Calcium electroporation (CaEP) is a novel local ablative method for tumor treatment, where high concentrations of calcium are used for induction of cell death, and electroporation is used as a delivery method. The effectiveness of CaEP was already tested in several clinical studies; however, its mechanisms are not yet completely clarified. In this study, the effectiveness of CaEP was studied on three levels. First, the direct cytotoxic effect on endothelial and tumor cell lines was tested, as well as the effects on cytoskeleton and cell-cell junctions of endothelial cells. In in vivo studies was shown, CaEP has a vessel disrupting effect, where tumor vasculature was shown to be more sensitive to CaEP than normal. Besides that, we tested immunological consequences of CaEP on two different tumor models, murine melanoma B16-F10 and murine carcinoma 4T1. We showed in B16-F10 tumor model that induction of immunogenic cell death occurs after CaEP, as excretion of HMGB1 and ATP transpires, as well as translocation of HSP70 to the cell surface, in ex vivo experiments, co-association of calreticulin with membranes was confirmed. We also evaluated the effectiveness of different concentrations of calcium, where we showed dose-dependent survival of cells with similar sensitivity of both cell lines to CaEP; however, in subcutaneous tumors in vivo, this was not the case. Here, 4T1 tumor model proved to be more sensitive to CaEP as B16-F10. A combination of CaEP with gene electrotransfer of plasmid encoding IL-12 was also evaluated, which proved to be dependent on tumor type as well as on the effectiveness of CaEP alone. Combined therapy showed significantly enhanced survival in B16-F10 model, whereas, in 4T1 model, calcium injection alone resulted in tumor cures and IL-12 did not improve the outcome. Electrochemotherapy was used as a positive control in all experiments. The results of this study will importantly contribute to the understanding of CaEP mechanisms.
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