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- ItemPlasma treatment limits human melanoma spheroid growth and metastasis independent of the ambient gas composition(Basel : MDPI AG, 2020) Hasse, Sybille; Meder, Tita; Freund, Eric; Woedtke, Thomas von; Bekeschus, SanderMelanoma skin cancer is still a deadly disease despite recent advances in therapy. Previous studies have suggested medical plasma technology as a promising modality for melanoma treatment. However, the efficacy of plasmas operated under different ambient air conditions and the comparison of direct and indirect plasma treatments are mostly unexplored for this tumor entity. Moreover, exactly how plasma treatment affects melanoma metastasis has still not been explained. Using 3D tumor spheroid models and high-content imaging technology, we addressed these questions by utilizing one metastatic and one non-metastatic human melanoma cell line targeted with an argon plasma jet. Plasma treatment was toxic in both cell lines. Modulating the oxygen and nitrogen ambient air composition (100/0, 75/25, 50/50, 25/75, and 0/100) gave similar toxicity and reduced the spheroid growth for all conditions. This was the case for both direct and indirect treatments, with the former showing a treatment time-dependent response while the latter resulted in cytotoxicity with the longest treatment time investigated. Live-cell imaging of in-gel cultured spheroids indicated that plasma treatment did not enhance metastasis, and flow cytometry showed a significant modulation of S100A4 but not in any of the five other metastasis-related markers (β-catenin, E-cadherin, LEF1, SLUG, and ZEB1) investigated. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemIdentification of two kinase inhibitors with synergistic toxicity with low-dose hydrogen peroxide in colorectal cancer cells in vitro(Basel : MDPI AG, 2020) Freund, Eric; Liedtke, Kim-Rouven; Miebach, Lea; Wende, Kristian; Heidecke, Amanda; Kaushik, Nagendra Kumar; Choi, Eun Ha; Partecke, Lars-Ivo; Bekeschus, SanderColorectal carcinoma is among the most common types of cancers. With this disease, diffuse scattering in the abdominal area (peritoneal carcinosis) often occurs before diagnosis, making surgical removal of the entire malignant tissue impossible due to a large number of tumor nodules. Previous treatment options include radiation and its combination with intraperitoneal heat-induced chemotherapy (HIPEC). Both options have strong side effects and are often poor in therapeutic efficacy. Tumor cells often grow and proliferate dysregulated, with enzymes of the protein kinase family often playing a crucial role. The present study investigated whether a combination of protein kinase inhibitors and low-dose induction of oxidative stress (using hydrogen peroxide, H2O2) has an additive cytotoxic effect on murine, colorectal tumor cells (CT26). Protein kinase inhibitors from a library of 80 substances were used to investigate colorectal cancer cells for their activity, morphology, and immunogenicity (immunogenic cancer cell death, ICD) upon mono or combination. Toxic compounds identified in 2D cultures were confirmed in 3D cultures, and additive cytotoxicity was identified for the substances lavendustin A, GF109203X, and rapamycin. Toxicity was concomitant with cell cycle arrest, but except HMGB1, no increased expression of immunogenic markers was identified with the combination treatment. The results were validated for GF109203X and rapamycin but not lavendustin A in the 3D model of different colorectal (HT29, SW480) and pancreatic cancer cell lines (MiaPaca, Panc01). In conclusion, our in vitro data suggest that combining oxidative stress with chemotherapy would be conceivable to enhance antitumor efficacy in HIPEC. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemNon-thermal plasma modulates cellular markers associated with immunogenicity in a model of latent HIV-1 infection(San Francisco, California, US : PLOS, 2021) Mohamed, Hager; Clemen, Ramona; Freund, Eric; Lackmann, Jan-Wilm; Wende, Kristian; Connors, Jennifer; Haddad, Elias K.; Dampier, Will; Wigdahl, Brian; Miller, Vandana; Bekeschus, Sander; Krebs, Fred C.; Kashanchi, FatahEffective 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.
