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- 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.
- ItemCombined In Vitro Toxicity and Immunogenicity of Cold Plasma and Pulsed Electric Fields(Basel : MDPI, 2022) Wolff, Christina M.; Kolb, Juergen F.; Bekeschus, SanderIn modern oncology, therapies are based on combining monotherapies to overcome treatment resistance and increase therapy precision. The application of microsecond-pulsed electric fields (PEF) is approved to enhance local chemotherapeutic drug uptake within combination electrochemotherapy regimens. Reactive oxygen species (ROS) have been implicated in anticancer effects, and cold physical plasma produces vast amounts of ROS, which have recently been shown to benefit head and neck cancer patients. PEF and cold plasma technology have been linked to immunogenic cell death (ICD) induction, a regulated cell death accompanied by sterile inflammation that promotes antitumor immunity. To this end, we investigated the combined effect of both treatments regarding their intracellular ROS accumulation, toxicity, ICD-related marker expression, and optimal exposure sequence in a leukemia model cell line. The combination treatment substantially increased ROS and intracellular glutathione levels, leading to additive cytotoxic effects accompanied by a significantly increased expression of ICD markers, such as the eat-me signal calreticulin (CRT). Preconditioned treatment with cold plasma followed by PEF exposure was the most potent treatment sequence. The results indicate additive effects of cold plasma and PEF, motivating further studies in skin and breast tumor models for the future improvement of ECT in such patients.
- ItemPancreatic Cancer Cells Undergo Immunogenic Cell Death upon Exposure to Gas Plasma-Oxidized Ringers Lactate(Basel : MDPI, 2023) Miebach, Lea; Mohamed, Hager; Wende, Kristian; Miller, Vandana; Bekeschus, SanderSurvival 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.
- ItemOxidized Proteins Differentially Affect Maturation and Activation of Human Monocyte-Derived Cells(Basel : MDPI, 2022) Clemen, Ramona; Arlt, Kevin; Miebach, Lea; von Woedtke, Thomas; Bekeschus, SanderIn cancer, antigen-presenting cells (APC), including dendritic cells (DCs), take up and process proteins to mount adaptive antitumor immune responses. This often happens in the context of inflamed cancer, where reactive oxygen species (ROS) are ubiquitous to modify proteins. However, the inflammatory consequences of oxidized protein uptake in DCs are understudied. To this end, we investigated human monocyte-derived cell surface marker expression and cytokine release profiles when exposed to oxidized and native proteins. Seventeen proteins were analyzed, including viral proteins (e.g., CMV and HBV), inflammation-related proteins (e.g., HO1 and HMGB1), matrix proteins (e.g., Vim and Coll), and vastly in the laboratory used proteins (e.g., BSA and Ova). The multifaceted nature of inflammation-associated ROS was mimicked using gas plasma technology, generating reactive species cocktails for protein oxidation. Fourteen oxidized proteins led to elevated surface marker expression levels of CD25, CD40, CD80, CD86, and MHC-II as well as strongly modified release of IL6, IL8, IL10, IL12, IL23, MCP-1, and TNFα compared to their native counterparts. Especially IL8, heme oxygenase 2, and vimentin oxidation gave pronounced effects. Furthermore, protein kinase phospho-array studies in monocyte-derived cells pulsed with native vs. oxidized IL8 and insulin showed enhanced AKT and RSK2 phosphorylation. In summary, our data provide for the first time an overview of the functional consequences of oxidized protein uptake by human monocyte-derived cells and could therefore be a starting point for exploiting such principle in anticancer therapy in the future.
- 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.
- ItemGas plasma–oxidized sodium chloride acts via hydrogen peroxide in a model of peritoneal carcinomatosis(Washington, DC : National Acad. of Sciences, 2022) Miebach, Lea; Freund, Eric; Clemen, Ramona; Kersting, Stephan; Partecke, Lars-Ivo; Bekeschus, SanderGas plasma technology generates reactive oxygen and nitrogen species (ROS/RNS), inducing lethal oxidative damage in tumor cells. The transfer of gas plasma–derived ROS/RNS into liquids has been proposed as an innovative anti-cancer strategy targeting peritoneal carcinomatosis (PC). However, the mechanism of action is under debate. To this end, we compared gas plasma–oxidized medical-grade sodium chloride (oxNaCl) with a concentration-matched control (cmc) of NaCl enriched with equivalent concentrations of H2O2 and NO32 in several cell lines and models of PC. Strikingly, oxNaCl and cmc performed equally well in oxidation and cytotoxic activity in tumor cells in two-dimensional cultures, three-dimensional (3D) tumor spheroids, vascularized 3D tumors grown on chicken-embryo chorioallantoic membranes, and a syngeneic PC mouse model in vivo. Given the importance of immunotherapies in oncology today, we focused on immunological consequences of the treatment. Again, to a similar extent, oxNaCl and cmc increased tumor cell immunogenicity and enhanced uptake by and maturation of peripheral blood monocyte–derived dendritic cells together with an inflammatory secretion profile. Furthermore, NanoString gene expression profiling revealed immune system processes and unfolded protein response-related pathways as being linked to the observed anti-tumor effects for both oxNaCl and cmc. In conclusion, gas plasma–generated oxNaCl and cmc showed equal therapeutic efficacy in our PC-related models. In light of the many promising anti-cancer studies of gas plasma–oxidized liquids and the convenient production of corresponding cmcs in large quantities as needed in clinics, our findings may spur research lines based on low-dose oxidants in peritoneal cancer therapy.
