesion (area of cell death) to develop for each cell type. These results indicate the presence of secondary processes within a cell that induce further cell death in the border of the lesion and cause the lesion to increase in size several hours after treatment. The use of high-frequency irreversible electroporation (H-FIRE)—comprised of short bursts of high-intensity, bipolar (both positive and negative polarity) pulses—can selectively treat cancer cells while keeping healthy cells in the neighboring areas alive. We show that H-FIRE pulses can target tumor-initiating cells (TICs) and late-stage, malignant cancer cells over non-malignant cells using a mouse ovarian cancer model representative of different stages of disease progression. To further explore the mechanisms that drive this difference in response to IRE and H-FIRE, we used more complex tumor models. Spheroids are a type of 3D cell culture model characterized by the aggregation of one or more types of cells within a single compact structure; when embedded in collagen gels, these provide cell-to-cell contact and cell-to-matrix adhesion by interactions of cells with the collagen fibers (closely mimicking the tumor microenvironment). The parameters for successful ablation with IRE and H-FIRE can be further optimized with the use of these models and the underlying mechanisms driving the response to PEFs at the cellular level can be revealed.

Regulation of cancer cell phenotype by the tumor microenvironment has motivated further investigation into how microenvironmental factors could contribute to tumor initiation, development, and therapeutic resistance. Analyzing how the microenvironment drives tumor development and cancer cell heterogeneity is particularly important in cancers such as glioblastoma multiforme (GBM) that have no known risk factors and are characterized by a high degree of heterogeneity. GBM patients have a median survival of 15 months and therefore are in great need of more effective therapeutic options. The goal of this research is to generate in vitro models of the heterogeneous brain tumor microenvironment, with a focus on vascular dynamics, to probe the impact of microenvironmental cues on tumor progression and to integrate the tumor models with highly sensitive analytical tools to characterize the epigenome of discrete phenotypic subpopulations that contribute to intratumoral cellular heterogeneity. As GBM tumors are characterized by a dense vasculature, we delved into microenvironmental factors that may be promoting angiogenesis. The correlations emerging between inflammation and cancer led to analysis of the inflammatory molecule lipopolysaccharide (LPS). We utilized 3D micro-tissue models to simulate vascular exposure to ultra-low chronic inflammatory levels of LPS and observed an increase in vascular formation when brain endothelial cells were exposed to ultra-low doses of LPS. We also utilized our micro-tissue models to analyze histone methylation changes across the epigenome in response to microenvironmental cues, namely culture dimensionality and oxygen status. The H3K4me3 modification we analyzed is associated with increased gene transcription, therefore the alterations we observed in H3K4me3 binding across the genome could be a mechanism by which the tumor microenvironment is regulating cancer cell phenotype. Lastly, we developed a microfluidic platform in which vascular dynamics along with microenvironmental heterogeneities can be modeled in a more physiologically relevant context. We believe the studies presented in this dissertation provide insight into how vasculature primed by chronic inflammation and epigenetic alterations in tumor cells could both contribute to enhanced tumor development. Modeling these biological processes in our advanced microfluidic platform further enables us to better understand microenvironmental regulation of tumor progression, uncovering new potential therapeutic targets. / PHD / Regulation of cancer cell behavior by the tumor microenvironment, which includes the surrounding extracellular matrix, native healthy cells, and signaling molecules, has motivated further investigation into how microenvironmental factors could contribute to tumor initiation, development, and therapeutic resistance. Analyzing how the microenvironment drives tumor development and heterogeneity in cancer cell behavior is particularly important in cancers such as glioblastoma multiforme (GBM) that have no known risk factors and are characterized by a high degree of heterogeneity. GBM patients have a median survival of 15 months and therefore are in great need of more effective therapeutic options. The goal of this research is to generate models of the heterogeneous brain tumor microenvironment with a focus on how microenvironmental cues impact blood vessel development, which facilitates tumor progression. We will also use these tumor models, along with sensitive analytical tools, to characterize epigenetic modifications that potentially contribute to tumor cell heterogeneity. As GBM tumors are characterized by a dense vasculature, we delved into microenvironmental factors that may promote blood vessel growth. The correlations emerging between inflammation and cancer led to analysis of the inflammatory molecule lipopolysaccharide (LPS). We utilized 3D tumor models to simulate blood vessel exposure to ultra-low chronic inflammatory levels of LPS and observed an increase in blood vessel formation when brain endothelial cells were exposed to ultra-low doses of LPS. We also utilized our tissue models to analyze histone methylation changes across the epigenome in response to microenvironmental cues, namely culture dimensionality and oxygen status. The histone methylation changes we observed across the genome could be a mechanism by which the tumor microenvironment is regulating cancer cell v behavior. Lastly, we developed a microfluidic platform in which blood vessel development along with microenvironmental heterogeneities can be modeled in a more physiologically relevant context. We believe the studies presented in this dissertation provide insight into how blood vessel exposure to chronic inflammatory factors and epigenetic alterations in tumor cells could both contribute to enhanced tumor development. Modeling these biological processes in our advanced microfluidic platform further enables us to better understand microenvironmental regulation of tumor progression, uncovering new potential therapeutic targets.

