Investigation of the Bystander Effect on Cell Viability After Application of Combined Electroporation-Based Methods

Electrochemotherapy (ECT) uses electroporation to enhance drug delivery into tumor cells, triggering bystander effects like immunogenicity and cell death. This study investigated bystander effects in vitro in 4T1 breast cancer cells following various electroporation treatments: reversible (1400 V/cm, 100 µs) bleomycin electrotransfer, irreversible (2800 V/cm, 100 µs) bleomycin electrotransfer, and calcium electroporation, including combinations. Conditioned media from treated cells (12-72 h incubation) were transferred to untreated cells, and viability was assessed via metabolic activity, cell count, and colony formation. A scratch assay evaluated wound healing. The bystander effect dramatically reduced colony formation, reaching 0% after bleomycin and calcium electrotransfer, and 2.37 ± 0.74% after irreversible electroporation (IRE). Metabolic activity decreased to 18.05 ± 6.77% and 11.62 ± 3.57% after bleomycin and calcium electrotransfer, respectively, and 56.21 ± 0.74% after IRE. Similarly, cell viability measured by flow cytometry was 10.00 ± 1.44%, 3.67 ± 0.32%, and 24.96 ± 1.37% after bleomycin electrotransfer, calcium electrotransfer, and IRE, respectively. Combined analysis of these effects yielded comparable results. Conditioned media, particularly from bleomycin electrotransfer and calcium electroporation, significantly reduced cell number, metabolic activity, and colony formation, demonstrating a strong bystander effect. Wound healing was also significantly delayed in groups exposed to conditioned media.