This study investigated the use of glutathione as a marker to establish a correlation between plasma parameters and the resultant liquid chemistry from two distinct sources to predefined biological outcomes. Two different plasma sources were operated at parameters that resulted in similar biological responses: cell viability, mitochondrial activity, and the cell surface display of calreticulin. Specific glutathione modifications appeared to be associated with biological responses elicited by plasma. These modifications were more pronounced with increased treatment time for the European Cooperation in Science and Technology Reference Microplasma Jet (COST-Jet) and increased frequency for the dielectric barrier discharge and were correlated with more potent biological responses. No correlations were found when cells or glutathione were exposed to exogenously added long-lived species alone. This implied that short-lived species and other plasma components were required for the induction of cellular responses, as well as glutathione modifications. These results showed that comparisons of medical plasma sources could not rely on measurements of long-lived chemical species; rather, modifications of biomolecules (such as glutathione) might be better predictors of cellular responses to plasma exposure.

Survival rates among patients with pancreatic cancer, the most lethal gastrointestinal cancer, have not improved compared to other malignancies. Early tumor dissemination and a supportive, cancer-promoting tumor microenvironment (TME) limit therapeutic options and consequently impede tumor remission, outlining an acute need for effective treatments. Gas plasma-oxidized liquid treatment showed promising preclinical results in other gastrointestinal and gynecological tumors by targeting the tumor redox state. Here, carrier solutions are enriched with reactive oxygen (ROS) and nitrogen (RNS) species that can cause oxidative distress in tumor cells, leading to a broad range of anti-tumor effects. Unfortunately, clinical relevance is often limited, as many studies have forgone the use of medical-grade solutions. This study investigated the efficacy of gas plasma-oxidized Ringer’s lactate (oxRilac), a physiological solution often used in clinical practice, on two pancreatic cancer cell lines to induce tumor toxicity and provoke immunogenicity. Tumor toxicity of the oxRilac solutions was further confirmed in three-dimensional tumor spheroids monitored over 72 h and in ovo using stereomicroscope imaging of excised GFP-expressing tumors. We demonstrated that cell death signaling was induced in a dose-dependent fashion in both cell lines and was paralleled by the increased surface expression of key markers of immunogenic cell death (ICD). Nuclear magnetic resonance (NMR) spectroscopy analysis suggested putative reaction pathways that may cause the non-ROS related effects. In summary, our study suggests gas plasma-deposited ROS in clinically relevant liquids as an additive option for treating pancreatic cancers via immune-stimulating and cytotoxic effects.

Non-thermal plasma application to cancer cells is known to induce oxidative stress, cytotoxicity and indirect immunostimulatory effects on antigen presenting cells (APCs). The purpose of this study was to evaluate the responses of two leukemic cell lines—Jurkat T lymphocytes and THP-1 monocytes—to NTP-generated reactive oxygen and nitrogen species (RONS). Both cell types depleted hydrogen peroxide, but THP-1 cells neutralized it almost immediately. Jurkat cells transiently blunted the frequency-dependent increase in nitrite concentrations in contrast to THP-1 cells, which exhibited no immediate effect. A direct relationship between frequency-dependent cytotoxicity and mitochondrial superoxide was observed only in Jurkat cells. Jurkat cells were very responsive to NTP in their display of calreticulin and heat shock proteins 70 and 90. In contrast, THP-1 cells were minimally responsive or unresponsive. Despite no NTP-dependent decrease in cell surface display of CD47 in either cell line, both cell types induced migration of and phagocytosis by APCs. Our results demonstrate that cells modulate the RONS-mediated changes in liquid chemistry, and, importantly, the resultant immunomodulatory effects of NTP can be independent of NTP-induced cytotoxicity.

In people living with HIV-1 (PLWH), antiretroviral therapy (ART) eventually becomes necessary to suppress the emergence of human immunodeficiency virus type 1 (HIV-1) replication from latent reservoirs because HIV-1-specific immune responses in PLWH are suboptimal. Immunotherapies that enhance anti-HIV-1 immune responses for better control of virus reemergence from latent reservoirs are postulated to offer ART-free control of HIV-1. Toward the goal of developing an HIV-1-specific immunotherapy based on non-thermal plasma (NTP), the early immunological responses to NTP-exposed latently infected T lymphocytes were examined. Application of NTP to the J-Lat T-lymphocyte cell line (clones 10.6 and 15.4) stimulated monocyte recruitment and macrophage maturation, which are key steps in initiation of an immune response. In contrast, CD8+ T lymphocytes in a mixed lymphocyte reaction assay were not stimulated by the presence of NTP-exposed J-Lat cells. Furthermore, co-culture of NTP-exposed J-Lat cells with mature phagocytes did not modulate their antigen presentation to primary CD8+ T lymphocytes (cross-presentation). However, reactivation from latency was stimulated in a clone-specific manner by NTP. Overall, these studies, which demonstrated that ex vivo application of NTP to latently infected lymphocytes can stimulate key immune cell responses, advance the development of an NTP-based immunotherapy that will provide ART-free control of HIV-1 reactivation in PLWH.