Microsystems are a broad category of engineered technologies in the micro and nano scale that have a diverse range of applications. They are emerging as a powerful tool in the field of biomedical research, drug discovery, as well as clinical diagnostics and prognostics, especially with regards to cancer. One of the major challenges in precision and personalized medicine in cancer lies in the technical difficulties of ex-vivo cell culture and propagation of the limited number of primary cells derived from patients. Therefore, our aims are to 1. Develop a biologically relevant platform for culturing cancer cells and characterize how it influences the cell growth and phenotype compared to conventional 2-dimensional(2D) cell culturing techniques, 2. Isolate secondary metabolites from endophytic fungi and screen them on the platform for potential anticancer properties in a preliminary drug discovery pipeline, 3. Design and develop biosensors for quantifying cell responses in real-time within these systems. Several biomaterial scaffolds with microscale architectures have been utilized for engineering the tumor extracellular matrix, but very few studies have thoroughly characterized the phenotypic changes in their cell models, which is critical for translational applications of biomaterial systems. The overall objective of these studies is to engineer a biomimetic platform for the culture of breast cancer cells in vitro and to quantify and profile their phenotypic changes. In order to do this, we first evaluated a blank-slate matrix consisting of thiolated collagen, hyaluronic acid and heparin, cross-linked chemically via Michael addition reaction using diacrylate functionalized poly (ethylene glycol). The hydrogel network was used with triple-negative breast cancer cells and showed significant changes in characteristics, with cells self-assembling to form a 3D spheroid morphology, with higher viability, and exhibiting significantly lower cell death upon chemotherapy treatment, as well as had a decrease in proliferation. Furthemore, the transcriptomic changes quantified using RNA-Seq and Next-Gen Sequencing showed the dramatic changes in some of the commonly targeted pathways in cancer therapy. Furthermore, we were able to show the importance of our biomimetic platform in the process of drug discovery using fungal endophytes and their secondary metabolites as the source for potential anticancer molecules. Additionally, we developed gold nanoparticle and antibody-based (ICAM1 and CD11b) sensors to quantify cell responses spatiotemporally on our platform. We were able to show quenching of the green fluorescent fluorophores due to the Förster Resonance Energy Transfer mechanism between the fluorophore and the gold nanometal surface. We also observed antigen-dependent recovery of fluorescence and inhibition of energy transfer upon the antibody binding to the cell-surface receptors. Future efforts are directed towards incorporating the hydrogel system with antigen-dependent sensors in a conceptually-designed microfluidic platform to spatiotemporally quantify the expression of surface proteins in various cells of the tumor stroma. This includes the migration,infiltration, and polarization of specific immune cells. This approach will provide further insight into the heterogeneity of cells at the single-cell resolution in defined spaces within the 3D microfluidic platform. / Doctor of Philosophy / Microsystems are a broad category of engineered technologies in the micro and nano scale that have a diverse range of applications. They are emerging as a powerful tool in the field of biomedical research, drug discovery, as well as clinical diagnostics and prognostics, especially with regards to cancer. However, a major challenge in being able to offer personalized medicine to cancer patients comes from the difficulty of growing cells from the patient's tumor biopsy in a laboratory for further screening and analysis. There are also limited resources available for real-time expression of proteins on cell-surfaces, that could be potential biomarkers and targets for treatment. Various natural and synthetic polymers are biocompatible and have been used widely in engineering the tumor extracellular matrix. However, the effect of hydrogels derived from these polymers on the specific tumor cells are not always well characterized. Our studies explore the influence of a biohybrid hydrogel on breast cancer cells and our results show that the microscale architecture of the hydrogel platform works as a suitable scaffold for recapitulating the 3-dimensional(3D) breast tumor microenvironment, and can also be employed in the drug discovery process. Additionally, we developed a nano-scale biosensor to enable the quantification of specific cell-surface proteins in real-time. Ongoing and future efforts are focused on designing and fabricating a microfluidic device with precise control over the design of space and special chambers for cell culture. These will be used for studying interactions of various cells in the tumor microenvironment that influence cancer progression. Integrating these micro-scale systems, including sensors will allow researchers to quantify cell behavior in response to the variable factors they are exposed to, as well as provide insight to answer fundamental questions about cancer biology that are limited by the conventional 2D cell culture systems.