Glioblastoma Multiforme (GBM) is a highly aggressive and prevalent brain tumor with an average 5-year survival rate of approximately 6.9%. Its complex pathophysiology, characterized by the capacity to invade surrounding tissues beyond the visible tumor margin, intratumor heterogeneity, hypoxic core, and the presence of the blood-brain barrier (BBB) that restricts the penetration of large therapeutic agents, all pose formidable challenges for effective therapeutic intervention. The standard of care for GBM has thus far exhibited limited success, and patients often face a poor prognosis. Electroporation-based therapies, such as irreversible electroporation (IRE), have emerged as promising alternatives to conventional treatments. By utilizing high amplitude pulsed electric fields, IRE is able to permeabilize cells, disrupt the BBB, and induce non thermal ablation of soft tissues. However, IRE is oftentimes accompanied by undesirable secondary effects such as muscle contractions, complex anesthetic protocols, and susceptibility to electrical heterogeneities, which have impeded its clinical translation. To address these limitations, high-frequency IRE (H-FIRE) was developed. H-FIRE employs short bursts of bipolar pulses, similar in duration to the cell charging time constant, enabling the desired tissue ablation while minimizing nerve excitation and muscle contractions. Additionally, H-FIRE reduces susceptibility to electrical heterogeneities, allowing for more predictable treatment volumes, thus enhancing the feasibility of clinical translation. This dissertation investigates H-FIRE for targeting malignant gliomas while looking into improved efficacy when administering the therapy in conjunction with other treatment forms and technologies. Specifically, the presented work focuses on several key areas: (1) determining the effect of pulsing protocol and geometric configuration selection on the biological outcomes from electroporation; (2) using a tumor bearing rodent glioma model to evaluate the effects of H-FIRE as a standalone therapy and as a combinatorial therapy with liposomal doxorubicin; (3) investigating the effects of waveform shape on biological outcomes; (4) utilizing real-time Fourier Analysis SpecTroscopy (FAST) to accurately model rises in temperature during treatment; and (5) modifying real-time FAST methods to determine treatment endpoints for safe and effective ablation volumes. / Doctor of Philosophy / Glioblastoma Multiforme (GBM) is one of the deadliest tumors, with an overall five-year survival rate of approximately 6.9%. Unfortunately, it also holds the position of being the most prevalent malignant brain tumor, constituting nearly 50.1% of all primary malignant brain tumor diagnoses. Despite its widespread occurrence, there has been limited success in improving survival rates. The tumor's infiltrative nature and its location behind the blood-brain barrier (BBB), which often screens out large drug molecules like chemotherapeutics, contribute significantly to these unfavorable treatment outcomes. This dissertation explores the potential of high-frequency irreversible electroporation (H-FIRE) as a solution to these challenges. H-FIRE employs bursts of pulsed electric fields to induce nanoscale defects in the cell membrane. The response to these defects may involve temporary pores that facilitate the uptake of therapeutic molecules into the cell, or larger and longer lasting pores that disrupt cell homeostasis, ultimately leading to cell death. Furthermore, this pulsed field therapy has shown success in enabling molecules to bypass the BBB. Thus, this dissertation aims to elucidate the various biophysical phenomena associated with H-FIRE, shedding light on how to manipulate treatment protocols to maximize BBB disruption and enhance therapy when used in conjunction with combinatorial agents. Additionally, this work aims to further develop technologies to provide real-time feedback, ensuring the safe and effective delivery of the treatment. Through these efforts, this dissertation aspires to offer valuable insights into optimizing H-FIRE for the treatment of malignant gliomas and advancing the understanding of combinatorial therapies in this specific context.

Glioblastoma (GBM) is the most common and deadliest of the malignant primary brain tumors in humans, with a reported 5-year survival rate of only 6.8% despite years of extensive research. Failure to improve local tumor control rates and overall patient outcome is attributed to GBM's inherent therapeutic resistance. Marked heterogeneity, extensive local invasion within the brain parenchyma, and profound immunosuppression within the tumor microenvironment (TME) are some of the unique features that drive GBM therapeutic resistance. Furthermore, tumor cells are sequestered behind the blood-brain barrier (BBB), limiting delivery of effective therapeutics and immune cell infiltration into the local tumor. Electroporation-based therapies, such as irreversible electroporation (IRE) and second generation, high-frequency IRE (H-FIRE) represent attractive alternative approaches to standard GBM therapy given their ability to induce transient BBB disruption (BBBD), achieve non-thermal tumor cell ablation and stimulate local and systemic anti-tumor immune responses without significant morbidity. The following work explores the use of H-FIRE to overcome GBM-induced therapeutic resistance and improve treatment success. Chapter 1 opens with an overview of GBM and known barriers to treatment success. Here, we emphasize the utility of spontaneous canine gliomas as an ideal translational model for investigations into novel treatment approaches. Chapter 2 introduces novel ablation methods (i.e. IRE/H-FIRE) capable of targeting treatment-resistant cancer stem cells. The focus of Chapter 3 is to highlight IRE applications in a variety of spontaneous tumor types. In Chapter 4, we investigate the feasibility and local immunologic response of percutaneous H-FIRE for treatment of primary liver tumors using a spontaneous canine hepatocellular carcinoma (HCC) model. In chapter 5, we characterize the mechanisms of H-FIRE-mediated BBBD in an in vivo healthy rodent model. In Chapter 6, we characterize the local and systemic immune responses to intracranial H-FIRE in rodent and canine glioma models to enhance the translational value of our work. Collectively, our work demonstrates the potential for H-FIRE to overcome therapeutic resistance in GBM, thereby supporting its use as a novel, alternative treatment approach to standard therapy. / Doctor of Philosophy / Glioblastoma (GBM) is the most common and deadliest form of primary brain cancer in humans, with only 6.8% of people surviving 5-years after their diagnosis. GBM is characterized by a number of unique features that make it resistant to standard treatments, such as surgery, radiation and chemotherapy. Examples include: (1) extensive invasion of tumor cells into the brain, making complete removal via surgery very difficult; (2) tumor cells are protected by a structure called the blood-brain barrier (BBB), which restricts the entry of most drugs (i.e. chemotherapy) and many immune cells, into the brain, thereby preventing them from reaching tumor cells; (3) tumor cells produce substances that block the immune system from being able to detect the tumor itself, which allows it to continue to grow undetected. High-frequency irreversible electroporation (H-FIRE) represents a new approach for the treatment of GBM. H-FIRE uses electric pulses to temporarily or permanently injure cell membranes without the use of heat, which allows for very precise treatment. The following work explores the ways in which H-FIRE can interfere with specific GBM features that drive its resistance to treatment. Here, we demonstrate that H-FIRE is capable of temporarily disrupting the BBB and characterize the mechanisms by which this occurs. This allows for drugs and immune cells within the blood to enter the brain and access the tumor cells, particularly those extending beyond the visible tumor mass and invading the brain. We also illustrate the potential for H-FIRE treatment within the brain to stimulate local and systemic immune responses by causing the release of proteins from injured cells. Similar to a vaccine, these proteins are recognized by the immune system, which becomes primed to help fight off cancer cells within the body. The end result is an anti-tumor immune response. Collectively, this work supports the use of H-FIRE as an alternative treatment approach to standard therapy for GBM given its potential to overcome certain causes of treatment resistance.