Exploring Interactions Between Malignant Brain Cancer Cells and the Tumor Microenvironment Following High-Frequency Irreversible Electroporation

Abstract

High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation therapeutic that applies bipolar, high-frequency pulsed electric fields to tumors, triggering the formation of irreversible membrane pores and to induce tumor cell death. H-FIRE has demonstrated pre-clinical and clinical utility as a therapeutic for brain tumors, including gliomas. H-FIRE has been shown to induce precise, uniform ablation within the tumor tissue, as well as local changes to the tumor microenvironment and systemic changes to the immune landscape. Namely, disruption of the peritumoral blood-brain barrier (BBB) following H-FIRE ablation of brain tumors, and infiltration and activation of the innate immune system are clinically observed following H-FIRE tumor ablation. Such effects persist long after death of the treated tumor, and therefore an understanding of the mechanisms underlying these local and systemic changes are critical for the development of H-FIRE. Using in vitro models of glioma and lung carcinoma-derived brain metastases, we investigate the interactions between cancer cells that have been ablated with H-FIRE and the brain tumor microenvironments. Specifically, we demonstrate that H-FIRE-treated cancer cells can recover treatment-induced damage and proliferative capacity after treatment with specific electric field doses, while higher doses inhibit such recovery. This suggests that after H-FIRE ablation of brain tumors, tumor cells can still secrete factors to trigger alterations in their local and systemic environments. We then specifically investigate the role of tumor-derived extracellular vesicles (TDEVs) in mediating these changes, namely pBBB disruption and changes in innate immunity. We find that, following H-FIRE ablation of brain cancer cells, treated cells immediately release TDEVs that disrupt the blood-brain barrier (BBB) endothelium in vitro, and are uniquely internalized by cerebral endothelial cells in vitro, despite reduced release of TDEVs after H-FIRE. We further demonstrate that H-FIRE significantly alters the proteomic payloads of TDEVs. When TDEVs released by sham- and H-FIRE-treated glioma cells are delivered to healthy rats, only TDEVs released by H-FIRE-ablated cells are retained in the brain, suggesting changes to TDEV organotropism after H-FIRE ablation of glioma. Further, once retained in the brain, these post-H-FIRE TDEVs cluster near cerebral endothelial cells, similarly to in vitro. Although the TDEVs released by H-FIRE ablated glioma cells do not disrupt the BBB in vivo, Iba1+ cells were increased in the brains of rats that received TDEVs released by H-FIRE-ablated glioma cells. Together, these data suggest that H-FIRE immediately alters the secretion and proteome of TDEVs, facilitating changes in TDEV organotropism and cellular tropism and immune cell recruitment to the tumor microenvironment. Together, this research indicates mechanisms by which tumor cells continue to modulate their local and systemic environments via the action of TDEVs, which is critical information for the continued development of H-FIRE and its optimization with adjuvant therapeutics for the treatment of malignant brain tumors.