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- ItemBiological Risk Assessment of Three Dental Composite Materials following Gas Plasma Exposure(Basel : MDPI, 2022) Bekeschus, Sander; Miebach, Lea; Pommerening, Jonas; Clemen, Ramona; Witzke, KatharinaGas plasma is an approved technology that generates a plethora of reactive oxygen species, which are actively applied for chronic wound healing. Its particular antimicrobial action has spurred interest in other medical fields, such as periodontitis in dentistry. Recent work has indicated the possibility of performing gas plasma-mediated biofilm removal on teeth. Teeth frequently contain restoration materials for filling cavities, e.g., resin-based composites. However, it is unknown if such materials are altered upon gas plasma exposure. To this end, we generated a new in-house workflow for three commonly used resin-based composites following gas plasma treatment and incubated the material with human HaCaT keratinocytes in vitro. Cytotoxicity was investigated by metabolic activity analysis, flow cytometry, and quantitative high-content fluorescence imaging. The inflammatory consequences were assessed using quantitative analysis of 13 different chemokines and cytokines in the culture supernatants. Hydrogen peroxide served as the control condition. A modest but significant cytotoxic effect was observed in the metabolic activity and viability after plasma treatment for all three composites. This was only partially treatment time-dependent and the composites alone affected the cells to some extent, as evident by differential secretion profiles of VEGF, for example. Gas plasma composite modification markedly elevated the secretion of IL6, IL8, IL18, and CCL2, with the latter showing the highest correlation with treatment time (Pearson’s r > 0.95). Cell culture media incubated with gas plasma-treated composite chips and added to cells thereafter could not replicate the effects, pointing to the potential that surface modifications elicited the findings. In conclusion, our data suggest that gas plasma treatment modifies composite material surfaces to a certain extent, leading to measurable but overall modest biological effects.
- ItemRisk Evaluation of EMT and Inflammation in Metastatic Pancreatic Cancer Cells Following Plasma Treatment(Lausanne : Frontiers Media, 2020) Freund, Eric; Spadola, Chiara; Schmidt, Anke; Privat-Maldonado, Angela; Bogaerts, Annemie; Woedtke, Thomas von; Weltmann, Klaus-Dieter; Heidecke, Claus-Dieter; Partecke, Lars-Ivo; Käding, André; Bekeschus, SanderThe requirements for new technologies to serve as anticancer agents go far beyond their toxicity potential. Novel applications also need to be safe on a molecular and patient level. In a broader sense, this also relates to cancer metastasis and inflammation. In a previous study, the toxicity of an atmospheric pressure argon plasma jet in four human pancreatic cancer cell lines was confirmed and plasma treatment did not promote metastasis in vitro and in ovo. Here, these results are extended by additional types of analysis and new models to validate and define on a molecular level the changes related to metastatic processes in pancreatic cancer cells following plasma treatment in vitro and in ovo. In solid tumors that were grown on the chorion-allantois membrane of fertilized chicken eggs (TUM-CAM), plasma treatment induced modest to profound apoptosis in the tissues. This, however, was not associated with a change in the expression levels of adhesion molecules, as shown using immunofluorescence of ultrathin tissue sections. Culturing of the cells detached from these solid tumors for 6d revealed a similar or smaller total growth area and expression of ZEB1, a transcription factor associated with cancer metastasis, in the plasma-treated pancreatic cancer tissues. Analysis of in vitro and in ovo supernatants of 13 different cytokines and chemokines revealed cell line-specific effects of the plasma treatment but a noticeable increase of, e.g., growth-promoting interleukin 10 was not observed. Moreover, markers of epithelial-to-mesenchymal transition (EMT), a metastasis-promoting cellular program, were investigated. Plasma-treated pancreatic cancer cells did not present an EMT-profile. Finally, a realistic 3D tumor spheroid co-culture model with pancreatic stellate cells was employed, and the invasive properties in a gel-like cellular matrix were investigated. Tumor outgrowth and spread was similar or decreased in the plasma conditions. Altogether, these results provide valuable insights into the effect of plasma treatment on metastasis-related properties of cancer cells and did not suggest EMT-promoting effects of this novel cancer therapy. © Copyright © 2020 Freund, Spadola, Schmidt, Privat-Maldonado, Bogaerts, von Woedtke, Weltmann, Heidecke, Partecke, Käding and Bekeschus.
