Browsing by Author "Freund, Eric"
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- ItemThe Anticancer Efficacy of Plasma-Oxidized Saline (POS) in the Ehrlich Ascites Carcinoma Model In Vitro and In Vivo(Basel : MDPI, 2021) Brito, Walison Augusto Silva; Freund, Eric; Nascimento, Thiago Daniel Henrique do; Pasqual-Melo, Gabriella; Sanches, Larissa Juliani; Dionísio, Joyce Hellen Ribeiro; Fumegali, William Capellari; Miebach, Lea; Cecchini, Alessandra Lourenço; Bekeschus, SanderCold physical plasma, a partially ionized gas rich in reactive oxygen species (ROS), is receiving increasing interest as a novel anticancer agent via two modes. The first involves its application to cells and tissues directly, while the second uses physical plasma-derived ROS to oxidize liquids. Saline is a clinically accepted liquid, and here we explored the suitability of plasma-oxidized saline (POS) as anticancer agent technology in vitro and in vivo using the Ehrlich Ascites Carcinoma (EAC) model. EAC mainly grows as a suspension in the peritoneal cavity of mice, making this model ideally suited to test POS as a putative agent against peritoneal carcinomatosis frequently observed with colon, pancreas, and ovarium metastasis. Five POS injections led to a reduction of the tumor burden in vivo as well as in a decline of EAC cell growth and an arrest in metabolic activity ex vivo. The treatment was accompanied by a decreased antioxidant capacity of Ehrlich tumor cells and increased lipid oxidation in the ascites supernatants, while no other side effects were observed. Oxaliplatin and hydrogen peroxide were used as controls and mediated better and worse outcomes, respectively, with the former but not the latter inducing profound changes in the inflammatory milieu among 13 different cytokines investigated in ascites fluid. Modulation of inflammation in the POS group was modest but significant. These results promote POS as a promising candidate for targeting peritoneal carcinomatosis and malignant ascites and suggest EAC to be a suitable and convenient model for analyzing innovative POS approaches and combination therapies.
- ItemAntitumor Effects in Gas Plasma-Treated Patient-Derived Microtissues—An Adjuvant Therapy for Ulcerating Breast Cancer?(Basel : MDPI, 2021) Akbari, Zahra; Saadati, Fariba; Mahdikia, Hamed; Freund, Eric; Abbasvandi, Fereshteh; Shokri, Babak; Zali, Hakimeh; Bekeschus, SanderDespite global research and continuous improvement in therapy, cancer remains a challenging disease globally, substantiating the need for new treatment avenues. Medical gas plasma technology has emerged as a promising approach in oncology in the last years. Several investigations have provided evidence of an antitumor action in vitro and in vivo, including our recent work on plasma-mediated reduction of breast cancer in mice. However, studies of gas plasma exposure on patient-derived tumors with their distinct microenvironment (TME) are scarce. To this end, we here investigated patient-derived breast cancer tissue after gas plasma-treated ex vivo. The tissues were disjoint to pieces smaller than 100 µm, embedded in collagen, and incubated for several days. The viability of the breast cancer tissue clusters and their outgrowth into their gel microenvironment declined with plasma treatment. This was associated with caspase 3-dependent apoptotic cell death, paralleled by an increased expression of the anti-metastatic adhesion molecule epithelial (E)-cadherin. Multiplex chemokine/cytokine analysis revealed a marked decline in the release of the interleukins 6 and 8 (IL-6, IL-8) and monocyte-chemoattractant-protein 1 (MCP) known to promote a cancer-promoting milieu in the TME. In summary, we provide here, for the first time, evidence of a beneficial activity of gas plasma exposure on human patient-derived breast cancer tissue.
