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Increasing evidence suggests the role of reactive oxygen and nitrogen species (RONS) in regulating antitumor immune effects and immunosuppression. RONS modify biomolecules and induce oxidative post-translational modifications (oxPTM) on proteins that can alarm phagocytes. However, it is unclear if and how protein oxidation by technical means could be a strategy to foster antitumor immunity and therapy. To this end, cold gas plasma technology producing various RONS simultaneously to oxidize the two melanoma-associated antigens MART and PMEL is utilized. Cold plasma-oxidized MART (oxMART) and PMEL (oxPMEL) are heavily decorated with oxPTMs as determined by mass spectrometry. Immunization with oxidized MART or PMEL vaccines prior to challenge with viable melanoma cells correlated with significant changes in cytokine secretion and altered T-cell differentiation of tumor-infiltrated leukocytes (TILs). oxMART promoted the activity of cytotoxic central memory T-cells, while oxPMEL led to increased proliferation of cytotoxic effector T-cells. Similar T-cell results are observed after incubating splenocytes of tumor-bearing mice with B16F10 melanoma cells. This study, for the first time, provides evidence of the importance of oxidative modifications of two melanoma-associated antigens in eliciting anticancer immunity.
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BACKGROUND: Oral leukoplakia (OL) is an unfavorable oral disease often resistant to therapy. To this end, cold physical plasma technology was explored as a novel therapeutic agent in an experimental setup. METHODS: Biopsies with a diameter of 3 mm were obtained from non-diseased and OL tissues. Subsequently, cold atmospheric pressure plasma (CAP) exposure was performed ex vivo in the laboratory. After 20 h of incubation, biopsies were cryo-conserved, and tissue sections were quantified for lymphocyte infiltrates, discriminating between naïve and memory cytotoxic and T-helper cells. In addition, the secretion pattern related to inflammation was investigated in the tissue culture supernatants by quantifying 10 chemokines and cytokines. RESULTS: In CAP-treated OL tissue, significantly decreased overall lymphocyte numbers were observed. In addition, reduced levels were observed when discriminating for the T-cell subpopulations but did not reach statistical significance. Moreover, CAP treatment significantly reduced levels of C-X-C motif chemokine 10 (CXCL10) and granulocyte-macrophage colony-stimulating factor in the OL biopsies' supernatants. In idiopathically inflamed tissues, ex vivo CAP exposure reduced T-cells and CXCL10 as well but also led to markedly increased interleukin-1ß secretion. CONCLUSION: Our findings suggest CAP to have immuno-modulatory properties, which could be of therapeutic significance in the therapy of OL. Future studies should investigate the efficacy of CAP therapy in vivo in a larger cohort.
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Citocinas , Inflamación , Humanos , Biopsia , Leucoplasia Bucal/terapiaRESUMEN
Strategies to improve activity and selectivity are major goals in oncological drug development. Medical gas plasma therapy has been subject to intense research in dermatooncology recently. Based on partial gas ionization, this approach is exceptional in generating a variety of reactive oxygen species simultaneously that can be applied locally at the tumor side. It is hypothesized that combined gas plasma treatment can potentiate drug responses in the treatment of melanoma. Using a plasma jet approved as medical device in Europe, a systematic screening of 46 mitochondria-targeted drugs identifies five agents synergizing in vitro and in vivo. Increased intratumoral leucocyte infiltration points to immunomodulatory aspects of the treatment, motivating to investigate responses to immune checkpoint blockade in combination with plasma. Tumor growth is monitored based on bioluminescent imaging, and single-cell suspensions are retrieved from each tumor to characterize tumor-infiltrating leucocytes using multicolor flow cytometry. Gene expression profiling is done using a validated NanoString panel targeting 770 genes specifically designed for immuno-oncological research. Cell type abundancies are characterized from bulk RNA samples using the CIBERSORT computational framework. Collectively, the results indicate that local application of medical gas plasma technology synergizes with mitochondria-targeted drugs and anti-PD1 checkpoint therapy in treating melanoma.
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INTRODUCTION: 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.
