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This thesis project serves to fill experimental gaps needed to advance the goal of performing pre-clinical trials using an orthotopic rat glioblastoma model to evaluate the efficacy of high-frequency electroporation (H-FIRE) and QUAD-CTX tumor receptor-targeted cytotoxic conjugate therapies, individually and in combination, in selectively and thoroughly treating glioblastoma multiforme. In order to achieve this, an appropriate model must be developed and characterized. I have transduced F98 rat glioma cells to express red-shifted firefly luciferase, which will facilitate longitudinal tumor monitoring in vivo through bioluminescent imaging. I have characterized their response to H-FIRE relative to DI TNC1 rat astrocytes. I have demonstrated the presence of the molecular targets of QUAD in F98 cells. The in vitro characterization of this model has enabled preclinical studies of this promising glioblastoma therapy in an immunocompetent rat model, an important step before advancing ultimately to clinical human trials.

Neurofibromatosis type 1 (NF1) syndrome is one of the most common inherited neurological disorders, affecting about one in every three thousand individuals. The disease typically manifests in childhood and can result in significant morbidity and a shortened lifespan. Despite decades of research, there is still no cure for NF1, and treatment is largely symptomatic. This is due in part to the multisystemic nature of the disease and a lack of representative, translational animal models. Murine models of NF1 mimic individual aspects of the syndrome, but none fully represents the complexity of disease seen in human patients. There is a need for new models of NF1 to complement the mouse and improve success in clinical trials. The objective of this dissertation was to fill this need by developing and characterizing the first genetically engineered NF1+/- minipig and determining its utility as a preclinical disease model for human NF1. Using targeted gene editing and somatic cell nuclear transfer cloning, we generated NF1+/- minipigs harboring a specific human disease allele associated with severe phenotypes in NF1 patients. We enrolled cohorts of NF1+/- minipigs and wild-type litter mate controls in a longitudinal phenotyping study and assessed for manifestations of NF1 syndrome. We performed gross assessments by physical examination, radiography, and magnetic resonance imaging over time. We also assessed the histological, molecular and biochemical defects associated with NF1 using tissues and primary cells isolated from lesions in NF1+/- minipigs. We evaluated several targeted therapies currently in clinical trials for NF1-associated neoplasia using pharmacokinetic and pharmacodynamic analyses in blood and clinically relevant tissues from NF1+/- minipigs. The results of these studies show that our NF1 minipig model offers a predictive preclinical disease model that will be crucial for developing and validating new NF1-targeted therapies as well as improved imaging and surgical modalities for early detection of nervous system tumors. Furthermore, the methods presented provide a blueprint for using minipig tissues to evaluate pharmacodynamics of new targeted therapies and using primary cells from minipigs to uncover novel targets and cellular interactions within the neurofibroma microenvironment.

Programmed cell death 1 (PD-1; CD279) is a type 1 transmembrane protein expressed by T, B and natural killer (NK) cells following activation. Its ligand programmed cell death ligand 1 (PD-L1; B7-H1; CD274) is constitutively expressed on specific tumour and immune cells, including activated B and T cells, dendritic cells (DCs) and macrophages. Binding of PD-L1 to PD-1 can impede T cell activation, disrupt cytokine production, and induce the apoptosis or exhaustion of effector T cells, resulting in tumour growth. Retrospective studies have observed that PD-L1 expression frequently correlates with significantly worse overall prognosis and survival . However, little research has been done investigating the prognostic capabilities of PD-L1 in thyroid cancer. We therefore constructed tissue microarrays (TMAs) comprised of PTC patient biopsies to assess the prognostic capabilities of PDL1 and tumour infiltrating lymphocytes (TILs). A systematic review and meta-analysis were also undertaken to validate these findings. Moreover, serum and plasma levels of soluble PD-L1 (sPD-L1) were evaluated in a cohort of PTCs to investigate its potential as a non-invasive biomarker. Checkpoint inhibitors targeting the PD-1/PD-L1 signalling pathway have produced impressive responses in select patients across several tumour types. These antibodies may serve as an innovative and effective option for the treatment of aggressive thyroid cancers unresponsive to standard therapies. However, despite these encouraging results, only a fraction of patients currently benefit from checkpoint blockade therapy. Increasing evidence suggests that mutations in key oncogenic pathways can impede immune cell infiltration and hinder their anti-neoplastic function within the tumour microenvironment. Targeting these oncogenic pathways may be essential in re-establishing immune surveillance and sensitising patients to immune checkpoint blockade. However, in thyroid cancers, the relationship between oncogenic drivers and PD-L1 expression has yet to be examined. We therefore aimed to investigate the molecular signature of thyroid cancer in order to identify novel biomarkers which can be used to influence treatment direction and optimise patient benefit from novel antitumour agents in thyroid cancer. Specifically, we focused on the influence of the phosphatidyl-inositol-3-kinase (PI3K)/ protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) pathway and epithelial-tomesenchymal transition (EMT) on PD-L1 expression. We went on to perform an indepth analysis of a patient with BRAF-negative stage IVC ATC treated with the anti- PD-1 monoclonal antibody, pembrolizumab, following radiographic progression on chemoradiation. We demonstrate how serial biopsies can provide an ongoing picture of an individual patient’s molecular and microbiome profile prior to and throughout the course of treatment.