- ItemArgon Plasma Exposure Augments Costimulatory Ligands and Cytokine Release in Human Monocyte-Derived Dendritic Cells(Basel : Molecular Diversity Preservation International (MDPI), 2021) Bekeschus, Sander; Meyer, Dorothee; Arlt, Kevin; von Woedtke, Thomas; Miebach, Lea; Freund, Eric; Clemen, RamonaCold physical plasma is a partially ionized gas expelling many reactive oxygen and nitrogen species (ROS/RNS). Several plasma devices have been licensed for medical use in dermatology, and recent experimental studies suggest their putative role in cancer treatment. In cancer therapies with an immunological dimension, successful antigen presentation and inflammation modulation is a key hallmark to elicit antitumor immunity. Dendritic cells (DCs) are critical for this task. However, the inflammatory consequences of DCs following plasma exposure are unknown. To this end, human monocyte-derived DCs (moDCs) were expanded from isolated human primary monocytes; exposed to plasma; and their metabolic activity, surface marker expression, and cytokine profiles were analyzed. As controls, hydrogen peroxide, hypochlorous acid, and peroxynitrite were used. Among all types of ROS/RNS-mediated treatments, plasma exposure exerted the most notable increase of activation markers at 24 h such as CD25, CD40, and CD83 known to be crucial for T cell costimulation. Moreover, the treatments increased interleukin (IL)-1α, IL-6, and IL-23. Altogether, this study suggests plasma treatment augmenting costimulatory ligand and cytokine expression in human moDCs, which might exert beneficial effects in the tumor microenvironment.
- ItemBiocompatible Gas Plasma Treatment Affects Secretion Profiles but Not Osteogenic Differentiation in Patient-Derived Mesenchymal Stromal Cells(Basel : Molecular Diversity Preservation International (MDPI), 2022) Fischer, Maximilian; Schoon, Janosch; Freund, Eric; Miebach, Lea; Weltmann, Klaus-Dieter; Bekeschus, Sander; Wassilew, Georgi I.Cold physical plasma (CPP), a partially ionized gas that simultaneously generates reactive oxygen and nitrogen species, is suggested to provide advantages in regenerative medicine. Intraoperative CPP therapy targeting pathologies related to diminished bone quality could be promising in orthopedic surgery. Assessment of a clinically approved plasma jet regarding cellular effects on primary bone marrow mesenchymal stromal cells (hBM-MSCs) from relevant arthroplasty patient cohorts is needed to establish CPP-based therapeutic approaches for bone regeneration. Thus, the aim of this study was to derive biocompatible doses of CPP and subsequent evaluation of human primary hBM-MSCs’ osteogenic and immunomodulatory potential. Metabolic activity and cell proliferation were affected in a treatment-time-dependent manner. Morphometric high content imaging analyses revealed a decline in mitochondria and nuclei content and increased cytoskeletal compactness following CPP exposure. Employing a nontoxic exposure regime, investigation on osteogenic differentiation did not enhance osteogenic capacity of hBM-MSCs. Multiplex analysis of major hBM-MSC cytokines, chemokines and growth factors revealed an anti-inflammatory, promatrix-assembling and osteoclast-regulating secretion profile following CPP treatment and osteogenic stimulus. This study can be noted as the first in vitro study addressing the influence of CPP on hBM-MSCs from individual donors of an arthroplasty clientele.
