Evaluation of unipolar and bipolar nanosecond pulses for calcium electrochemotherapy and immune response

Calcium electrochemotherapy (CaECT) is an effective alternative to standard chemotherapeutic treatments, utilizing intracellular delivery of supraphysiological calcium concentrations to induce cell death. High-frequency sub-microsecond bursts can be successfully used for CaECT, however, bipolar waveforms have not yet been characterized in this context, potentially offering better impedance mitigation and more uniform treatment when compared to unipolar procedures. Therefore, this study evaluated the feasibility of unipolar and symmetric bipolar sub-microsecond pulses (7 kV/cm × 300 ns × 250, 1 MHz) for CaECT including their capacity to modulate antitumor immunity in a moderately immunogenic murine breast cancer model. Standard microsecond protocol (1.5 kV/cm × 100 μs × 8 pulses at 1 Hz) was used as a reference. In vitro data revealed that a bipolar cancellation phenomenon when symmetric bipolar pulses were applied, whereas this effect was not detected in vivo. CaECT treatment induced systemic immune alterations across electroporation groups, including increased CD4⁺ and CD8⁺ memory T-cell populations in the spleen and reduced CD4⁺ regulatory T cells and myeloid-derived suppressor cells in tumor-draining lymph nodes. Unipolar nanosecond pulses showed a clearer increase in central memory T-cell populations, while bipolar pulses were associated with pronounced modulation of lymph-node immune composition. It is shown that bipolar cancellation phenomenon is not necessarily triggered in vivo, which was predicted by in vitro data.