Project 3 Summary This project will develop a combinatorial therapy that enhances high-frequency irreversible electroporation (H- FIRE) focal ablation with targeting agents to selectively target tumor cells which infiltrate beyond the tumor margin, a current challenge to successfully treating glioblastoma (GBM). H-FIRE is a new, minimally invasive ablation technique that involves delivering a series of low energy (intense, but short) bursts of electric pulses to targeted tissue for approximately 5 minutes. These pulses destabilize the cell membranes of the targeted tissue, inducing cell death without causing thermal damage. H-FIRE creates complete and predictable cell ablation with sharp transition between normal and necrotic tissue. Furthermore, H-FIRE only affects a single component of the treated volume, the cell membrane, and preserves important tissue components such as scaffolds, myelin sheaths, blood vessels, connective tissue, and nerves. We hypothesize that infiltrative cells (beyond the H-FIRE ablated zone) can be selectively killed using a low dose of a targeted anti-GBM agent in combination with H-FIRE, resulting in complete regression of tumors while preventing further infiltration beyond the treatment margins. For tumor cells outside the zone of direct tissue ablation, there is a non-destructive increase in blood-brain barrier (BBB) permeability, making these cells more susceptible to the administered agents and thus, making the combination of IRE and adjuvant agents synergistic. By focusing on brain cancer, we will be directly addressing the need to develop alternative approaches to radiation and chemotherapy, both of which have adverse side effects and limited efficacy. This Project 3 of our P01 proposal has three Specific Aims. In Aim 1, we will characterize the electrical properties of brain tumor tissues in response to H-FIRE pulses at both the cellular and tissue scales, using a combination of engineered 3D tumor models, as well as excised healthy (canine) and malignant (human, canine) brain tissue. We will also characterize cellular responses to H-FIRE in combination with QUAD-CTX/Pep-1-L-CTX from Project 1 of this P01. The data collected from this study will help accurately predict treatment volume for future H-FIRE treatments. In Aim 2, we will quantify the effects of H-FIRE treatment in rats to assess BBB breakdown in vivo. In Aim 3, we will assess QUAD-CTX/Pep-1-L-CTX coupled with H-FIRE to treat spontaneous brain tumors in canine patients. If successful, this study will provide the foundation for a new form of cancer therapy capable of improving conventional treatments for both tumors and the infiltrative GBM cells beyond the tumor margin to eliminate the likelihood of tumor recurrence, while preserving the vital healthy surrounding tissue.