- ItemGas plasma irradiation of breast cancers promotes immunogenicity, tumor reduction, and an abscopal effect in vivo(Abingdon : Taylor & Franics, 2021) Mahdikia, Hamed; Saadati, Fariba; Freund, Eric; Gaipl, Udo S.; Majidzadeh-A, Keivan; Shokri, Babak; Bekeschus, SanderWhile many new and emerging therapeutic concepts have appeared throughout the last decades, cancer still is fatal in many patients. At the same time, the importance of immunology in oncotherapy is increasingly recognized, not only since the advent of checkpoint therapy. Among the many types of tumors, also breast cancer has an immunological dimension that might be exploited best by increasing the immunogenicity of the tumors in the microenvironment. To this end, we tested a novel therapeutic concept, gas plasma irradiation, for its ability to promote the immunogenicity and increase the toxicity of breast cancer cells in vitro and in vivo. Mechanistically, this emerging medical technology is employing a plethora of reactive oxygen species being deposited on the target cells and tissues. Using 2D cultures and 3D tumor spheroids, we found gas plasma-irradiation to drive apoptosis and immunogenic cancer cell death (ICD) in vitro, as evidenced by an increased expression of calreticulin, heat-shock proteins 70 and 90, and MHC-I. In 4T1 breast cancer-bearing mice, the gas plasma irradiation markedly decreased tumor burden and increased survival. Interestingly, non-treated tumors injected in the opposite flank of mice exposed to our novel treatment also exhibited reduced growth, arguing for an abscopal effect. This was concomitant with an increase of apoptosis and tumor-infiltrating CD4+ and CD8+ T-cells as well as dendritic cells in the tissues. In summary, we found gas plasma-irradiated murine breast cancers to induce toxicity and augmented immunogenicity, leading to reduced tumor growth at a site remote to the treatment area.
- ItemPlasma, cancer, immunity(Bristol : IOP Publ., 2022) Bekeschus, Sander; Clemen, RamonaAlbeit heavily investigated for several decades already, the importance of the immune system in targeting cancer has received wide clinical attention only in recent years. This is partly because of long-standing rather traditional concepts on tumor biology on the one hand and the complexity of the immune system and its processes on the other. The viewpoint of evaluating existing and emerging approaches in oncology based on toxicity to tumors and the ability to engage antitumor-immunity is gaining ground across several disciplines. Along those lines, cold physical plasma was suggested as potential anticancer tool more than a decade ago, but solid evidence of the immune system playing a role in plasma cancer treatment only emerged in recent years. Moreover, plasma may support cancer immunotherapies in the future. Cancer immunotherapies are systemic treatments with biologicals that were reported to synergize with existing local physical modalities before, such as radiotherapy and photodynamic therapy. This review outlines key concepts in oncology, immunology, and tumor therapy, links them to plasma research, and discusses immuno-oncological consequences. Finally, promising future clinical applications are summarized. Synoptically, first scientific evidence supports an immuno-oncological dimension of plasma cancer treatment in selected instances, but robust clinical evidence is still lacking. More basic and clinical research is needed to determine the immuno-molecular mechanisms and detailed plasma application modalities to facilitate real patient benefit in the long term.
- ItemZebrafish larvae as a toxicity model in plasma medicine(Hoboken, NJ : Wiley Interscience, 2021) Gandhirajan, Rajesh K.; Endlich, Nicole; Bekeschus, SanderPlasma technology has emerged as a promising tool in medicine that, however, requires not only efficacy but also toxicological assessments. Traditional cell culture systems are fast and economical, but they lack in vivo relevance; however, rodent models are highly complex and necessitate extended facilities. Zebrafish larvae bridge this gap, and many larvae can be analyzed in well plates in a single run, giving results in 1–2 days. Using the kINPen, we found plasma exposure to reduce hedging rates and viability in a dose-dependent manner, accompanied with an increase in reactive oxygen species and a decrease of glutathione in plasma-treated fish. Modest growth alterations were also observed. Altogether, zebrafish larvae constitute a fast, reliable, and relevant model for testing the toxicity of plasma sources.
- ItemArgon Humidification Exacerbates Antimicrobial and Anti-MRSA kINPen Plasma Activity(Basel : MDPI, 2023) Clemen, Ramona; Singer, Debora; Skowski, Henry; Bekeschus, SanderGas plasma is a medical technology with antimicrobial properties. Its main mode of action is oxidative damage via reactive species production. The clinical efficacy of gas plasma-reduced bacterial burden has been shown to be hampered in some cases. Since the reactive species profile produced by gas plasma jets, such as the kINPen used in this study, are thought to determine antimicrobial efficacy, we screened an array of feed gas settings in different types of bacteria. Antimicrobial analysis was performed by single-cell analysis using flow cytometry. We identified humidified feed gas to mediate significantly greater toxicity compared to dry argon and many other gas plasma conditions. The results were confirmed by inhibition zone analysis on gas-plasma-treated microbial lawns grown on agar plates. Our results may have vital implications for clinical wound management and potentially enhance antimicrobial efficacy of medical gas plasma therapy in patient treatment.
- ItemCombined toxicity of indirubins with cold physical plasma in skin cancer cells in vitro(Bristol : IOP Publ., 2022) Berner, Julia; Bekeschus, SanderCold physical plasma is a partially ionized gas that generates various components identified as potential anticancer compounds. Due to its topical application, cold plasmas are suitable, especially in dermatological applications. We, therefore, tested the cold plasma effects in skin cancer cells in vitro. An atmospheric pressure argon plasma jet was used as the plasma source. The plasma exposure alone reduced the metabolic activity and induced lethal effects in a treatment time-dependent fashion in both cell lines investigated. This was accompanied by executioner caspases 3 and 7, cleavage indicative of apoptosis and reduced cell migration and proliferation. Recent research also indicated roles of novel indirubin derivatives with potent anticancer effects. Three candidates were tested, and reduced metabolic activity and viability in a dose-dependent manner were found. Strikingly, one compound exerted notable synergistic toxicity when combined with plasma in skin cancer cells, which may be promising for future in vivo experiments.
