Glioblastoma is the most common and deadly primary brain tumor with an average survival of 15 months following diagnosis. Characterized as highly infiltrative with diffuse tumor margins, complete resection and annihilation of tumor cells is impossible following current standard of care therapies. Thus, tumor recurrence is inevitable. Interstitial fluid surrounds all of the cells in the body and has been linked to elevated invasion in glioma, which highlights the importance of this understudied fluid compartment in the brain. The primary objective of this dissertation was to identify specific interstitial fluid transport behaviors associated with elevated invasion surrounding glioma tumors. We first describe our methods to measure interstitial fluid flow in the brain using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), a clinically used, non-invasive imaging modality. We highlight the versatility of the technique and the possibilities that could arise from widespread adoption into existing perfusion-based imaging protocols. Using this method, we examined transport associated with invasion in a murine GL261 cell line. We found that elevated interstitial fluid velocity magnitudes, decreased diffusion coefficients and regions with accumulating flow were significantly associated with invasion. We tested the validity of our invasive trends by extending our analysis to multiple, clinically-relevant tumor locations in the brain. Interestingly, we found invasion did not follow the same trends across brain regions indicating location-specific structures may drive both interstitial flow and corresponding invasion heterogeneities. Lastly, we aimed to manipulate flow by engaging with the meningeal lymphatics, an established pathway for interstitial fluid drainage. Over-expression of VEGF-C in the tumor microenvironment neither enhanced drainage nor altered invasion in comparison to our control, indicating other tumor-secreted growth factors, such as VEGF-A, may play a larger role in mediating flow and invasion. Taken together, by identifying specific transport factors associated with invasion, we may be better equipped to target and treat infiltrative tumor margins, ultimately extending survival in patients diagnosed with this devastating disease. / Doctor of Philosophy / Glioblastoma is the most common and deadly primary brain tumor with an average survival of 15 months following diagnosis. Characterized as highly infiltrative with diffuse tumor margins, complete resection and annihilation of tumor cells is impossible following current standard of care therapies. Thus, tumor recurrence is inevitable. Interstitial fluid surrounds all of the cells in the body and has been linked to elevated invasion in glioma, which highlights the importance of this understudied fluid compartment in the brain. The primary objective of this dissertation was to identify specific interstitial fluid transport behaviors associated with elevated invasion surrounding glioma tumors. We first describe our methods to measure interstitial fluid flow in the brain using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), a clinically used, non-invasive imaging modality. We highlight the versatility of the technique and the possibilities that could arise from widespread adoption into existing imaging projects. Using this method, we examined transport associated with cancer cell invasion in a mouse tumor cell line. We found that interstitial fluid speeds were elevated while diffusion was decreased in regions of invasion. Further, regions that had interstitial fluid flow congregation were significantly associated with invasion. We tested the validity of these invasive trends by extending our analysis to multiple, clinically-relevant tumor locations in the brain. Interestingly, we found invasion did not follow the same trends across brain regions, indicating location-specific structures may drive both interstitial flow and invasion differences. Lastly, we aimed to manipulate flow by engaging with the meningeal lymphatics, an established pathway for interstitial fluid drainage. Following administration of a meningeal lymphatic-relevant protein, we saw no changes in flow or invasion in comparison to our untreated control, indicating other tumor-secreted proteins may play a larger role in these responses. Taken together, by identifying specific transport factors associated with invasion, we may be better equipped to target and treat infiltrative tumor margins, ultimately extending survival in patients diagnosed with this devastating disease.

The human brain is a complex organ that is responsible for regulating all the physiological processes in the body, ranging from memory to movement. As humans age, the brain goes through a variety of changes including a reduction in glymphatic waste clearance and increase in glial reactivity. Two neurological conditions that affect individuals over the age of 65 include glioblastoma (GBM) and Alzheimer's disease (AD). Interestingly, patients with GBM do not present with AD and vice versa. Both conditions are characterized by a disruption in interstitial fluid flow (IFF) and an increase in neuroinflammation. Throughout the following dissertation, we examined the role of IFF in AD and GBM progression using a three-sided approach (in vivo, in vitro, and in silico). Increased IFF underlies glioma invasion into the surrounding tumor microenvironment (TME) in GBM. We used a 3D hydrogel model of the GBM TME to examine potential pathways by which astrocytes and microglia contribute to glioma invasion. A reduction in IFF contributes to accumulation of the toxic protein amyloid beta (Aβ) in AD. We sought to create a novel, patient-inspired model of the AD hippocampus for examination of the relationship between IFF and Aβ clearance. Human AD and unaffected control hippocampal brain samples were stained for markers of neurons, astrocytes, microglia and Aβ. The percentage of each cell population in the CA1 region of the hippocampus was calculated. We also analyzed the amount and characteristics of the Aβ aggregates present in this hippocampal region. Pearson correlation analysis was completed to assess the relationships between the various cell populations, Aβ load, and patient descriptors. The cell ratios gleaned from the patient samples were incorporated into a novel, 3D hydrogel model of the AD hippocampus. This model features a hydrogel mixture like the native brain extracellular matrix (ECM) and allows for the application of IFF and Aβ. To our knowledge, we are the first group to create a patient-specific triculture model of the AD hippocampus, which is the main site of Aβ aggregation in the AD brain. We used this model to examine the relationship between IFF-mediated Aβ clearance and glial reactivity. The last aim of this dissertation was to create a computational model for examining Aβ binding within the ECM and the effects of IFF on Aβ clearance. In vitro experiments were conducted to generate 3D renderings of glial cells and to determine relevant parameters for our model. Throughout this work, we discuss the relationship between disruption in IFF and glial reactivity in the context of GBM and AD. / Doctor of Philosophy / The human brain is a complex organ that is responsible for regulating all the physiological processes in the body, ranging from memory to movement. As humans age, the brain goes through a variety of changes including a reduction in brain waste removal and an increase in inflammation. Two neurological conditions that affect individuals over the age of 65 include glioblastoma (GBM) and Alzheimer's disease (AD). Interestingly, patients with GBM do not present with AD and vice versa. Both conditions are characterized by a disruption in brain interstitial fluid flow (IFF) and an increase in neuroinflammation. Throughout the following dissertation, we examined the role of IFF in AD and GBM progression using a three-sided approach including analysis of mouse and human tissues, engineered cell models, and computational methods. Specific interactions between brain cell types and their relationships with glioma invasion were examined using a 3D cell model that mimics the brain. Through the work presented here, we also sought to create a novel cell model of the hippocampus region located in the AD brain. We quantified the various cell types in the hippocampus of AD patient samples and incorporated this information into our hydrogel model. The resulting model features three brain cell types (astrocytes, microglia, and neurons) that are added at patient relevant ratios, a matrix that mimics the native brain scaffold, and allows for the application of IFF. In the AD brain there is a reduction in brain waste removal that leads to accumulation of the toxic protein amyloid beta (Aβ). We were successfully able to incorporate this protein within our model so we could assess the relationship between IFF and Aβ removal from the brain. We further studied this relationship using a new computational model of Aβ accumulation in the brain. Throughout this work, we discuss the connection between disrupted IFF and neuroinflammation in the context of GBM and AD.

