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. 2021 Oct 23;22(21):11446.
doi: 10.3390/ijms222111446.

Patient-Derived Human Basal and Cutaneous Squamous Cell Carcinoma Tissues Display Apoptosis and Immunomodulation following Gas Plasma Exposure with a Certified Argon Jet

Fariba Saadati  1 , Juliane Moritz  1 , Julia Berner  1   2 , Eric Freund  1   3 , Lea Miebach  1   3 , Iris Helfrich  4   5   6   7 , Ingo Stoffels  4   5   6 , Steffen Emmert  8 , Sander Bekeschus  1
Affiliations

Patient-Derived Human Basal and Cutaneous Squamous Cell Carcinoma Tissues Display Apoptosis and Immunomodulation following Gas Plasma Exposure with a Certified Argon Jet

Fariba Saadati et al. Int J Mol Sci. .

Abstract

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.

Keywords: ROS; chemokines; cold physical plasma; cytokines; reactive oxygen species; skin cancer.

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Conflict of interest statement

The authors report no conflict of interest.

Figures

Figure 1
Figure 1
Gas plasma treatment in vitro: (a) image of the metabolic activity assay with higher (control) and lower (bottom) resazurin transformation, respectively; (b,c) quantitative analysis of gas plasma treatment effects in four cell lines exposed in suspension (b) or adherent (c) states, where grey boxes indicate IC25 values; (d) representative brightfield and fluorescence (caspase 3/7, green) overlay images of cells exposed to various conditions, catalase (cat) and argon gas treatment (Arg) served as controls; (e) viability of the four cell lines exposed in adherent state to several plasma treatment times (15 s, 30 s, 45 s, 60 s and 90 s); (f) representative overlay histogram of the DAPI intensity of control and gas plasma-treated SCC13 samples; (g) quantification of cell cycle phases in fixed and permeabilized DAPI-stained SCC13 and A431 cells after acquisition using flow cytometry and analysis of individual cell cycle phases using mathematical modeling according to the Michael H. Fox algorithm employed in the Kaluza analysis software; (h) G1/G2 ratios calculated from cell cycle data; (i,j) representative flow cytometry overlay histograms (i) and normalized quantification of three oxidative stress-related markers (j). Data were normalized to control and are displayed as the mean with SEM of three experiments. Statistical analysis was performed using t-test with p < 0.001 (***). Scale bar is 100 µm.
Figure 2
Figure 2
Human tumor tissue sampling and treatment scheme: (a) excised skin tumors were retrieved and used to generate punch biopsies of identical sizes. The punch biopsies were subsequently added to microtiter well plates for standardized gas plasma treatment. Afterward, cell culture medium was added to each well and the samples were incubated for 24 h, before supernatants were collected and tissues were cryo-sectioned and stained. Supernatants were analyzed using multiplex flow cytometry, and cryo-sections were stained with antibodies, followed by quantitative immunofluorescence imaging; (b) quantification of ROS (hydrogen peroxide, H2O2) and RNS (nitrite, NO2−; nitrate, NO3−) in plasma-treated liquids (200 µL of PBS). Gas refers to 120 s of exposure of the liquid with argon gas only (i.e., plasma = off).
Figure 3
Figure 3
Immunofluorescence analysis: (a) representative immunofluorescence images of DAPI, TUNEL, CD86, CD206, and FOXP3 staining in gas plasma and argon gas (control)-treated BCC tissues; (b) quantitative imaging data for all markers in BCC samples; (c) quantitative imaging data for all markers in SCC samples. Data show boxplots from 6–8 patients, and statistical analysis was performed using t-test with p < 0.05 (*) and p < 0.001 (***). Scale bars are 100 µm.
Figure 4
Figure 4
Secretion profile of BCCs: quantification of chemokines, cytokines, and growth factors in pg/mL in BCC tissue culture supernatants 24 h after exposure to gas plasma or argon gas. Data show boxplots from 6–8 patients, and statistical analysis was performed using t-test with p < 0.05 (*) and p < 0.01 (**).
Figure 5
Figure 5
Secretion profile of SCCs: quantification of chemokines, cytokines, and growth factors in pg/mL in BCC tissue culture supernatants 24 h after exposure to gas plasma or argon gas. Data show boxplots from 6–8 patients, and statistical analysis was performed using t-test with p < 0.05 (*).
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References

    1. Didona D., Paolino G., Bottoni U., Cantisani C.J.B. Non melanoma skin cancer pathogenesis overview. Biomedicines. 2018;6:6. doi: 10.3390/biomedicines6010006. - DOI - PMC - PubMed
    1. Schafer M., Semmler M.L., Bernhardt T., Fischer T., Kakkassery V., Ramer R., Hein M., Bekeschus S., Langer P., Hinz B., et al. Small molecules in the treatment of squamous cell carcinomas: Focus on indirubins. Cancers (Basel) 2021;13:1770. doi: 10.3390/cancers13081770. - DOI - PMC - PubMed
    1. Apalla Z., Lallas A., Sotiriou E., Lazaridou E., Ioannides D. Epidemiological trends in skin cancer. Derm. Pr. Concept. 2017;7:2. doi: 10.5826/dpc.0702a01. - DOI - PMC - PubMed
    1. Georgescu S.R., Mitran C.I., Mitran M.I., Caruntu C., Caruntu A., Lupu M., Matei C., Constantin C., Neagu M. Tumour microenvironment in skin carcinogenesis. Tumor Microenviron. Organs. 2020;1226:123–142. - PubMed
    1. Ahmed F., Haass N.K. Microenvironment-driven dynamic heterogeneity and phenotypic plasticity as a mechanism of melanoma therapy resistance. Front. Oncol. 2018;8:173. doi: 10.3389/fonc.2018.00173. - DOI - PMC - PubMed

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