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. 2022 Feb 5;14(3):813.
doi: 10.3390/cancers14030813.

Gas Plasma Exposure of Glioblastoma Is Cytotoxic and Immunomodulatory in Patient-Derived GBM Tissue

Sander Bekeschus  1 , Mikael Ispirjan  1   2 , Eric Freund  1   3 , Frederik Kinnen  1   2 , Juliane Moritz  1 , Fariba Saadati  1   4 , Jacqueline Eckroth  1 , Debora Singer  1 , Matthias B Stope  5 , Kristian Wende  1 , Christoph A Ritter  6 , Henry W S Schroeder  2 , Sascha Marx  2   7
Affiliations

Gas Plasma Exposure of Glioblastoma Is Cytotoxic and Immunomodulatory in Patient-Derived GBM Tissue

Sander Bekeschus et al. Cancers (Basel). .

Abstract

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-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.

Keywords: brain tumor; chemokines; cold physical plasma; cytokines; reactive oxygen species.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Metabolic activity decline and cell cycle rest of glioblastoma monocultures in vitro. (a) study scheme; (b) H2O2 generation by the DBD and the jet operated with dry and humid (wet) argon gas; (c) cellular oxidation as indicated by DCF fluorescence upon exposure to plasma-treated media; (d,e) representative macroscopic image of the resazurin assay (d) and metabolic activity quantified and normalized (e) against each untreated control for HaCaT keratinocytes, HaCaTs with a history of plasma exposure and passaging (LT) and malignant T98G GBM cells; (f,g) representative flow cytometry overlay histograms of propidium iodide (PI) fluorescence (left), quantification of cell cycle phases using mathematically modeling (middle), and spaghetti plots of the same data visualizing G1 to G2 ratios for T98G (f) and HaCaT (g) cells. Data are representative of boxplot or mean and standard error of three experiments; statistical analysis was performed using unpaired, two-tailed t-test with p < 0.05 (*) and p < 0.001 (***) differing significantly or non-significantly (ns).
Figure 2
Figure 2
Monocyte surface marker expression and cytokine release profiles in GBM cultures. (a) Study protocol; (b) absolute viable monocyte counts and median fluorescence intensities of monocytes (M), gas plasma-treated monocytes (MP), monocyte–GBM co-cultures (MG), gas plasma-treated monocytes co-cultured with GBM (MPG), and gas plasma-treated GBM cells co-cultured with monocytes (MGP) as determined using multicolor flow cytometry; (c) cytokine, chemokine, and growth factor release quantification in monocyte monoculture and co-culture supernatants. Data are mean and standard error of monocytes from four donors; statistical analysis was performed using two-tailed Wilcoxon rank test with p < 0.05 (*) differing significantly or non-significantly (ns).
Figure 3
Figure 3
Patient-derived GBM tumor sample analysis. (a) Study protocol; (b) examples of highly apoptotic (TUNEL+, green) patient-derived glioblastoma multiforme tissue ultrathin cryosections following gas plasma exposure (nuclei counterstained with DAPI in blue); (c) algorithm-driven quantitative image analysis of tissue sections for TUNEL+ (apoptotic) cells per area. Data are box plots (Tukey) from 16 patient samples; statistical analysis was performed using two-tailed Mann–Whitney test with p < 0.001 (***) differing significantly. Scale bar is 150 µm.
Figure 4
Figure 4
Secretion profiling of patient-derived GBM tissues. A total of 11 cytokines, chemokines, and growth factors were analyzed in the supernatants of untreated or gas plasma-treated patient-derived glioblastoma multiforme (GBM) tumor biopsies cultured for 24 h. Data are from 15–16 donors and show mean of each donor and control and treatment group (red: increase, blue: decrease) with mean delta (Δ) given in gray boxes; statistical analysis was performed based on normality distribution using either a paired t-test or two-tailed Wilcoxon rank test with p < 0.05 (*) differing significantly or non-significantly (ns).
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