Calcium electroporation may offer a simple general tool for anticancer therapy. Transient permeabilization of cancer cell membranes created by applying short, high-voltage pulses in tumors enables high calcium influxes that trigger cell death. In this study, we compared the relative sensitivity of different human tumor models and normal tissues to calcium electroporation. Plasma membrane Ca-ATPase (PMCA) protein expression was confirmed in all cancer cell lines and normal primary dermal fibroblasts studied. In all tumor types tested, calcium electroporation effectively induced necrosis, with a range of sensitivities observed (36%-88%) 2 days after treatment. Necrosis was induced using calcium concentrations of 100-500 mmol/L and injection volumes 20%-80% of tumor volume. Notably, only limited effects were seen in normal tissue. Calcium content increased >7-fold in tumor and skin tissue after calcium electroporation but decreased in skin tissue 4 hours after treatment to levels comparable with untreated controls, whereas calcium content endured at high levels in tumor tissue. Mechanistic experiments indicated that calcium influx was similar in fibroblasts and cancer cells. However, we observed decreased PMCA expression in cancer cells compared with fibroblasts, offering a potential explanation for the different calcium content in tumor cells versus normal tissues. Overall, our results suggest that calcium electroporation can elicit a rapid and selective necrosis of solid tumors, with limited deleterious effects on surrounding normal tissues..

Electroporation is used in cancer treatment because of its ability to increase local cytotoxicity of e.g. bleomycin (electrochemotherapy) and calcium (calcium electroporation). Calcium electroporation is a novel anticancer treatment that selectively kills cancer cells by necrosis, a cell death pathway that stimulates the immune system due to high release of antigens and "danger signals." In this exploratory study, we aimed to investigate whether calcium electroporation could initiate an anticancer immune response similar to electrochemotherapy. To this end, we treated immunocompetent balb/c mice with CT26 colon tumors with calcium electroporation, electrochemotherapy, or ultrasound-based delivery of calcium or bleomycin. High treatment efficiency was observed with 100% complete remission in all four groups (12/12 with complete remission in each treatment group). In addition, none of the surviving mice from these groups formed new tumors when re-challenged with CT26 cancer cells 100-d post treatment, whereas mice challenged with different cancer cells (4T1 breast cancer) all developed tumors. Treatment of immunodeficient mice with calcium electroporation and electrochemotherapy showed no long-lasting tumor response. Calcium electroporation and electrochemotherapy was associated with a release of High Mobility Group Box 1 protein (HMGB1) ( = 0.029) and a significant increase of the overall systemic level of pro-inflammatory cytokines in serum from the treated mice ( < 0.003). These findings indicate that calcium electroporation as well as electrochemotherapy could have a role as immune stimulators in future treatments.

Depending on the initiating stimulus, cancer cell death can be immunogenic or non-immunogenic. Inducers of immunogenic cell death (ICD) rely on endoplasmic reticulum (ER) stress for the trafficking of danger signals such as calreticulin (CRT) and ATP. We found that nanosecond pulsed electric fields (nsPEF), an emerging new modality for tumor ablation, cause the activation of the ER-resident stress sensor PERK in both CT-26 colon carcinoma and EL-4 lymphoma cells. PERK activation correlates with sustained CRT exposure on the cell plasma membrane and apoptosis induction in both nsPEF-treated cell lines. Our results show that, in CT-26 cells, the activity of caspase-3/7 was increased fourteen-fold as compared with four-fold in EL-4 cells. Moreover, while nsPEF treatments induced the release of the ICD hallmark HMGB1 in both cell lines, extracellular ATP was detected only in CT-26. Finally, in vaccination assays, CT-26 cells treated with nsPEF or doxorubicin equally impaired the growth of tumors at challenge sites eliciting a protective anticancer immune response in 78% and 80% of the animals, respectively. As compared to CT-26, both nsPEF- and mitoxantrone-treated EL-4 cells had a less pronounced effect and protected 50% and 20% of the animals, respectively. These results support our conclusion that nsPEF induce ER stress, accompanied by bona fide ICD.