- 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.
- ItemMedical Gas Plasma—A Potent ROS-Generating Technology for Managing Intraoperative Bleeding Complications(Basel : MDPI, 2022) Miebach, Lea; Poschkamp, Broder; van der Linde, Julia; Bekeschus, SanderCold medical gas plasmas are under pre-clinical investigation concerning their hemostatic activity and could be applied for intra-operative bleeding control in the future. The technological leap innovation was their generation at body temperature, thereby causing no thermal harm to the tissue and ensuring tissue integrity. This directly contrasts with current techniques such as electrocautery, which induces hemostasis by carbonizing the tissue using a heated electrode. However, the necrotized tissue is prone to fall, raising the risk of post-operative complications such as secondary bleedings or infection. In recent years, various studies have reported on the ability of medical gas plasmas to induce blood coagulation, including several suggestions concerning their mode of action. As non-invasive and gentle hemostatic agents, medical gas plasmas could be particularly eligible for vulnerable tissues, e.g., colorectal surgery and neurosurgery. Further, their usage could be beneficial regarding the prevention of post-operative bleedings due to the absence or sloughing of eschar. However, no clinical trials or individual healing attempts for medical gas plasmas have been reported to pave the way for clinical approvement until now, despite promising results in experimental animal models. In this light, the present mini-review aims to emphasize the potential of medical gas plasmas to serve as a hemostatic agent in clinical procedures. Providing a detailed overview of the current state of knowledge, feasible application fields are discussed, and possible obstacles are addressed.
- ItemLow-Dose Oxidant Toxicity and Oxidative Stress in Human Papillary Thyroid Carcinoma Cells K1(Basel : MDPI, 2022) Lens, Hannah Hamada Mendonça; Lopes, Natália Medeiros Dias; Pasqual-Melo, Gabriella; Marinello, Poliana Camila; Miebach, Lea; Cecchini, Rubens; Bekeschus, Sander; Cecchini, Alessandra LourençoMedical gas plasmas are of emerging interest in pre-clinical oncological research. Similar to an array of first-line chemotherapeutics and physics-based therapies already approved for clinical application, plasmas target the tumor redox state by generating a variety of highly reactive species eligible for local tumor treatments. Considering internal tumors with limited accessibility, medical gas plasmas help to enrich liquids with stable, low-dose oxidants ideal for intratumoral injection and lavage. Pre-clinical investigation of such liquids in numerous tumor entities and models in vitro and in vivo provided evidence of their clinical relevance, broadening the range of patients that could benefit from medical gas plasma therapy in the future. Likewise, the application of such liquids might be promising for recurrent BRAF(V600E) papillary thyroid carcinomas, resistant to adjuvant administration of radioiodine. From a redox biology point of view, studying redox-based approaches in thyroid carcinomas is particularly interesting, as they evolve in a highly oxidative environment requiring the capability to cope with large amounts of ROS/RNS. Knowledge on their behavior under different redox conditions is scarce. The present study aimed to clarify resistance, proliferative activity, and the oxidative stress response of human papillary thyroid cancer cells K1 after exposure to plasma-oxidized DMEM (oxDMEM). Cellular responses were also evaluated when treated with different dosages of hydrogen peroxide and the RNS donor sodium nitroprusside (SNP). Our findings outline plasma-oxidized liquids as a promising approach targeting BRAF(V600E) papillary thyroid carcinomas and extend current knowledge on the susceptibility of cells to undergo ROS/RNS-induced cell death.
- ItemUnspecific CTL Killing Is Enhanced by High Glucose via TNF-Related Apoptosis-Inducing Ligand(Lausanne : Frontiers Media, 2022) Yang, Wenjuan; Denger, Andreas; Diener, Caroline; Küppers, Frederic; Soriano-Baguet, Leticia; Schäfer, Gertrud; Yanamandra, Archana K.; Zhao, Renping; Knörck, Arne; Schwarz, Eva C.; Hart, Martin; Lammert, Frank; Roma, Leticia Prates; Brenner, Dirk; Christidis, Grigorios; Helms, Volkhard; Meese, Eckart; Hoth, Markus; Qu, BinTNF-related apoptosis inducing ligand (TRAIL) is expressed on cytotoxic T lymphocytes (CTLs) and TRAIL is linked to progression of diabetes. However, the impact of high glucose on TRAIL expression and its related killing function in CTLs still remains largely elusive. Here, we report that TRAIL is substantially up-regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo. Non-mitochondrial reactive oxygen species, NFκB and PI3K/Akt are essential in HG-induced TRAIL upregulation in CTLs. TRAILhigh CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Treatment with metformin and vitamin D reduces HG-enhanced expression of TRAIL in CTLs and coherently protects 1.4E7 cells from TRAIL-mediated apoptosis. Our work suggests that HG-induced TRAILhigh CTLs might contribute to the destruction of pancreatic beta cells in a hyperglycemia condition.