Abstract 4917: High-frequency irreversible electroporation ablation of glioma alters extracellular vesicles and disrupts the blood-brain barrier endothelium
Authors: Murphy Kelsey , Aycock Kenneth , Marsh Spencer , Hay Alayna , Chang Christine , Bracha Shay , Gourdie Robert , Davalos Rafael , Rossmeisl John H. , Dervisis Nikolaos 
Published: April 2023

Abstract
High-frequency irreversible electroporation (H-FIRE) is a nonthermal tumor ablation technology that kills cancer cells via pulsed electric fields. H-FIRE has been shown to precisely and nonthermally ablate brain tumors while transiently disrupting the peritumoral blood-brain barrier (BBB). The enhanced BBB permeability post-tumor ablation can be exploited to enhance therapeutic delivery to invasive tumor margins, but the mechanisms of H-FIRE-induced BBB disruption remain incompletely characterized. We hypothesize that bystander effects of H-FIRE tumor cell ablation, specifically the release of tumor-derived extracellular vesicles (TDEVs), mediate peritumoral BBB endothelium disruption. F98 glioma and bEnd.3 cerebral endothelial cell lines modelled primary brain cancer and the BBB endothelium, respectively. F98 glioma cells were treated in vitro with sham treatment, a sub-ablative (1,500 V/cm), or an ablative (3,000 V/cm) electric field dose of H-FIRE, and TDEVs were isolated from the supernatants via filtration and ultracentrifugation. Post-H-FIRE TDEVs were characterized via nanoparticle tracking analysis and transmission electron microscopy, with ablative doses of H-FIRE resulting in decreased release of TDEVs compared to sub-ablative H-FIRE and sham treatment. Post-H-FIRE TDEVs were then applied to Transwell models of the BBB endothelium to evaluate the effect of H-FIRE-induced TDEVs on BBB endothelium permeability. TDEVs released after the ablative H-FIRE dose (3,000 V/cm) significantly increased permeability of the BBB endothelium in vitro compared to TDEVs released after sub-ablative and sham H-FIRE treatment. TDEVs were then fluorescently labelled with CFSE, and confocal microscopy was used to characterize internalization of post-H-FIRE TDEVs by cerebral endothelial cells. The TDEVs released after ablative doses of H-FIRE were significantly internalized by bEnd.3 cerebral endothelial cells, while TDEVs released after sub-ablative and sham treatment were not internalized. The proteomic payloads of the post-H-FIRE TDEVs were characterized using mass spectrometry, and clustering analysis demonstrated that the in vitro BBB-modulatory TDEVs released after 3,000 V/cm H-FIRE had distinct proteomic payloads compared to the non-BBB-modulatory TDEV populations released after sham and 1,500 V/cm H-FIRE treatment. Proteins associated with proteasomal degradation, cellular adhesion, and modulation of junctional integrity were increased in the BBB-modulatory group of TDEVs. Taken together, our results suggest that H-FIRE ablation of glioma significantly alters the proteomic cargo of TDEVs to increase internalization of TDEVs by cerebral endothelial cells and increase permeability of the BBB endothelium.
Citation Format: Kelsey Murphy, Kenneth Aycock, Spencer Marsh, Alayna Hay, Christine Chang, Shay Bracha, Robert Gourdie, Rafael Davalos, John H. Rossmeisl, Nikolaos Dervisis. High-frequency irreversible electroporation ablation of glioma alters extracellular vesicles and disrupts the blood-brain barrier endothelium. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4917.