Molecular mechanisms in the development and progression of thyroid carcinomas are still not fully understood. In the present thesis the highly malignant anaplastic thyroid carcinoma (ATC) was used to study regulation of apoptosis and tumor interstitial fluid pressure (IFP). Addition of a natural estrogen metabolite, 2-Methoxyestradiol (2-ME), induced a G2/M cell cycle arrest and apoptosis in five out of six human ATC cell lines. Treatment with 2-ME induced DNA-fragmentation as well as activation of caspase-3. Inhibitors of JNK and p38 MAPKs activity decreased the effect of 2-ME suggesting involvement in the induction of apoptosis. Solid tumors have an elevated IFP. High IFP forms or reflects a barrier for exchange of molecules between microvessels and surrounding tissue. The mechanisms for the generation of the high IFP were investigated using a specific TGF-β inhibitor in an ATC model in athymic mice. Tumor IFP was lowered in TGF-β inhibitor-treated compared to control mice. Affymetrix microarray analysis showed a decreased expression of macrophage-associated genes in treated tumors. Furthermore, the number and activity of tumor-associated macrophages was reduced after TGF-β inhibition. A decreased protein leakage together with an increased coverage of α-smooth-muscle actin (SMA)-expressing cells indicated vessel normalization. An adjuvant treatment with the TGF-β inhibitor resulted in an increased treatment efficacy of doxorubicin. Thus, TGF-β inhibitor-treatment suggests improved microvessel function which results in a lowering of tumor IFP and increased tumor drug uptake. To create a model for specific inactivation of genes in the thyroid, a transgenic mouse with a thyrocyte-specific expression of Cre recombinase was generated. The thyroglobulin promoter together with an inducible Cre recombinase (creERT2) was used. Two transgenic founder lines were identified expressing cre mRNA solely in the thyroid. Functional activity of the CreERT2 protein was demonstrated by using a ROSA26-LacZ reporter mouse.

High interstitial fluid pressure (IFP) in tumours is recognized as a barrier to drug delivery resulting in reduced efficacy. High IFP impedes the normal process of convective fluid flow (CFF) from blood vessels into the interstitium. The aim of this study was to develop an in vitro model that could be used to measure CFF and to study its effects on drug delivery. The model consists of a transwell cell culture insert which supports the growth of multicell layers (MCL) on collagen coated membranes. A graduated tube is inserted into the transwell and a pressure gradient is applied across the membrane by raising the volume of medium in the tube above that of the bottom chamber. CFF is determined by measuring the weight of medium in the bottom chamber as a function of time. CFF was inversely proportional to MCL thickness and 41.1±3.6µm thick MCL has completely stopped CFF. Using a physiologically relevant hydrostatic pressure of 28mmHg, a CFF of 21µL/min was recorded using a DLD-1 MCL that was 12.21±3.2µm thick. Under these conditions, the rates of penetration of doxorubicin, imatinib and gefitinib were respectively 42, 26 and 13 folds greater than when no CFF exists. Reversing the CFF so that it opposed the drug diffusion gradient significantly impairs drug penetration. In conclusion, a novel in vitro model for assessing the impact of CFF on drug delivery has been developed. This model could be used to evaluate strategies designed to increase drug delivery to solid tumours by modifying the CFF.

