A Comparison of Floating-Electrode DBD and kINPen Jet: Plasma Parameters to Achieve Similar Growth Reduction in Colon Cancer Cells Under Standardized Conditions

A comparative study of two plasma sources (floating-electrode dielectric barrier discharge, DBD, Drexel University; atmospheric pressure argon plasma jet, kINPen, INP Greifswald) on cancer cell toxicity was performed. Cell culture protocols, cytotoxicity assays, and procedures for assessment of hydrogen peroxide (H2O2) were standardized between both labs. The inhibitory concentration 50 (IC50) and its corresponding H2O2 deposition was determined for both devices. For the DBD, IC50 and H2O2 generation were largely dependent on the total energy input but not pulsing frequency, treatment time, or total number of cells. DBD cytotoxicity could not be replicated by addition of H2O2 alone and was inhibited by larger amounts of liquid present during the treatment. Jet plasma toxicity depended on peroxide generation as well as total cell number and amount of liquid. Thus, the amount of liquid present during plasma treatment in vitro is key in attenuating short-lived species or other physical effects from plasmas. These in vitro results suggest a role of liquids in or on tissues during plasma treatment in a clinical setting. Additionally, we provide a platform for correlation between different plasma sources for a predefined cellular response

Activation of murine immune cells upon co-culture with plasma-treated B16F10 melanoma cells

Recent advances in melanoma therapy increased median survival in patients. However, death rates are still high, motivating the need of novel avenues in melanoma treatment. Cold physical plasma expels a cocktail of reactive species that have been suggested for cancer treatment. High species concentrations can be used to exploit apoptotic redox signaling pathways in tumor cells. Moreover, an immune-stimulatory role of plasma treatment, as well as plasma-killed tumor cells, was recently proposed, but studies using primary immune cells are scarce. To this end, we investigated the role of plasma-treated murine B16F10 melanoma cells in modulating murine immune cells' activation and marker profile. Melanoma cells exposed to plasma showed reduced metabolic and migratory activity, and an increased release of danger signals (ATP, CXCL1). This led to an altered cytokine profile with interleukin-1β (IL-1β) and CCL4 being significantly increased in plasma-treated mono- and co-cultures with immune cells. In T cells, plasma-treated melanoma cells induced extracellular signal-regulated Kinase (ERK) phosphorylation and increased CD28 expression, suggesting their activation. In monocytes, CD115 expression was elevated as a marker for activation. In summary, here we provide proof of concept that plasma-killed tumor cells are recognized immunologically, and that plasma exerts stimulating effects on immune cells alone. © 2019 by the authors

GSH modification as a marker for plasma source and biological response comparison to plasma treatment

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. © 2020 by the authors

Non-thermal plasma modulates cellular markers associated with immunogenicity in a model of latent HIV-1 infection

Effective control of infection by human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), requires continuous and life-long use of anti-retroviral therapy (ART) by people living with HIV-1 (PLWH). In the absence of ART, HIV-1 reemergence from latently infected cells is ineffectively suppressed due to suboptimal innate and cytotoxic T lymphocyte responses. However, ART-free control of HIV-1 infection may be possible if the inherent immunological deficiencies can be reversed or restored. Herein we present a novel approach for modulating the immune response to HIV-1 that involves the use of non-thermal plasma (NTP), which is an ionized gas containing various reactive oxygen and nitrogen species (RONS). J-Lat cells were used as a model of latent HIV-1 infection to assess the effects of NTP application on viral latency and the expression of pro-phagocytic and pro-chemotactic damage-associated molecular patterns (DAMPs). Exposure of J-Lat cells to NTP resulted in stimulation of HIV-1 gene expression, indicating a role in latency reversal, a necessary first step in inducing adaptive immune responses to viral antigens. This was accompanied by the release of pro-inflammatory cytokines and chemokines including interleukin-1β (IL-1β) and interferon-γ (IFN-γ); the display of pro-phagocytic markers calreticulin (CRT), heat shock proteins (HSP) 70 and 90; and a correlated increase in macrophage phagocytosis of NTP-exposed J-Lat cells. In addition, modulation of surface molecules that promote or inhibit antigen presentation was also observed, along with an altered array of displayed peptides on MHC I, further suggesting methods by which NTP may modify recognition and targeting of cells in latent HIV-1 infection. These studies represent early progress toward an effective NTP-based ex vivo immunotherapy to resolve the dysfunctions of the immune system that enable HIV-1 persistence in PLWH

Non-Thermal Plasma-Induced Immunogenic Cell Death in Cancer: A Topical Review.