- ItemCharacterization of antimicrobial effects of Plasma-Treated Water (PTW) produced by Microwave-Induced Plasma (MidiPLexc) on pseudomonas fluorescens biofilms(Basel : MDPI, 2020) Handorf, Oliver; Pauker, Viktoria Isabella; Schnabel, Uta; Weihe, Thomas; Freund, Eric; Bekeschus, Sander; Riedel, Katharina; Ehlbeck, JörgFor the decontamination of surfaces in the food production industry, plasma-generated compounds such as plasma-treated water or plasma-processed air offer many promising possibilities for future applications. Therefore, the antimicrobial effect of water treated with microwave-induced plasma (MidiPLexc) on Pseudomonas fluorescens biofilms was investigated. A total of 10 mL deionized water was treated with the MidiPLexc plasma source for 100, 300 and 900 s (pretreatment time) and the bacterial biofilms were exposed to the plasma-treated water for 1, 3 and 5 min (post-treatment time). To investigate the influence of plasma-treated water on P. fluorescens biofilms, microbiological assays (colony-forming units, fluorescence and XTT assay) and imaging techniques (fluorescence microscopy, confocal laser scanning microscopy, and atomic force microscopy) were used. The colony-forming units showed a maximum reduction of 6 log10 by using 300 s pretreated plasma water for 5 min. Additionally, a maximum reduction of 81% for the viability of the cells and a 92% reduction in the metabolic activity of the cells were achieved by using 900 s pretreated plasma water for 5 min. The microscopic images showed evident microbial inactivation within the biofilm even at the shortest pretreatment (100 s) and post-treatment (1 min) times. Moreover, reduction of the biofilm thickness and increased cluster formation within the biofilm was detected. Morphologically, the fusion of cell walls into a uniform dense cell mass was detectable. The findings correlated with a decrease in the pH value of the plasma-treated water, which forms the basis for the chemically active components of plasma-treated water and its antimicrobial effects. These results provide valuable insights into the mechanisms of inactivation of biofilms by plasma-generated compounds such as plasma-treated water and thus allow for further parameter adjustment for applications in food industry. © 2020 by the authors.
- 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.
- 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.
- ItemGas Plasma Exposure of Glioblastoma Is Cytotoxic and Immunomodulatory in Patient-Derived GBM Tissue(Basel : MDPI, 2022) Bekeschus, Sander; Ispirjan, Mikael; Freund, Eric; Kinnen, Frederik; Moritz, Juliane; Saadati, Fariba; Eckroth, Jacqueline; Singer, Debora; Stope, Matthias B.; Wende, Kristian; Ritter, Christoph A.; Schroeder, Henry W. S.; Marx, SaschaGlioblastoma multiforme (GBM) is the most common primary malignant adult brain tumor. Therapeutic options for glioblastoma are maximal surgical resection, chemotherapy, and radiotherapy. Therapy resistance and tumor recurrence demand, however, new strategies. Several experimental studies have suggested gas plasma technology, a partially ionized gas that generates a potent mixture of reactive oxygen species (ROS), as a future complement to the existing treatment arsenal. However, aspects such as immunomodulation, inflammatory consequences, and feasibility studies using GBM tissue have not been addressed so far. In vitro, gas plasma generated ROS that oxidized cells and led to a treatment time-dependent metabolic activity decline and G2 cell cycle arrest. In addition, peripheral blood-derived monocytes were co-cultured with glioblastoma cells, and immunomodulatory surface expression markers and cytokine release were screened. Gas plasma treatment of either cell type, for instance, decreased the expression of the M2-macrophage marker CD163 and the tolerogenic molecule SIGLEC1 (CD169). In patient-derived GBM tissue samples exposed to the plasma jet kINPen ex vivo, apoptosis was significantly increased. Quantitative chemokine/cytokine release screening revealed gas plasma exposure to significantly decrease 5 out of 11 tested chemokines and cytokines, namely IL-6, TGF-β, sTREM-2, b-NGF, and TNF-α involved in GBM apoptosis and immunomodulation. In summary, the immuno-modulatory and proapoptotic action shown in this study might be an important step forward to first clinical observational studies on the future discovery of gas plasma technology’s potential in neurosurgery and neuro-oncology especially in putative adjuvant or combinatory GBM treatment settings.
- 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.