The lymphatic and blood vascular systems are two important vessel networks that serve different roles in healthy states and in cancer. In breast cancer the most common cancer amongst women, mortality remains high despite increased treatment response due to metastatic spread, preferentially through the lymphatics. One aggressive subtype, triple negative breast cancer (TNBC) contributing to 15 to 30 percent of cases and is characterized by the absence of expression of three therapeutic biomarkers. As targeted therapy is limited, treatment relies on standard of care via surgery, radiotherapy, and chemotherapy with limited efficacy and increase in survival. Chemotherapies negatively alter the lymphatic vasculature benefiting the tumor, through lymphangiogenesis. This dissertation seeks to understand how the mechanisms of commonly used chemotherapeutics, like carboplatin, and a novel 2nd generation ablative therapy called High Frequency Irreversible Electroporation (H-FIRE), which utilizes electric pulses to ablate tumor cells, affect the lymphatic and blood microvasculature in the tumor, surrounding fat pad, tumor draining lymph node (TDLN) using multiple analysis methods. This occurred through three main methods 1) identification of oxidative stress effects of chemotherapeutic application of carboplatin on lymphatic endothelial cells in vitro, 2) characterization of lymphatic and blood microvascular dynamics in a 4T1 breast cancer mouse model treated with sub-ablative H-FIRE, 3) through the development of a novel habitat imaging method to identify treatment specific changes in the tumor draining lymph node, and the development of a hybrid agent-based model (ABM) to test cancer cell flow mediated invasion in brain cancer. Herin the work showed that carboplatin induced lymphatic phenotypic changes occurred through generation of reactive oxygen species dependent on VEGFR3 and was reversed through treatment with the antioxidant N-acetylcysteine. In the 4T1 model, sub ablation with H-FIRE induced temporal remodeling of the lymphatic and blood vasculature within the viable tumor, in the surrounding fat pad, and in the tumor draining lymph node over seven days, suggesting an optimal time of application of adjuvant therapy. The development of a habitat imaging analysis method to identify TDLN vascular habitats and the perturbation to treatment in a retrospective analysis of prior work. Lastly, the development of a hybrid ABM through the incorporation of experimentally measured fluid flow fields from dynamic contrast enhanced MRI imaging building upon existing work, and showing the usefulness in comparing mechanisms of cancer cell invasion mediated fluid flow. Altogether, this work presents novel insight into the lymphatic system in cancer within various treatments contexts and new methods of quantifying changes due to treatment. Hopefully, these findings can be used to further inform the field towards a more comprehensive understanding of treatment effects in breast cancer. / Doctor of Philosophy / The lymphatic and blood vascular systems are two important vessel networks that serve different purposes in healthy states and in the disease called cancer. In breast cancer , a common form of cancer in women , spread of this cancer tends towards the lymphatic vasculature and eventually to other parts of the body. Triple negative breast cancer (TNBC) a less common, but more aggressive form, relies on clinical standard treatments with anti-tumor drugs called chemotherapies. These chemotherapies negatively alter the lymphatic vasculature to the tumors benefit, leaving a lack new methods of treatment. This dissertation seeks to understand how the mechanisms of commonly used chemotherapeutics and a new promising pulsed electric field therapy , High frequency Irreversible Electroporation (H-FIRE), change the lymphatic and blood vessels over time and in different locations using different tools. This occurred through three main methods 1) the effects on lymphatic vascular cells treated with chemotherapy, 2) in a breast cancer mouse model treated with H-FIRE, 3) in math models of the draining lymphatic organ, called the lymph node and an agent-based math model (ABM) of cancer cell movement due to fluid flow. The work showed that in the lymphatic cells, carboplatin a type of chemotherapeutic used to treat breast cancer, changed lymphatic vasculature through generating stress through oxidation and was reversed through treatment with an anti-oxidant. In the breast cancer mouse model, incomplete ablation with H-FIRE caused time dependent changes to the lymphatic and blood vasculature in the tumor, in the surrounding tissue, and in the lymph node over seven days. This work shows the novel findings of pulsed electric field therapy causing changes to the lymphatic vasculature. The creation of a new method of identifying habitats of the lymph node was used to compare changes to the lymphatic and blood vasculature to treatment. Lastly, the creation of an ABM added measured fluid flow maps from medical imaging methods to build upon existing work, and showed the usefulness in comparing mechanisms of cancer cell invasion due to fluid flow. Altogether, this work presents novel insight into the lymphatic system in cancer within after various treatments are applied and new methods of measuring these changes because of treatment using multiple methods. It is our hope that these findings can be used to further inform the field towards a more comprehensive understanding of treatment effects in breast cancer.