Glioblastoma (GBM) is the most aggressive and malignant glioma. It accounts for 48.6% of all primary, malignant gliomas with a median survival of 15 months. Infiltration into the surrounding parenchyma is a hallmark of GBM. Radiotherapy is used to address the invasion; however, recent studies have implicated that radiation contributes to increased invasiveness of glioma. Although the effect of radiation on cells has been studied extensively, its effect on the transport of fluid is not well characterized. Transport in the brain which has significant roles in physiology, GBM pathophysiology, and GBM treatment. Thus, understanding the effect of radiation on transport within the lesion and surrounding interstitium will be beneficial in characterizing the effects of radiotherapy in GBM patients. This dissertation seeks to explore the relationship between radiation, transport, and movement of glioma cells and includes the following: 1) Characterizing in vitro motility metrics of glioma stem cell lines in and relating them to in vivo invasion. 2) Studying the effect of radiation on motility, flow-mediated invasion, extracellular matrix components, and transport within the lesion and interstitium. 3) Assessing transport in clinical images and relating transport parameters to progression of GBM. 4) Developing a novel pipeline for applying vector field topology to the study of interstitial fluid flow in glioma. Surprisingly, we found that motility metrics in vitro have a negative correlation trend with in vivo invasion. Next, we found that radiation causes a transient increase in advective flow, and a more sustained decrease in diffusivity in a murine glioma model. Tenascin C was found to correlate significantly with invasion and diffusivity, indicating that it might be a link between radiation, transport, and invasion. Furthermore, interstitial fluid flow was calculated and assessed in clinical images. This showed that interstitial fluid flow velocity magnitude in the tumor correlates with overall survival in GBM patients. Lastly, vector field topology was introduced as a novel method of studying transport that provides more detailed information to identify potential drivers of transport within a flow field. Altogether, this work presents novel insight into the effects of radiation on invasion and transport in GBM. Hopefully, this work can provide a foundation to build upon in efforts of improving treatment planning and clinical outcomes for GBM patients. / Doctor of Philosophy / Glioblastoma (GBM) is the most aggressive glioma. It accounts for 48.6% of all primary, malignant gliomas with a median survival of 15 months. The movement of cancer cells into the surrounding tissue is a defining factor of GBM. Radiotherapy is used after surgery to treat the remaining cancer cells in tissue surrounding the tumor; however, recent studies have implicated that radiation contributes to increased movement of glioma into surrounding tissue. Although the effect of radiation on cells has been studied extensively, its effect on transport of fluid is not well characterized. Interstitial fluid flow in the brain has significant roles in healthy bodily functions, GBM disease state, and GBM treatment. Thus, understanding the effect of radiation on transport within the tumor and surrounding tissue is beneficial in better characterizing the effects of radiotherapy. This dissertation seeks to explore the relationship between radiation, transport, and movement of glioma cells and includes the following: 1) Characterizing in vitro motility metrics of glioma cells in and relating them to in vivo movement into healthy tissue. 2) Studying the effect of radiation on motility, flow-mediated infiltration into healthy tissue, tissue matrix components, and fluid flow within the tumor and surrounding tissue. 3) Assessing transport in clinical images and relating transport parameters to progression of GBM. 4) Developing a novel pipeline for applying vector field topology to the study of interstitial fluid flow in glioma. Surprisingly, we found that motility metrics in vitro have a negative correlation trend with in vivo invasion. Next, we found that radiation causes a transient increase in flow velocity magnitude, and a more sustained decrease in diffusivity in a murine glioma model. Tenascin C, a component of the tissue matrix, was found to correlate significantly with invasion and diffusivity. This indicates that Tenascin C might be a link between radiation, transport, and invasion. Furthermore, interstitial fluid flow was calculated and assessed in clinical images which showed that interstitial fluid flow velocity magnitude in the tumor correlates with survival. Lastly, vector field topology was introduced as a novel method of studying fluid flow in glioma that provides more detailed information regarding the flow field. Altogether, this work presents novel insight into the effects of radiation on fluid flow and cellular movement in GBM. Hopefully, this work can provide a foundation to build upon in efforts of improving treatment planning and clinical outcomes for GBM patients.

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.

<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>