- ItemGas Plasma Technology Augments Ovalbumin Immunogenicity and OT-II T Cell Activation Conferring Tumor Protection in Mice(Weinheim : Wiley-VCH, 2021) Clemen, Ramona; Freund, Eric; Mrochen, Daniel; Miebach, Lea; Schmidt, Anke; Rauch, Bernhard H.; Lackmann, Jan‐Wilm; Martens, Ulrike; Wende, Kristian; Lalk, Michael; Delcea, Mihaela; Bröker, Barbara M.; Bekeschus, SanderReactive oxygen species (ROS/RNS) are produced during inflammation and elicit protein modifications, but the immunological consequences are largely unknown. Gas plasma technology capable of generating an unmatched variety of ROS/RNS is deployed to mimic inflammation and study the significance of ROS/RNS modifications using the model protein chicken ovalbumin (Ova vs oxOva). Dynamic light scattering and circular dichroism spectroscopy reveal structural modifications in oxOva compared to Ova. T cells from Ova-specific OT-II but not from C57BL/6 or SKH-1 wild type mice presents enhanced activation after Ova addition. OxOva exacerbates this activation when administered ex vivo or in vivo, along with an increased interferon-gamma production, a known anti-melanoma agent. OxOva vaccination of wild type mice followed by inoculation of syngeneic B16F10 Ova-expressing melanoma cells shows enhanced T cell number and activation, decreased tumor burden, and elevated numbers of antigen-presenting cells when compared to their Ova-vaccinated counterparts. Analysis of oxOva using mass spectrometry identifies three hot spots regions rich in oxidative modifications that are associated with the increased T cell activation. Using Ova as a model protein, the findings suggest an immunomodulating role of multi-ROS/RNS modifications that may spur novel research lines in inflammation research and for vaccination strategies in oncology.
- 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.
- ItemGas Plasma-Oxidized Liquids for Cancer Treatment: Preclinical Relevance, Immuno-Oncology, and Clinical Obstacles(New York, NY : IEEE, 2021) Freund, Eric; Bekeschus, SanderGas plasmas, often referred to as cold physical plasma, are currently being investigated for their potential to serve as anticancer agents. Along similar lines, gas plasma-oxidized liquids as a carrier for reactive oxygen species have found their way into preclinical research. This review focuses on in vivo studies that utilized such gas plasma-oxidized liquids for cancer therapies. These preclinical tumor models, treatment modalities, and types of liquids that were used are summarized and critically discussed. Among these studies, significant results were observed, indicating the potential of oxidative liquids to serve as an anticancer treatment. However, several steps have to be taken to enhance the quality and translational capacities of this approach in order to gain clinical acceptance for possible future cancer therapies. The most crucial steps include not only a careful selection of suitable liquids, with respect to their approval as medical products, but also the consideration of orthotopic and immunocompetent animal tumor models. This would increase the relevance of such studies and simultaneously allow studying the contribution of the most potent of all anticancer effectors, the immune system.
- ItemGas plasma-treated prostate cancer cells augment myeloid cell activity and cytotoxicity(Basel : MDPI, 2020) Bekeschus, Sander; Ressel, Verena; Freund, Eric; Gelbrich, Nadine; Mustea, Alexander; Stope, Matthias B.Despite recent improvements in cancer treatment, with many of them being related to foster antitumor immunity, tumor-related deaths continue to be high. Novel avenues are needed to complement existing therapeutic strategies in oncology. Medical gas plasma technology recently gained attention due to its antitumor activity. Gas plasmas act via the local deposition of a plethora of reactive oxygen species (ROS) that promote the oxidative cancer cell death. The immunological consequences of plasma-mediated tumor cell death are only poorly understood, however. To this end, we exposed two prostate cancer cell lines (LNCaP, PC3) to gas plasma in vitro, and investigated the immunomodulatory effects of the supernatants in as well as of direct co-culturing with two human myeloid cell lines (THP-1, HL-60). After identifying the cytotoxic action of the kINPen plasma jet, the supernatants of plasma-treated prostate cancer cells modulated myeloid cell-related mitochondrial ROS production and their metabolic activity, proliferation, surface marker expression, and cytokine release. Direct co-culture amplified differentiation-like surface marker expression in myeloid cells and promoted their antitumor-toxicity in the gas plasma over the untreated control conditions. The results suggest that gas plasma-derived ROS not only promote prostate cancer cell death but also augment myeloid cell activity and cytotoxicity. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- 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.
