The application of cold atmospheric plasma (CAP) in cancer therapy could be one of the new anticancer strategies. In the current work, we used cold atmospheric plasma jet for the treatment of cultured cells and mice. We showed that CAP induced the death of MX−7 mouse rhabdomyosarcoma cells with the hallmarks of immunogenic cell death (ICD): calreticulin and heat shock protein 70 (HSP70) externalization and high-mobility group box 1 protein (HMGB1) release. The intensity of HMGB1 release after the CAP treatment correlated directly with the basal extracellular HMGB1 level. Releasing from dying cells, HMGB1 can act as a proinflammatory cytokine. Our in vivo study demonstrated that cold atmospheric plasma induces a short-term two-times increase in serum HMGB1 level only in tumor-bearing mice with no effect in healthy mice. These findings support our hypothesis that CAP-dependent HMGB1 release from dying cancer cells can change the serum HMGB1 level. At the same time, we showed a weak cytokine response to CAP irradiation in healthy mice that can characterize CAP as an immune-safety physical antitumor approach.

Cold atmospheric plasma (CAP) is an intensively-studied approach for the treatment of malignant neoplasms. Various active oxygen and nitrogen compounds are believed to be the main cytotoxic effectors on biotargets; however, the comprehensive mechanism of CAP interaction with living cells and tissues remains elusive. In this study, we experimentally determined the optimal discharge regime (or semi-selective regime) for the direct CAP jet treatment of cancer cells, under which lung adenocarcinoma A549, A427 and NCI-H23 cells demonstrated substantial suppression of viability, coupled with a weak viability decrease of healthy lung fibroblasts Wi-38 and MRC-5. The death of CAP-exposed cancer and healthy cells under semi-selective conditions was caspase-dependent. We showed that there was an accumulation of lysosomes in the treated cells. The increased activity of lysosomal protease Cathepsin D, the transcriptional upregulation of autophagy-related MAPLC3B gene in cancer cells and the changes in autophagy-related proteins may have indicated the activation of autophagy. The addition of the autophagy inhibitor chloroquine (CQ) after the CAP jet treatment increased the death of A549 cancer cells in a synergistic manner and showed a low effect on the viability of CAP-treated Wi-38 cells. Downregulation of Drp1 mitochondrial protein and upregulation of PINK1 protein in CAP + CQ treated cells indicated that CQ increased the CAP-dependent destabilization of mitochondria. We concluded that CAP weakly activated pro-survival autophagy in irradiated cells, and CQ promoted CAP-dependent cell death due to the destabilization of autophagosomes formation and mitochondria homeostasis. To summarize, the combination of CAP treatment with CQ could be useful for the development of cold plasma-based antitumor approaches for clinical application.

The effect of the presence or absence of a grounded substrate beneath dielectric targets, including cancer cells, during exposure to the cold atmospheric plasma jet is studied in the experiments and in fluid model simulations for the discharge parameters typical for the medical applications. It is shown that the dynamics of streamers generated in each positive cycle of ac voltage depends on the grounded substrate position. The streamers approach the target more often if the grounded substrate is beneath the target, that provides more intensive plasma-target interaction. In this case, the measured spectrum of plasma jet emission near the target demonstrates much higher intensity compared to an electrically isolated target case. The calculated and measured discharge currents with time demonstrate a mismatch of frequencies of the ac voltage and current over the target. The viability of A431 human skin carcinoma and MX7 mouse rhabdomyosarcoma cells treated by cold atmospheric plasma jet with/without the grounded substrate is measured with MTT assay 24 h after. The results show an enhanced suppression of the cell viability when using the grounded substrate for both cell lines. Achieving effective death of tumor cells with a shorter irradiation time can be considered an advantage of using a grounded electrode beneath the bio target.

The interaction of cold atmospheric plasma (CAP) with biotargets is accompanied by chemical reactions on their surfaces and insides, and it has great potential as an anticancer approach. This study discovers the molecular mechanisms that may explain the selective death of tumor cells under CAP exposure. To reach this goal, the transcriptional response to CAP treatment was analyzed in A549 lung adenocarcinoma cells and in lung-fibroblast Wi-38 cells. We found that the CAP treatment induced the common trend of response from A549 and Wi-38 cells—the p53 pathway, KRAS signaling, UV response, TNF-alpha signaling, and apoptosis-related processes were up-regulated in both cell lines. However, the amplitude of the response to CAP was more variable in the A549 cells. The CAP-dependent death of A549 cells was accompanied by DNA damage, cell-cycle arrest in G2/M, and the dysfunctional response of glutathione peroxidase 4 (GPx4). The activation of the genes of endoplasmic reticulum stress and ER lumens was detected only in the A549 cells. Transmission-electron microscopy confirmed the alteration of the morphology of the ER lumens in the A549 cells after the CAP exposure. It can be concluded that the responses to nuclear stress and ER stress constitute the main differences in the sensitivity of tumor and healthy cells to CAP exposure.

The desirable properties of a cold atmospheric plasma jet (CAPJ) for medical applications are high efficiency of the treatment and low thermal effects. To meet these requirements, we propose to use the CAPJ generated with the positive pulsed (PP) voltage. In our experiments and fluid model simulations, it is shown that a change of the pulse duration allows us to control precisely the intensity of interaction of the plasma jet with the target and the heating of the contact zone. The helium CAPJ is formed with 13–30 kHz PP voltage frequencies with the pulse duration of 7–20 µ s. The enhancement of plasma-target interaction is analyzed with the measured discharge current and calculated ionization rate near the target surface. The temperature measurements in the plasma-target active zone show that the heating by CAPJ excited with the PP voltage is generally less than in the case of AC voltage. The efficacy of treatment is tested on A549 cancer cells exposed to CAPJ ignited with the positively pulsed voltage with different pulse duration and with AC voltage.

The treatment of solid tumors by a cold atmospheric plasma (CAP) jet is an innovative approach that has been actively developed only in the last decade. As a result, studies aimed at identifying the conditions for selective effect on tumor cells, including as parts of 3D models that mimic malignant neoplasms are pertinent. It is known that the main cytotoxic effects of a CAP are caused by reactive oxygen and nitrogen species in the plasma jet. Their availability for cells in classical 2D and 3D models of cell cultivation may be different. Here, we used multicellular spheroids of MCF7–EGFR cells with overexpression of the epidermal growth-factor receptor (EGFR), cells of parental breast adenocarcinoma MCF7 cell line, and nontransformed human breast cells MCF10A. Irradiation of MCF7–EGFR spheroids induced destruction of multicellular 3D structures into individual cells and activated cell death. It has been shown that the cells of irradiated spheroids undergo phagocytosis by activated macrophages. Comparison of a direct CAP irradiation of MCF7–EGFR spheroids and indirect treatment with plasma-activated medium (PAM) revealed a higher content of reactive oxygen and nitrogen species in spheroid cells cultivated in the irradiated medium. It produced a higher cytotoxic effect than direct irradiation. It has been shown that the cytotoxic properties of PAM are better preserved when such a medium is stored at 4° than at –20°C. Thus, the death of tumor cells in spheroids was more effective with addition of the culture medium irradiated with a CAP than direct irradiation of cells.