- ItemPlasma-Treated Water Affects Listeria monocytogenes Vitality and Biofilm Structure(Lausanne : Frontiers Media, 2021) Handorf, Oliver; Pauker, Viktoria Isabella; Weihe, Thomas; Schäfer, Jan; Freund, Eric; Schnabel, Uta; Bekeschus, Sander; Riedel, Katharina; Ehlbeck, JörgBackground: Plasma-generated compounds (PGCs) such as plasma-processed air (PPA) or plasma-treated water (PTW) offer an increasingly important alternative for the control of microorganisms in hard-to-reach areas found in several industrial applications including the food industry. To this end, we studied the antimicrobial capacity of PTW on the vitality and biofilm formation of Listeria monocytogenes, a common foodborne pathogen. Results: Using a microwave plasma (MidiPLexc), 10 ml of deionized water was treated for 100, 300, and 900 s (pre-treatment time), after which the bacterial biofilm was exposed to the PTW for 1, 3, and 5 min (post-treatment time) for each pre-treatment time, separately. Colony-forming units (CFU) were significantly reduced by 4.7 log10 ± 0.29 log10, as well as the metabolic activity decreased by 47.9 ± 9.47% and the cell vitality by 69.5 ± 2.1%, compared to the control biofilms. LIVE/DEAD staining and fluorescence microscopy showed a positive correlation between treatment and incubation times, as well as reduction in vitality. Atomic force microscopy (AFM) indicated changes in the structure quality of the bacterial biofilm. Conclusion: These results indicate a promising antimicrobial impact of plasma-treated water on Listeria monocytogenes, which may lead to more targeted applications of plasma decontamination in the food industry in the future.
- ItemTumor cytotoxicity and immunogenicity of a novel V-jet neon plasma source compared to the kINPen(London : Nature Publishing Group, 2021) Miebach, Lea; Freund, Eric; Horn, Stefan; Niessner, Felix; Sagwal, Sanjeev Kumar; von Woedtke, Thomas; Emmert, Steffen; Weltmann, Klaus-Dieter; Clemen, Ramona; Schmidt, Anke; Gerling, Torsten; Bekeschus, SanderRecent research indicated the potential of cold physical plasma in cancer therapy. The plethora of plasma-derived reactive oxygen and nitrogen species (ROS/RNS) mediate diverse antitumor effects after eliciting oxidative stress in cancer cells. We aimed at exploiting this principle using a newly designed dual-jet neon plasma source (Vjet) to treat colorectal cancer cells. A treatment time-dependent ROS/RNS generation induced oxidation, growth retardation, and cell death within 3D tumor spheroids were found. In TUM-CAM, a semi in vivo model, the Vjet markedly reduced vascularized tumors' growth, but an increase of tumor cell immunogenicity or uptake by dendritic cells was not observed. By comparison, the argon-driven single jet kINPen, known to mediate anticancer effects in vitro, in vivo, and in patients, generated less ROS/RNS and terminal cell death in spheroids. In the TUM-CAM model, however, the kINPen was equivalently effective and induced a stronger expression of immunogenic cancer cell death (ICD) markers, leading to increased phagocytosis of kINPen but not Vjet plasma-treated tumor cells by dendritic cells. Moreover, the Vjet was characterized according to the requirements of the DIN-SPEC 91315. Our results highlight the plasma device-specific action on cancer cells for evaluating optimal discharges for plasma cancer treatment.
- ItemHypochlorous acid selectively promotes toxicity and the expression of danger signals in human abdominal cancer cells(Athens : Spandidos Publ., 2021) Freund, Eric; Miebach, Lea; Stope, Matthias; Bekeschus, SanderTumors of the abdominal cavity, such as colorectal, pancreatic and ovarian cancer, frequently metastasize into the peritoneum. Large numbers of metastatic nodules hinder cura- tive surgical resection, necessitating lavage with hyperthermic intraperitoneal chemotherapy (HIPEC). However, HIPEC not only causes severe side effects but also has limited therapeutic efficacy in various instances. At the same time, the age of immunotherapies such as biological agents, checkpoint- inhib- itors or immune-cell therapies, increasingly emphasizes the critical role of anticancer immunity in targeting malignancies. The present study investigated the ability of three types of long-lived reactive species (oxidants) to inactivate cancer cells and potentially complement current HIPEC regimens, as well as to increase tumor cell expression of danger signals that stimulate innate immunity. The human abdominal cancer cell lines HT-29, Panc-01 and SK-OV-3 were exposed to different concentrations of hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and peroxynitrite (ONOO-). Metabolic activity was measured, as well as determination of cell death and danger signal expression levels via flow cytometry and detection of intracellular oxidation via high-content microscopy. Oxidation of tumor decreased intracellular levels of the antioxidant glutathione and induced oxidation in mitochondria, accompa- nied by a decrease in metabolic activity and an increase in regulated cell death. At similar concentrations, HOCl showed the most potent effects. Non-malignant HaCaT keratinocytes were less affected, suggesting the approach to be selective to some extent. Pro-immunogenic danger molecules were investi- gated by assessing the expression levels of calreticulin (CRT), and heat-shock protein (HSP)70 and HSP90. CRT expression was greatest following HOCl and ONOO- treatment, whereas HOCl and H2O2 resulted in the greatest increase in HSP70 and HSP90 expression levels. These results suggested that HOCl may be a promising agent to complement current HIPEC regi- mens targeting peritoneal carcinomatosis.
