LAUSR

Simple Summary Standard microsecond electrochemotherapy (μsECT) is used in clinical trials for the elimination of tumours, while the first studies on nanosecond electrochemotherapy (nsECT) only started to appear recently. Nanosecond pulses enable more homogeneous treatment and better control of pulse burst energy, and thus the whole field of electroporation is moving towards the shorter pulse range. In order to ensure a full anticancer response and potentially prevent any metastases, the immunomodulatory effects should also be induced. Therefore, in this work, we used nsECT protocols based on kHz and MHz pulse bursts and characterized the response of the immune system to the novel modality of nano-electrochemotherapy. The results of this study are useful for the development of effective anticancer treatment strategies based on high frequency nanosecond electric fields. 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 Lin− 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.