High-Frequency Nanosecond Bleomycin Electrochemotherapy and its Effects on Changes in the Immune System and Survival

Abstract

In this work, a time-dependent and time-independent study on bleomycin-based high-frequency nsECT (3.5 kV/cm × 200 pulses) for the elimination of LLC1 tumours in C57BL/6J mice is performed. We show the efficiency of nsECT (200 ns and 700 ns delivered at 1 kHz and 1 MHz) for the elimination of tumours in mice and increase of their survival. The dynamics of the immunomodulatory effects were observed after electrochemotherapy by investigating immune cell populations and antitumour antibodies at different timepoints after the treatment. ECT treatment resulted in an increased percentage of CD4⁺ T, splenic memory B and tumour-associated dendritic cell subsets. Moreover, increased levels of antitumour IgG antibodies after ECT treatment were detected. Based on the time-dependent study results, nsECT treatment upregulated PD 1 expression on splenic CD4⁺ Tr1 cells, increased the expansion of splenic CD8⁺ T, CD4⁺CD8⁺ T, plasma cells and the proportion of tumour-associated pro inflammatory macrophages. The Linˉ population of immune cells that was increased in the spleens and tumour after nsECT was identified. It was shown that nsECT prolonged survival of the treated mice and induced significant changes in the immune system, which shows a promising alliance of nanosecond electrochemotherapy and immunotherapy.

Calcium Electrochemotherapy for Tumor Eradication and the Potential of High-Frequency Nanosecond Protocols

“…Additionally, ensuring a homogeneous treatment is challenging, especially with non-invasive electrodes and highly heterogeneous tissue structures [ 19 ]. To compensate, higher frequency pulses can be used for impedance mitigation and equivalent efficacy nanosecond pulse protocols can be derived, which was confirmed in vitro [ 20 , 21 , 22 , 23 ] and in vivo [ 19 , 24 , 25 , 26 , 27 , 28 ]. High-frequency short-duration protocols show potential advantages compared to standard EP treatment, such as minimized thermal damage [ 29 ], reduced neuromuscular stimulation [ 30 ], lower Joule heating [ 31 ], and most importantly, a more homogeneous response with high flexibility in parametric protocol design.…”

“…In order to compensate for the lack of significant electrophoretic components and increase the stability and size of the pores, a new modality of high-frequency electroporation (MHz range) can be used [ 45 , 46 ]. Previously, we have shown that high-frequency nsECT with bleomycin can be as good or superior to ESOPE protocols [ 27 ]. However, calcium electroporation is gaining more and more popularity due to accessibility and the capabilities to modulate a systemic immune response.…”

“…As a result, the electrotransfer of drugs or genes is significantly improved, enabling derivation of equivalent efficiency or even superior to ESOPE protocols [ 21 , 47 ]. The high potential of MHz burst of pulses was confirmed in vivo using bleomycin electrochemotherapy [ 27 , 48 ].…”