Recent advances in biomedical research in cancer immunotherapy have identified the use of an oxidative stress-based approach to treat cancers, which works by inducing immunogenic cell death (ICD) in cancer cells. Since the anti-cancer effects of non-thermal plasma (NTP) are largely attributed to the reactive oxygen and nitrogen species that are delivered to and generated inside the target cancer cells, it is reasonable to postulate that NTP would be an effective modality for ICD induction. NTP treatment of tumors has been shown to destroy cancer cells rapidly and, under specific treatment regimens, this leads to systemic tumor-specific immunity. The translational benefit of NTP for treatment of cancer relies on its ability to enhance the interactions between NTP-exposed tumor cells and local immune cells which initiates subsequent protective immune responses. This review discusses results from recent investigations of NTP application to induce immunogenic cell death in cancer cells. With further optimization of clinical devices and treatment protocols, NTP can become an essential part of the therapeutic armament against cancer

The efficacy and safety of enzalutamide with trastuzumab in patients with HER2+ and androgen receptor-positive metastatic or locally advanced breast cancer

Purpose: Androgen receptor (AR) expression occurs in up to 86% of human epidermal growth factor receptor 2-positive (HER2+) breast cancers. In vitro, AR inhibitors enhance antitumor activity of trastuzumab, an anti-HER2 antibody, in trastuzumab-resistant HER2+ cell lines. This open-label, single-arm, phase II study evaluated the efficacy and safety of enzalutamide, an AR-signaling inhibitor, in patients with advanced HER2+ AR+ breast cancer previously treated with trastuzumab. Methods: Eligible patients had measurable or non-measurable evaluable disease per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1, Eastern Cooperative Oncology Group status 64 1, no history of brain metastases, and previously received 65 1 anti-HER2 regimen for advanced disease. Patients received 160\ua0mg oral enzalutamide daily and 6\ua0mg/kg intravenous trastuzumab every 21\ua0days until disease progression or unacceptable toxicity. Primary end point was clinical benefit rate at 24\ua0weeks (CBR24); secondary end points included progression-free survival (PFS) and safety. Results: Overall, 103\ua0women were enrolled [median age 60\ua0years (range 34\u201383)]; 62% had received 65 3\ua0lines of prior anti-HER2 therapy. CBR24, comprising patients with confirmed partial responses (5%) and durable stable disease at 24\ua0weeks (19%), was 24% in the efficacy evaluable set (n = 89). CBR24 did not seem related to AR-expression levels or hormone receptor status. Median PFS was 3.4\ua0months (95% confidence interval 2.0\u20133.8). Overall, 97 (94%) patients experienced treatment-emergent adverse events (TEAEs), with fatigue most common (34%). Dyspnea (4%) and malignant neoplasm progression (3%) were the only TEAEs grade 65 3 reported in 65 3\ua0patients. 22 patients (21%) reported serious TEAEs. Four patients (4%) experienced fatal, non-drug-related TEAEs. Conclusions: Enzalutamide plus trastuzumab was well tolerated, and a subset of patients in this heavily pretreated population had durable disease control. Determination of biomarkers is needed to identify patients most likely to benefit from this combination. ClinicalTrials.gov number: NCT0209196

Genome Wide Analysis of Inbred Mouse Lines Identifies a Locus Containing Ppar-γ as Contributing to Enhanced Malaria Survival

The genetic background of a patient determines in part if a person develops a mild form of malaria and recovers, or develops a severe form and dies. We have used a mouse model to detect genes involved in the resistance or susceptibility to Plasmodium berghei malaria infection. To this end we first characterized 32 different mouse strains infected with P. berghei and identified survival as the best trait to discriminate between the strains. We found a locus on chromosome 6 by linking the survival phenotypes of the mouse strains to their genetic variations using genome wide analyses such as haplotype associated mapping and the efficient mixed-model for association. This new locus involved in malaria resistance contains only two genes and confirms the importance of Ppar-γ in malaria infection