Abstract 6572: High-frequency irreversible electroporation (H-FIRE) enhances extracellular vesicle release and promotes immune system interaction in cancer treatment

Abstract Background: High-Frequency Irreversible Electroporation (H-FIRE) is a non-thermal tissue ablation technique that induces cell death by destabilizing the cell membrane. This innovative approach has demonstrated significant potential in cancer treatment and has been associated with modulation of the tumor microenvironment in both in vivo and in vitro settings. Recent findings from our lab reveal that extracellular vesicles (EVs) released by cells lethally treated with H-FIRE exhibit a unique proteomic profile. These EVs may play a pivotal role in modulating the tumor microenvironment. Building on our preliminary experiments, we hypothesize that EVs released following H-FIRE-mediated tumor ablation actively engage with and influence the immune system. Methods: Established carcinoma cell lines of breast (4T1) and exocrine pancreas (PanO2) were used. Cells will be treated with sham, sublethal H-FIRE (1500V), and lethal H-FIRE (3000V). The supernatant of the treated cells was collected and EVs were isolated by ultracentrifugation. EVs were then assessed quantitatively via Nanosight. Mouse monocytes (J774a) were then exposed to EVs and evaluated for extracellular vesicle uptake via confocal imaging and evaluation of mean fluorescence intensity. Results: In quantitative analysis of the EVs from each treatment group, we found a significantly increased number of EVs present within the supernatant of lethally treated cells, in comparison to both the untreated and sublethal treatment groups (4T1 - sham 1.52e11/mL, sublethal 6.89e10/mL, lethal 3.25e11/mL, p<.0001 between all groups) (PanO2 - sham 2.18e11/mL, sublethal 1.1e11/mL, lethal 1.34e12/mL, p<.0001 when comparing lethal group to other groups). No significant difference in particle size was observed between the treatment groups. J774a macrophages were exposed to TDEVs from each of the treatment groups, and confocal imaging and evaluation of mean fluorescence intensity showed a qualitative and quantitative increase in macrophage uptake of TDEVs from both the lethal and sublethal groups relative to uptake of TDEVs from the sham group. Using TDEV from the 4T1 cell line, average mean intensity was increased in macrophages exposed to EVs from the lethal and sublethal groups relative to those from the sham group (lethal 2.50, sublethal 2.32, sham 2.04, p<0.0001 between sham and both treatment groups). Conclusion: Our study demonstrates that lethal treatment of carcinoma cells with H-FIRE results in a significant increase in extracellular vesicle (EV) release. Furthermore, we provide evidence that EVs derived from H-FIRE-treated cells exhibit enhanced uptake by macrophages. These findings are in support our working hypothesis that H-FIRE exerts some of its effects in cancer immune system modulation through EV release. Citation Format: David Ogburn, Irving Allen, Nikolaos Dervisis. High-frequency irreversible electroporation (H-FIRE) enhances extracellular vesicle release and promotes immune system interaction in cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6572.