- ItemHmox1 Upregulation Is a Mutual Marker in Human Tumor Cells Exposed to Physical Plasma-Derived Oxidants(Basel : MDPI, 2018-10-27) Bekeschus, Sander; Freund, Eric; Wende, Kristian; Gandhirajan, Rajesh; Schmidt, AnkeIncreasing numbers of cancer deaths worldwide demand for new treatment avenues. Cold physical plasma is a partially ionized gas expelling a variety of reactive oxygen and nitrogen species, which can be harnesses therapeutically. Plasmas and plasma-treated liquids have antitumor properties in vitro and in vivo. Yet, global response signatures to plasma treatment have not yet been identified. To this end, we screened eight human cancer cell lines to investigate effects of low-dose, tumor-static plasma-treated medium (PTM) on cellular activity, immune-modulatory properties, and transcriptional levels of 22 redox-related genes. With PTM, a moderate reduction of metabolic activity and modest modulation of chemokine/cytokine pattern and markers of immunogenic cell death was observed. Strikingly, the Nuclear factor (erythroid-derived 2)-like 2 (nrf2) target heme oxygenase 1 (hmox1) was upregulated in all cell lines 4 h post PTM-treatment. nrf2 was not changed, but its baseline expression inversely and significantly correlated with hmox1 expression after exposure to PTM. Besides awarding hmox1 a central role with plasma-derived oxidants, we present a transcriptional redox map of 22 targets and chemokine/cytokine secretion map of 13 targets across eight different human tumor cell lines of four tumor entities at baseline activity that are useful for future studies in this field.
- 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.
- 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.
- ItemImmunophenotyping of Circulating and Intratumoral Myeloid and T Cells in Glioblastoma Patients(Basel : MDPI, 2022) Marx, Sascha; Wilken, Fabian; Miebach, Lea; Ispirjan, Mikael; Kinnen, Frederik; Paul, Sebastian; Bien-Möller, Sandra; Freund, Eric; Baldauf, Jörg; Fleck, Steffen; Siebert, Nikolai; Lode, Holger; Stahl, Andreas; Rauch, Bernhard H.; Singer, Stephan; Ritter, Christoph; Schroeder, Henry W. S.; Bekeschus, SanderGlioblastoma is the most common and lethal primary brain malignancy that almost inevitably recurs as therapy-refractory cancer. While the success of immune checkpoint blockade (ICB) revealed the immense potential of immune-targeted therapies in several types of cancers outside the central nervous system, it failed to show objective responses in glioblastoma patients as of now. The ability of glioblastoma cells to drive multiple modes of T cell dysfunction while exhibiting low-quality neoepitopes, low-mutational load, and poor antigen priming limits anti-tumor immunity and efficacy of antigen-unspecific immunotherapies such as ICB. An in-depth understanding of the GBM immune landscape is essential to delineate and reprogram such immunosuppressive circuits during disease progression. In this view, the present study aimed to characterize the peripheral and intratumoral immune compartments of 35 glioblastoma patients compared to age- and sex-matched healthy control probands, particularly focusing on exhaustion signatures on myeloid and T cell subsets. Compared to healthy control participants, different immune signatures were already found in the peripheral circulation, partially related to the steroid medication the patients received. Intratumoral CD4+ and CD8+ TEM cells (CD62Llow/CD45ROhigh) revealed a high expression of PD1, which was also increased on intratumoral, pro-tumorigenic macrophages/microglia. Histopathological analysis further identified high PSGL-1 expression levels of the latter, which has recently been linked to increased metastasis in melanoma and colon cancer via P-selectin-mediated platelet activation. Overall, the present study comprises immunophenotyping of a patient cohort to give implications for eligible immunotherapeutic targets in neurooncology in the future.