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Carcinoma de Células Transicionales , Neoplasias de la Vejiga Urinaria , Humanos , Especies Reactivas de Oxígeno/metabolismo , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/genética , Apoptosis , Muerte CelularRESUMEN
Glioblastoma 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.
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Reactive 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.
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Gas 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 NO3- 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.
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Peróxido de Hidrógeno , Neoplasias Peritoneales , Cloruro de Sodio , Animales , Línea Celular Tumoral , Embrión de Pollo , Peróxido de Hidrógeno/química , Ratones , Neoplasias Peritoneales/tratamiento farmacológico , Gases em Plasma , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Cloruro de Sodio/farmacologíaRESUMEN
Glioblastoma 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-ß, 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.
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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.
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Diferenciación Celular , Citocinas/metabolismo , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Gases em Plasma/farmacología , Anciano , Núcleo Celular/metabolismo , Células Cultivadas , Citocinas/genética , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Mitocondrias/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismoRESUMEN
Gas plasma jet technology was recently identified as a potential adjuvant in the palliation of cancer patients. However, a practical point raised is if higher therapeutic efficacy is achieved with the gas plasma applied in direct contact to the tumor tissue (conducting) or during treatment with the remote cloud of reactive oxygen and nitrogen species (ROS/RNS) being expelled. In a bedside-to-bench study, this clinical question was translated into studying these two distinct treatment modalities using a three-dimensional tumor cell-matrix-hydrogel assay with subsequent quantitative confocal imaging. Z-resolved fluorescence analysis of two cancer cell lines revealed greater toxicity of the conducting mode. This result was re-iterated in the growth analysis of vascularized tumor tissue cultured on chicken embryos' CAM using in ovo bioluminescence imaging. Furthermore, for conducting compared to free mode, optical emission spectroscopy revealed stronger RNS signal lines in the gas phase, while both ROS/RNS deposition in the liquid was drastically exacerbated in the conducting mode. Altogether, our results are vital in understanding the importance of standardized treatment distances on the therapeutic efficacy of gas plasma exposure in clinical oncology and will help to give critical implications for clinicians involved in plasma onco-therapy in the future.
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Neoplasias , Gases em Plasma , Animales , Argón , Embrión de Pollo , Humanos , Neoplasias/terapia , Gases em Plasma/farmacología , Gases em Plasma/uso terapéutico , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Reactive oxygen species (ROS) have been subject of increasing interest in the pathophysiology and therapy of cancers in recent years. In skin cancer, ROS are involved in UV-induced tumorigenesis and its targeted treatment via, e.g., photodynamic therapy. Another recent technology for topical ROS generation is cold physical plasma, a partially ionized gas expelling dozens of reactive species onto its treatment target. Gas plasma technology is accredited for its wound-healing abilities in Europe, and current clinical evidence suggests that it may have beneficial effects against actinic keratosis. Since the concept of hormesis dictates that low ROS levels perform signaling functions, while high ROS levels cause damage, we investigated herein the antitumor activity of gas plasma in non-melanoma skin cancer. In vitro, gas plasma exposure diminished the metabolic activity, preferentially in squamous cell carcinoma cell (SCC) lines compared to non-malignant HaCaT cells. In patient-derived basal cell carcinoma (BCC) and SCC samples treated with gas plasma ex vivo, increased apoptosis was found in both cancer types. Moreover, the immunomodulatory actions of gas plasma treatment were found affecting, e.g., the expression of CD86 and the number of regulatory T-cells. The supernatants of these ex vivo cultured tumors were quantitatively screened for cytokines, chemokines, and growth factors, identifying CCL5 and GM-CSF, molecules associated with skin cancer metastasis, to be markedly decreased. These findings suggest gas plasma treatment to be an interesting future technology for non-melanoma skin cancer topical therapy.