Les acides gras polyinsaturés n-3 (acide docosahexaènoïque, DHA et acide eicosapentaènoïque, EPA) sensibilisent les tumeurs mammaires aux agents anticancéreux. Cette sensibilisation implique la régulation de la vascularisation tumorale. En effet, un régime nutritionnel EPA/DHA, associé à une chimiothérapie par le Docétaxel (Taxane) diminue la quantité de vascularisation (effet anti-angiogénique). Une amélioration de la qualité vasculaire est aussi observée par une diminution de la pression du liquide interstitiel, paramètre décrit comme un frein à la délivrance des drogues dans les tumeurs. Cette diminution est associée à une extravasation plus importante du bleu d’Evans, suggérant une meilleure distribution des agents anticancéreux au sein des tumeurs. L’effet antiangiogénique du DHA implique une diminution de la voie de signalisation VEGF/eNOS/NO. Ainsi, l’activation de la NO synthase endothéliale (eNOS) est diminuée dans des cellules endothéliales en culture et dans les tumeurs mammaires. Nos résultats suggèrent que l’apport d'acides gras EPA/DHA aux patients au cours de la chimiothérapie pourrait être une nouvelle approche thérapeutique pour normaliser la vascularisation tumorale et améliorer l’efficacité des traitements anticancéreux. / Polyunsaturated fatty acids n-3 (docosahexaenoic acid, DHA and eicosapentaenoic acid, EPA) sensitize mammary tumors to anticancer drugs. This sensitization involves the regulation of tumor vasculature. Indeed, a nutritional diet with EPA / DHA, in combination with taxane chemotherapy (docetaxel) decreases the vascular quantity (anti-angiogenic effect). An improvement of vascular quality is also observed by a decrease of interstitial fluid pressure, a parameter described as a barrier to drug delivery in tumors. This decrease improves extravasation of Evans blue, suggesting a better distribution of anticancer agents in tumors. The antiangiogenic effect of DHA involves a decrease of signaling pathway VEGF / eNOS / NO. Thus, activation of endothelial NO synthase (eNOS) is decreased in endothelial cells in culture and in mammary tumors. Our results suggest that intake of fatty acids EPA / DHA to patients during chemotherapy could be a new therapeutic approach to normalize tumor vasculature and improve the efficacy of cancer treatments.

Le liquide interstitiel est aujourd’hui considéré comme un candidat prometteur comme alternative, ou complément, à l’analyse sanguine pour la quantification de biomarqueurs. Localisé notamment dans la peau, sa composition demeure peu décrite dans la littérature. Cela peut s’expliquer par le fait que le prélèvement de liquide interstitiel reste problématique. En effet, les méthodes d’extraction actuelles sont chronophages, douloureuses et conduisent au prélèvement de volumes très faibles ne permettant pas toujours une analyse subséquente. L’utilisation de timbres de microaiguilles conçus en hydrogel superabsorbant représente une solution indolore, rapide et efficace pour le prélèvement du liquide interstitiel. Un premier type de timbre a été conçu par photopolymérisation, un processus de fabrication caractérisé par sa rapidité. Ce type de timbre de microaiguilles présente une capacité d’absorption très élevée et peut trouver une application dans l’élaboration des profils protéomique, métabolomique et lipidomique du liquide interstitiel dermique. Le second type de timbres de microaiguilles est obtenu par chauffage d’une formulation contenant des polymères superabsorbants. Ce procédé s’avère plus long mais conduit à un hydrogel superabsorbant riche en groupements chimiques permettant d’envisager une fonctionnalisation pour la capture et la détection in situ de biomarqueurs spécifiques du liquide interstitiel dermique. In fine, les timbres de microaiguilles développés pourraient donc permettre d’approfondir notre connaissance de la composition du liquide interstitiel; mais laissent également entrevoir la possibilité de développer des dispositifs médicaux portables permettant le diagnostic, ou la surveillance, rapide et indolore de certaines pathologies. Ces dispositifs pourraient diminuer les coûts normalement associés à ces pratiques et améliorer la prise en charge des patients. C’est le cas notamment de l’insuffisance cardiaque, dont la gestion pourrait être considérablement facilitée par le suivi à domicile du biomarqueur NT-proBNP. / Nowadays, interstitial fluid is considered a valid alternative for blood analysis and biomarker monitoring. However, its composition is scarcely described in the literature. Notably located in the skin, its collection remains a challenge as current methods are time-consuming, painful and the extracted volume limits subsequent analysis. Here we put forward the use of superabsorbant hydrogel-based microneedle patches to enable a painless, rapid and efficient sampling of dermal interstitial fluid. A first kind of microneedle patch was obtained using UV-curing, a rapid fabrication process. This type of microneedle patch enables the collection of a high volume of liquid and can therefore be utilized for subsequent proteomic, metabolomic and lipidomic analyses of the dermal interstitial fluid that had been extracted in a painless fashion. The second class of microneedle patch developed was fabricated from superabsorbant polymers using heating. Although time consuming, this process produced hydrogel-based microneedle patches that could be functionalized for the in situ detection of specific biomarkers in the dermal interstitial fluid. In fine, the aforementioned microneedle patches have the potential to broaden our understanding of the interstitial fluid composition, as well as be integrated in novel portable biosensing devices for a rapid and painless diagnosis, or for the monitoring of certain medical conditions. For example, quantifying the NT-proBNP biomarker in the dermal interstitial fluid could significantly improve the quality of life of heart failure patients.