- ItemInactivation of Cerebral Cavernous Malformation Genes Results in Accumulation of von Willebrand Factor and Redistribution of Weibel-Palade Bodies in Endothelial Cells(Lausanne : Frontiers, 2021) Much, Christiane D.; Sendtner, Barbara S.; Schwefel, Konrad; Freund, Eric; Bekeschus, Sander; Otto, Oliver; Pagenstecher, Axel; Felbor, Ute; Rath, Matthias; Spiegler, StefanieCerebral cavernous malformations are slow-flow thrombi-containing vessels induced by two-step inactivation of the CCM1, CCM2 or CCM3 gene within endothelial cells. They predispose to intracerebral bleedings and focal neurological deficits. Our understanding of the cellular and molecular mechanisms that trigger endothelial dysfunction in cavernous malformations is still incomplete. To model both, hereditary and sporadic CCM disease, blood outgrowth endothelial cells (BOECs) with a heterozygous CCM1 germline mutation and immortalized wild-type human umbilical vein endothelial cells were subjected to CRISPR/Cas9-mediated CCM1 gene disruption. CCM1−/− BOECs demonstrated alterations in cell morphology, actin cytoskeleton dynamics, tube formation, and expression of the transcription factors KLF2 and KLF4. Furthermore, high VWF immunoreactivity was observed in CCM1−/− BOECs, in immortalized umbilical vein endothelial cells upon CRISPR/Cas9-induced inactivation of either CCM1, CCM2 or CCM3 as well as in CCM tissue samples of familial cases. Observer-independent high-content imaging revealed a striking reduction of perinuclear Weibel-Palade bodies in unstimulated CCM1−/− BOECs which was observed in CCM1+/− BOECs only after stimulation with PMA or histamine. Our results demonstrate that CRISPR/Cas9 genome editing is a powerful tool to model different aspects of CCM disease in vitro and that CCM1 inactivation induces high-level expression of VWF and redistribution of Weibel-Palade bodies within endothelial cells.
- ItemMedical gas plasma augments bladder cancer cell toxicity in preclinical models and patient-derived tumor tissues(Amsterdam [u.a.] : Elsevier, 2022) Gelbrich, Nadine; Miebach, Lea; Berner, Julia; Freund, Eric; Saadati, Fariba; Schmidt, Anke; Stope, Matthias; Zimmermann, Uwe; Burchardt, Martin; Bekeschus, SanderIntroduction: Medical gas plasma therapy has been successfully applied to several types of cancer in preclinical models. First palliative tumor patients suffering from advanced head and neck cancer benefited from this novel therapeutic modality. The gas plasma-induced biological effects of reactive oxygen and nitrogen species (ROS/RNS) generated in the plasma gas phase result in oxidation-induced lethal damage to tumor cells. Objectives: This study aimed to verify these anti-tumor effects of gas plasma exposure on urinary bladder cancer. Methods: 2D cell culture models, 3D tumor spheroids, 3D vascularized tumors grown on the chicken chorion-allantois-membrane (CAM) in ovo, and patient-derived primary cancer tissue gas plasma-treated ex vivo were used. Results: Gas plasma treatment led to oxidation, growth retardation, motility inhibition, and cell death in 2D and 3D tumor models. A marked decline in tumor growth was also observed in the tumors grown in ovo. In addition, results of gas plasma treatment on primary urothelial carcinoma tissues ex vivo highlighted the selective tumor-toxic effects as non-malignant tissue exposed to gas plasma was less affected. Whole-transcriptome gene expression analysis revealed downregulation of tumor-promoting fibroblast growth factor receptor 3 (FGFR3) accompanied by upregulation of apoptosis-inducing factor 2 (AIFm2), which plays a central role in caspase-independent cell death signaling. Conclusion: Gas plasma treatment induced cytotoxicity in patient-derived cancer tissue and slowed tumor growth in an organoid model of urinary bladder carcinoma, along with less severe effects in non-malignant tissues. Studies on the potential clinical benefits of this local and safe ROS therapy are awaited.