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-12T13:07:39Z No. of bitstreams: 2 Dissertação - Carla Martí Castelló - 2017.pdf: 3945260 bytes, checksum: f2fe2f6e23c2e7bc3c77a3fa04a19181 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-12T13:33:15Z (GMT) No. of bitstreams: 2 Dissertação - Carla Martí Castelló - 2017.pdf: 3945260 bytes, checksum: f2fe2f6e23c2e7bc3c77a3fa04a19181 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-12T13:33:15Z (GMT). No. of bitstreams: 2 Dissertação - Carla Martí Castelló - 2017.pdf: 3945260 bytes, checksum: f2fe2f6e23c2e7bc3c77a3fa04a19181 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-03 / The research to prevent cancer, diagnose early, and find new therapies is one of the main challenges of current medicine, and in vivo tumor models are essential for this aim. Imaging techniques, such as ultrasound, assists the research by helping to obtain data that are more accurate and to reduce the number of animals necessary to obtain statistically significant results. Ehrlich carcinoma is one of the most widely used models but it has not an ultrasonographic description. In this study, serial ultrasound examinations were performed, in B-mode and Doppler, on Ehrlich solid carcinomas (ESC) inoculated in mice. From the measurements obtained by ultrasound, the growth patterns were analyzed and the tumors were separated in two groups depending on the specific growth rate (SGR). Ultrasonographic characteristics of capsule, margins, echotexture, vascular flow, and Doppler indices of Resistivity Index (RI) and Pulsability Index (PI) were compared between groups. ESC presents variable growth patterns; a capsule detectable by ultrasound, which sometimes present discontinuity; detectable flow in most of the exams; and the possibility of a central focus of necrosis or several necrosis focuses separated by tissue. In conclusion, tumors with high vascularization tend to have high SGR, while tumors that presented homogeneous echotexture and absence of blood flow tend to have lower SGR. The study also showed that changes in tumor vessels are reflected in Doppler indices, with significantly lower RI and PI, than normal vessels / A pesquisa para o diagnóstico precoce, prevenção e novos tratamentos do câncer é um dos principais desafios da medicina atual e os modelos tumorais in vivo são imprescindíveis para esse fim. Técnicas de imagem, como a ultrassonografia, auxiliam a obter dados mais precisos e diminuir o número de animais para obter resultados estatisticamente significativos. O carcinoma de Ehrlich é um dos modelos mais usados, mas não existem descrições ultrassonográficas dele. Nesse trabalho, foram realizados exames ultrassonográficos seriados, em modo-B e Doppler, em carcinomas sólidos de Ehrlich (ESC) inoculados em camundongos. A partir das medidas obtidas por ultrassom, foram analisados os padrões de crescimento e os tumores foram separados em dois grupos dependendo da taxa específica de crescimento (SGR). Foram comparadas as características ultrassonográficas da capsula, margens, ecotextura, distribuição e quantidade de fluxo e os índices Doppler, Índice de Resistividade (IR) e Índice de Pulsabilidade (IP), entre os grupos. Os ESCs apresentaram-se como tumores com padrões de crescimento variáveis; uma capsula detectável por ultrassonografia, mas que pode apresentar descontinuidade em alguns casos; fluxo detectável na maioria dos exames; e que podem apresentam um foco de necrose central ou vários focos de necrose separados. Conclui-se que os tumores que apresentam alta vascularização tendem a ter alta SGR enquanto os que apresentam ausência de fluxo e ecotextura homogênea estão correlacionados com baixa SGR. O estudo também demonstrou que as alterações e mudanças nos vasos tumorais se encontram refletidas nos índices Doppler, apresentando IR e IP significativamente menores as dos vasos extratumorais.

Immunotherapy became a very promising approach for cancer therapy. Tumor cells are eliminated using the body's own immune system with minimal negative effect on healthy tissue. This thesis is focused on immunotherapy based on activation of innate immunity, specifically on intratumoral application of ligands stimulating phagocytosis and Toll-like receptor ligands. This therapeutic approach was tested in several types of tumor mouse models, such as melanoma B16-F10, pancreatic adenocarcinoma, and pheochromocytoma. The composition of the therapeutic mixture as well as the application schedule were optimized in our studies. Subsequently the underlying mechanisms involved in tumor elimination during this therapy were investigated.