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Argón/farmacología , Carcinoma Basocelular/metabolismo , Gases em Plasma/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Carcinoma Basocelular/patología , Carcinoma Basocelular/terapia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/terapia , Línea Celular Tumoral , Humanos , Inmunomodulación/efectos de los fármacos , Inmunomodulación/fisiología , Fotoquimioterapia/efectos adversos , Gases em Plasma/metabolismo , Cultivo Primario de Células , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapiaRESUMEN
Cold 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.
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Cerebral 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.
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Macrophages and immuno-modulation play a dominant role in the pathology of pancreatic cancer. Gas plasma is a technology recently suggested to demonstrate anticancer efficacy. To this end, two murine cell lines were employed to analyze the inflammatory consequences of plasma-treated pancreatic cancer cells (PDA) on macrophages using the kINPen plasma jet. Plasma treatment decreased the metabolic activity, viability, and migratory activity in an ROS- and treatment time-dependent manner in PDA cells in vitro. These results were confirmed in pancreatic tumors grown on chicken embryos in the TUM-CAM model (in ovo). PDA cells promote tumor-supporting M2 macrophage polarization and cluster formation. Plasma treatment of PDA cells abrogated this cluster formation with a mixed M1/M2 phenotype observed in such co-cultured macrophages. Multiplex chemokine and cytokine quantification showed a marked decrease of the neutrophil chemoattractant CXCL1, IL6, and the tumor growth supporting TGFß and VEGF in plasma-treated compared to untreated co-culture settings. At the same time, macrophage-attractant CCL4 and MCP1 release were profoundly enhanced. These cellular and secretome data suggest that the plasma-inactivated PDA6606 cells modulate the inflammatory profile of murine RAW 264.7 macrophages favorably, which may support plasma cancer therapy.
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Reactive 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.
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Vacunas contra el Cáncer/inmunología , Inflamación/inmunología , Melanoma/tratamiento farmacológico , Ovalbúmina/inmunología , Gases em Plasma/química , Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Inflamación/metabolismo , Activación de Linfocitos/inmunología , Melanoma/inmunología , Melanoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/química , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Background: 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.
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Cold 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.
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Antígenos CD/metabolismo , Argón/farmacología , Citocinas/metabolismo , Células Dendríticas/metabolismo , Monocitos/metabolismo , Gases em Plasma/farmacología , Humanos , Ligandos , Microambiente Tumoral/efectos de los fármacosRESUMEN
Tumors of the abdominal cavity, such as colorectal, pancreatic and ovarian cancer, frequently metastasize into the peritoneum. Large numbers of metastatic nodules hinder curative 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 inhibitors or immunecell therapies, increasingly emphasizes the critical role of anticancer immunity in targeting malignancies. The present study investigated the ability of three types of longlived 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 HT29, Panc01 and SKOV3 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 highcontent microscopy. Oxidation of tumor decreased intracellular levels of the antioxidant glutathione and induced oxidation in mitochondria, accompanied by a decrease in metabolic activity and an increase in regulated cell death. At similar concentrations, HOCl showed the most potent effects. Nonmalignant HaCaT keratinocytes were less affected, suggesting the approach to be selective to some extent. Proimmunogenic danger molecules were investigated by assessing the expression levels of calreticulin (CRT), and heatshock 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 regimens targeting peritoneal carcinomatosis.
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Alarminas/metabolismo , Ácido Hipocloroso/farmacología , Neoplasias Peritoneales/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Femenino , Células HaCaT , Humanos , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/uso terapéutico , Quimioterapia Intraperitoneal Hipertérmica/métodos , Ácido Hipocloroso/uso terapéutico , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Oxidación-Reducción/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Neoplasias Peritoneales/secundario , Peritoneo/efectos de los fármacos , Peritoneo/patología , Ácido Peroxinitroso/farmacología , Ácido Peroxinitroso/uso terapéuticoRESUMEN
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-1ß (IL-1ß) 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.
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Infecciones por VIH/tratamiento farmacológico , Inmunidad/fisiología , Gases em Plasma/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Antirretrovirales/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , VIH-1/patogenicidad , Humanos , Inmunidad/efectos de los fármacos , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Gases em Plasma/metabolismo , Células THP-1 , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Recent 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.