<p>Subcutaneous administration has become a common approach for drug delivery of biotherapeutics, such as monoclonal antibodies, which is achieved mainly by absorption through the lymphatic system. This dissertation focuses on the computational modeling of the fluid flow and solute transport in the skin tissue and the quantitative investigation of lymphatic uptake. First, the various mechanisms governing drug transport and lymphatic uptake of biotherapeutics through subcutaneous injection are investigated quantitatively through high-fidelity numerical simulations, including lymphatic drainage, blood perfusion, binding, and metabolism. The tissue is modeled as a homogeneous porous medium using both a single-layered domain and a multi-layered domain, which includes the epidermis, dermis, hypodermis (subcutaneous tissue), and muscle layers. A systematic parameter study is conducted to understand the roles of different properties of the tissue in terms of permeability, porosity, and vascular permeability. The role of binding and metabolism on drug absorption is studied by varying the binding parameters for different macromolecules after coupling the transport equation with a pharmacokinetic equation. The interstitial pressure plays an essential role in regulating the absorption of unbound drug proteins during the injection, while the binding and metabolism of drug molecules reduce the total free drugs. </p> <p>  </p> <p>The lymphatic vessel network is essential to achieve the functions of the lymphatic system. Thus, the drug transport and lymphatic uptake through a three-dimensional hybrid discrete-continuum vessel network in the skin tissue are investigated through high-fidelity numerical simulations. The explicit heterogeneous vessel network is embedded into the continuum model to investigate the role of explicit heterogeneous vessel network in drug transport and absorption. The solute transport across the vessel wall is investigated under various transport conditions. The diffusion of the drug solutes through the explicit vessel wall affects the drug absorption after the injection, while the convection under large interstitial pressure dominates the drug absorption during the injection. The effect of diffusion cannot be captured by the previously developed continuum model. Furthermore, the effects of injection volume and depth on the lymphatic uptake are investigated in a multi-layered domain. The injection volume significantly affects lymphatic uptake through the heterogeneous vessel network, while the injection depth has little influence. At last, the binding and metabolism of drug molecules are studied to bridge the simulation to the experimentally measured drug clearance. </p> <p><br></p> <p>Convective transport of drug solutes in biological tissues is regulated by the interstitial fluid pressure, which plays a crucial role in drug absorption into the lymphatic system through the subcutaneous (SC) injection.  An approximate continuum poroelasticity model is developed to simulate the pressure evolution in the soft porous tissue during an SC injection. This poroelastic model mimics the deformation of the tissue by introducing the time variation of the interstitial fluid pressure. The advantage of this method lies in its computational time efficiency and simplicity, and it can accurately model the relaxation of pressure. The interstitial fluid pressure obtained using the proposed model is validated against both the analytical and the numerical solution of the poroelastic tissue model. The decreasing elasticity elongates the relaxation time of pressure, and the sensitivity of pressure relaxation to elasticity decreases with the hydraulic permeability, while the increasing porosity and permeability due to deformation alleviate the high pressure. </p> <p><br></p> <p>At last, an improved Kedem-Katchalsky model is developed to study solute transport across the lymphatic vessel network, including convection and diffusion in the multi-layered poroelastic tissue with a hybrid discrete-continuum vessel network embedded inside. The effect of different drug solutes with different Stokes radii and different structures of the lymphatic vessel network, such as fractal trees and Voronoi structure, on the lymphatic uptake is investigated. The drug solute with a small size has a larger partition coefficient and diffusivity across the openings of the lymphatic vessel wall, which favors drug absorption. The Voronoi structure is found to be more efficient in lymphatic uptake. </p> <p><br></p> <p>The systematic and quantitative investigation of subcutaneous absorption based on high-fidelity numerical simulations can provide guidance on the optimization of drug delivery systems and is valuable for the translation of bioavailability from the pre-clinical species to humans. We provide a novel approach to studying the diffusion and convection of drug molecules into the lymphatic system by developing the hybrid discrete-continuum vessel network. The study of the solute transport across the discrete lymphatic vessel walls further improves our understanding of lymphatic uptake. The novel and time-efficient computational model for solute transport across the lymphatic vasculature connects the microscopic properties of the lymphatic vessel membrane to macroscopic drug absorption. The comprehensive hybrid vessel network model developed here can be further used to improve our understanding of the diseases caused by the disturbed lymphatic system, such as lymphedema, and provide insights into the treatment of diseases caused by the malfunction of lymphatics.</p>