- 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.
- ItemCombination of Gas Plasma and Radiotherapy Has Immunostimulatory Potential and Additive Toxicity in Murine Melanoma Cells In Vitro(Basel : Molecular Diversity Preservation International, 2020) Pasqual-Melo, Gabriella; Sagwal, Sanjeev Kumar; Freund, Eric; Gandhirajan, Rajesh Kumar; Frey, Benjamin; von Woedtke, Thomas; Gaipl, Udo; Bekeschus, SanderDespite continuous advances in therapy, malignant melanoma is still among the deadliest types of cancer. At the same time, owing to its high plasticity and immunogenicity, melanoma is regarded as a model tumor entity when testing new treatment approaches. Cold physical plasma is a novel anticancer tool that utilizes a plethora of reactive oxygen species (ROS) being deposited on the target cells and tissues. To test whether plasma treatment would enhance the toxicity of an established antitumor therapy, ionizing radiation, we combined both physical treatment modalities targeting B16F10 murine melanoma cell in vitro. Repeated rather than single radiotherapy, in combination with gas plasma-introduced ROS, induced apoptosis and cell cycle arrest in an additive fashion. In tendency, gas plasma treatment sensitized the cells to subsequent radiotherapy rather than the other way around. This was concomitant with increased levels of TNFa, IL6, and GM-CSF in supernatants. Murine JAWS dendritic cells cultured in these supernatants showed an increased expression of cell surface activation markers, such as MHCII and CD83. For PD-L1 and PD-L2, increased expression was observed. Our results are the first to suggest an additive therapeutic effect of gas plasma and radiotherapy, and translational tumor models are needed to develop this concept further. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemGas plasma-conditioned ringer’s lactate enhances the cytotoxic activity of cisplatin and gemcitabine in pancreatic cancer in vitro and in ovo(Basel : MDPI AG, 2020) Liedtke, Kim-Rouven; Freund, Eric; Hermes, Maraike; Oswald, Stefan; Heidecke, Claus-Dieter; Partecke, Lars-Ivo; Bekeschus, SanderPancreatic cancer is one of the most aggressive tumor entities. Diffuse metastatic infiltration of vessels and the peritoneum restricts curative surgery. Standard chemotherapy protocols include the cytostatic drug gemcitabine with limited efficacy at considerable toxicity. In search of a more effective and less toxic treatment modality, we tested in human pancreatic cancer cells (MiaPaca and PaTuS) a novel combination therapy consisting of cytostatic drugs (gemcitabine or cisplatin) and gas plasma-conditioned Ringer’s lactate that acts via reactive oxygen species. A decrease in metabolic activity and viability, change in morphology, and cell cycle arrest was observed in vitro. The combination treatment was found to be additively toxic. The findings were validated utilizing an in ovo tumor model of solid pancreatic tumors growing on the chorionallantois membrane of fertilized chicken eggs (TUM-CAM). The combination of the drugs (especially cisplatin) with the plasma-conditioned liquid significantly enhanced the anti-cancer effects, resulting in the induction of cell death, cell cycle arrest, and inhibition of cell growth with both of the cell lines tested. In conclusion, our novel combination approach may be a promising new avenue to increase the tolerability and efficacy of locally applied chemotherapeutic in diffuse metastatic peritoneal carcinomatosis of the pancreas. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.