Increasing the selectivity of cancer treatments is attractive, as it has the potential to reduce side-effects of therapy. Cold atmospheric plasma (CAP) is a novel cancer treatment that disrupts the intracellular oxidative balance. Several reports claim CAP treatment to be selective, but retrospective analysis of these studies revealed discrepancies in several biological factors and culturing methods. Before CAP can be conclusively stated as a selective cancer treatment, the importance of these factors must be investigated. In this study, we evaluated the influence of the cell type, cancer type, and cell culture medium on direct and indirect CAP treatment. Comparison of cancerous cells with their non-cancerous counterparts was performed under standardized conditions to determine selectivity of treatment. Analysis of seven human cell lines (cancerous: A549, U87, A375, and Malme-3M; non-cancerous: BEAS-2B, HA, and HEMa) and five different cell culture media (DMEM, RPMI1640, AM, BEGM, and DCBM) revealed that the tested parameters strongly influence indirect CAP treatment, while direct treatment was less affected. Taken together, the results of our study demonstrate that cell type, cancer type, and culturing medium must be taken into account before selectivity of CAP treatment can be claimed and overlooking these parameters can easily result in inaccurate conclusions of selectivity.

Non-thermal plasma (NTP), an ionized gas generated at ambient pressure and temperature, has been an emerging technology for medical applications. Through controlled delivery of reactive oxygen and nitrogen species (ROS/RNS), NTP can elicit hormetic cellular responses, thus stimulating broad therapeutic effects. To enable clinical translation of the promising preclinical research into NTP therapy, a deeper understanding of NTP interactions with clinical substrates is profoundly needed. Since NTP-generated ROS/RNS will inevitably interact with blood in several clinical contexts, understanding their stability in this system is crucial. In this study, two medically relevant NTP delivery modalities were used to assess the stability of NTP-generated ROS/RNS in three aqueous solutions with increasing organic complexities: phosphate-buffered saline (PBS), blood plasma (BP), and processed whole blood. NTP-generated RNS collectively (NO₂⁻, ONOO⁻), H₂O₂, and ONOO⁻ exclusively were analyzed over time. We demonstrated that NTP-generated RNS and H₂O₂ were stable in PBS but scavenged by different components of the blood. While RNS remained stable in BP after initial scavenging effects, it was completely reduced in processed whole blood. On the other hand, H₂O₂ was completely scavenged in both liquids over time. Our previously developed luminescent probe europium(III) was used for precision measurement of ONOO⁻ concentration. NTP-generated ONOO⁻ was detected in all three liquids for up to at least 30 seconds, thus highlighting its therapeutic potential. Based on our results, we discussed the necessary considerations to choose the most optimal NTP modality for delivery of ROS/RNS to and via blood in the clinical context.

Non-thermal plasma (NTP) has shown promising anti-cancer effects, but there is still limited knowledge about the underlying cell death mechanisms induced by NTP and inherent differences between NTP treatment modalities. This study aimed to investigate four major regulated cell death (RCD) pathways, namely apoptosis, pyroptosis, necroptosis, and ferroptosis, in melanoma cancer cells following NTP treatment, and to provide an overview of molecular mechanistic differences between direct and indirect NTP treatment modalities. To discriminate which cell death pathways were triggered after treatment, specific inhibitors of apoptosis, pyroptosis, necroptosis, and ferroptosis were evaluated. RCD-specific molecular pathways were further investigated to validate the findings with inhibitors. Both direct and indirect NTP treatment increased caspase 3/7 and annexin V expression, indicative of apoptosis, as well as lipid peroxidation, characteristic of ferroptosis. Pyroptosis, on the other hand, was only induced by direct NTP treatment, evidenced by increased caspase 1 activity, whereas necroptosis was stimulated in a cell line-dependent manner. These findings highlight the molecular differences and implications of direct and indirect NTP treatment for cancer therapy. Altogether, activation of multiple cell death pathways offers advantages in minimizing treatment resistance and enhancing therapeutic efficacy, particularly in a combination setting. Understanding the mechanisms underlying NTP-induced RCD will enable the development of strategic combination therapies targeting multiple pathways to achieve cancer lethality.

Despite the promising results of non‐thermal plasma (NTP) devices for cancer therapy, the potential adverse effects of NTP irradiation have remained unexplored, including the effects on epithelial‐mesenchymal transition (EMT) and subsequent cancer metastasis. In this study, we investigate NTP‐induced EMT initiation and progression. A microsecond‐pulsed dielectric barrier discharge plasma device was used for NTP treatment, and chicken chorioallantoic membrane (CAM) and spheroids were used as 3D tumor models. NTP treatment reduced tumor volume in the CAM model, but a shift towards a mesenchymal‐like phenotype was also measured in melanoma tumors via analysis of the six EMT biomarkers, though changes in cancer cell migration to other organs were not significant. In the spheroid model, molecular analysis also indicated an EMT response following NTP treatment, and enhanced cell migration was measured in one cell line. EMT induction with NTP was dose‐dependent and transient; high NTP treatments caused significant EMT response and enhanced migration, but low NTP doses did not. Our findings highlight the important role of NTP parameters for cancer treatment and consequential EMT responses. The insights obtained here further build the foundation for clinical optimization, harnessing the cancer‐killing potential of NTP while safeguarding against undesirable EMT‐related outcomes.