- ItemSynergistic In Vitro Anticancer Toxicity of Pulsed Electric Fields and Glutathione(Basel : Molecular Diversity Preservation International (MDPI), 2022) Wolff, Christina M.; Bekeschus, SanderDespite continuous advancement in skin cancer therapy, the disease is still fatal in many patients, demonstrating the need to improve existing therapies, such as electrochemotherapy (ECT). ECT can be applied in the palliative or curative setting and is based on the application of pulsed electric fields (PEF), which by themselves exerts none to low cancer toxicity but become potently toxic when combined with low-dosed chemotherapeutics such as bleomycin and cisplatin. Albeit their favorable side-effect profiles, not all patients respond to standard ECT, and some responders experience tumor recurrence. To identify potential adjuvant or alternative agents to standard electrochemotherapy, we explored the possibility of combining PEF with a physiological compound, glutathione (GSH), to amplify anticancer toxicity. GSH is an endogenous antioxidant and is available as a dietary supplement. Surprisingly, neither GSH nor PEF mono treatment but GSH + PEF combination treatment exerted strong cytotoxic effects and declined metabolic activity in four skin cancer cell lines in vitro. The potential applicability to other tumor cells was verified by corroborating results in two leukemia cell lines. Strikingly, GSH + PEF treatment did not immediately increase intracellular GSH levels, while levels 24 h following treatment were enhanced. Similar tendencies were made for intracellular reactive oxygen species (ROS) levels, while extracellular ROS increased following combination treatment. ROS levels and the degree of cytotoxicity could be partially reversed by pre-incubating cells with the NADPH-oxidase (NOX) inhibitor diphenyleneiodonium (DPI) and the H2O2-degrading enzyme catalase. Collectively, our findings suggest a promising new “endogenous” drug to be combined with PEF for future anticancer research approaches.
- ItemConductive Gas Plasma Treatment Augments Tumor Toxicity of Ringer’s Lactate Solutions in a Model of Peritoneal Carcinomatosis(Basel : MDPI, 2022) Miebach, Lea; Freund, Eric; Cecchini, Alessandra Lourenço; Bekeschus, SanderReactive species generated by medical gas plasma technology can be enriched in liquids for use in oncology targeting disseminated malignancies, such as metastatic colorectal cancer. Notwithstanding, reactive species quantities depend on the treatment mode, and we recently showed gas plasma exposure in conductive modes to be superior for cancer tissue treatment. However, evidence is lacking that such a conductive mode also equips gas plasma-treated liquids to confer augmented intraperitoneal anticancer activity. To this end, employing atmospheric pressure argon plasma jet kINPen-treated Ringer’s lactate (oxRilac) in a CT26-model of colorectal peritoneal carcinomatosis, we tested repeated intraabdominal injection of such remotely or conductively oxidized liquid for antitumor control and immunomodulation. Enhanced reactive species formation in conductive mode correlated with reduced tumor burden in vivo, emphasizing the advantage of conduction over the free mode for plasma-conditioned liquids. Interestingly, the infiltration of lymphocytes into the tumors was equally enhanced by both treatments. However, significantly lower levels of interleukin (IL)4 and IL13 and increased levels of IL2 argue for a shift in intratumoral T-helper cell subpopulations correlating with disease control. In conclusion, our data argue for using conductively over remotely prepared plasma-treated liquids for anticancer treatment.
- ItemMedical Gas Plasma Jet Technology Targets Murine Melanoma in an Immunogenic Fashion(Weinheim : Wiley-VCH, 2020) Bekeschus, Sander; Clemen, Ramona; Nießner, Felix; Sagwal, Sanjeev Kumar; Freund, Eric; Schmidt, AnkeMedical technologies from physics are imperative in the diagnosis and therapy of many types of diseases. In 2013, a novel cold physical plasma treatment concept was accredited for clinical therapy. This gas plasma jet technology generates large amounts of different reactive oxygen and nitrogen species (ROS). Using a melanoma model, gas plasma technology is tested as a novel anticancer agent. Plasma technology derived ROS diminish tumor growth in vitro and in vivo. Varying the feed gas mixture modifies the composition of ROS. Conditions rich in atomic oxygen correlate with killing activity and elevate intratumoral immune-infiltrates of CD8+ cytotoxic T-cells and dendritic cells. T-cells from secondary lymphoid organs of these mice stimulated with B16 melanoma cells ex vivo show higher activation levels as well. This correlates with immunogenic cancer cell death and higher calreticulin and heat-shock protein 90 expressions induced by gas plasma treatment in melanoma cells. To test the immunogenicity of gas plasma treated melanoma cells, 50% of mice vaccinated with these cells are protected from tumor growth compared to 1/6 and 5/6 mice negative control (mitomycin C) and positive control (mitoxantrone), respectively. Gas plasma jet technology is concluded to provide immunoprotection against malignant melanoma both in vitro and in vivo.