Research ArticleExperimental Studies
Open Access

Cold Physical Plasma Reduces Motility of Various Bone Sarcoma Cells While Remodeling the Cytoskeleton

ANDREAS NITSCH, PAULINE MARTHALER, SARA QARQASH, MAXIMILIAN BEMMANN, SANDER BEKESCHUS, GEORGI I. WASSILEW and LYUBOMIR HARALAMBIEV
In Vivo July 2024, 38 (4) 1571-1578; DOI: https://doi.org/10.21873/invivo.13607

Abstract

Background/Aim: Cold physical plasma (CPP) has emerged as an effective therapy in oncology by inducing cytotoxic effects in various cancer cells, including chondrosarcoma (CS), Ewing’s sarcoma (ES), and osteosarcoma (OS). The current study investigated the impact of CPP on cell motility in CS (CAL-78), ES (A673), and OS (U2-OS) cell lines, focusing on the actin cytoskeleton. Materials and Methods: The CASY Cell Counter and Analyzer was used to study cell proliferation and determine the optimal concentrations of fetal calf serum to maintain viability without stimulation of cell proliferation. CellTiter-BlueCell viability assay was used to determine the effects of CPP on the viability of bone sarcoma cells. The Radius assay was used to determine cell migration. Staining for Deoxyribonuclease I, G-actin, and F-actin was used to assay for the effects on the cytoskeleton. Results: Reductions in cell viability and motility were observed across all cell lines following CPP treatment. CPP induced changes in the actin cytoskeleton, leading to decreased cell motility. Conclusion: CPP effectively reduces the motility of bone sarcoma cells by altering the actin cytoskeleton. These findings underscore CPP’s potential as a therapeutic tool for bone sarcomas and highlight the need for further research in this area.

Key Words:

Cold physical plasma (CPP) has emerged as a promising therapeutic tool in oncology due to its ability to induce cytotoxic effects in various cancer types by generating reactive oxygen and nitrogen species (RONS) (1-3). Previous studies have demonstrated the efficacy of CPP in reducing cell viability, proliferation, and motility, as well as inducing apoptosis in cancer cells (4-10).

Notably, these effects have also been observed in chondrosarcoma (CS) (8, 11), Ewing’s sarcoma (ES) (12), and osteosarcoma (OS) (10, 12-15), indicating the potential benefit of CPP in the treatment of bone sarcomas.

CPP has been demonstrated to promote cell motility in non-malignant cells by upregulating the expression of adhesion molecules like integrins and releasing FGF-2 (16-19). Conversely, CPP can reduce cell motility in malignant cells by disrupting cell adhesion and cell-cell interactions or by inducing cell death or cell cycle arrest, thereby limiting cell movement (20-24).

The cell’s actin cytoskeleton plays a central role in regulating its motility. As a dynamic network of actin filaments, the cytoskeleton is involved in cell movement, whether it be wound healing or cancer cell metastasis (25, 26). CPP treatment can induce the reorganization of the actin cytoskeleton, which also affects cell morphology (27, 28). Additionally, CPP has also been shown to affect the activity of proteins involved in actin polymerization and depolymerization (29, 30).

The aim of this study was to investigate the impact of CPP on cell motility in CS (CAL-78), ES (A673), and OS (U2-OS) cell lines. Given the particular importance of the actin cytoskeleton in cell motility and, thus, metastasis, the effects on the actin cytoskeleton were investigated.

Materials and Methods

Cell culture. Three different human bone sarcoma cell lines were used in this study. The chondrosarcoma cell line CAL-78 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) was cultured with RPMI 1640 media supplemented with 1% penicillin/streptomycin (P/S) and 20% fetal calf serum (FCS). The osteosarcoma cells U2-OS (Cell Lines Service, Eppelheim, Germany) and the Ewing’s sarcoma cell line A673 (American Type Culture Collection, Manassas, VA, USA) cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM) containing 1.0 g/l glucose, 10% fetal bovine serum, 1 mM sodium pyruvate, and 1 % penicillin/streptomycin (all reagents from PAN Biotech, Aidenbach, Germany). All cells were incubated at 37°C and 5% CO2.

Proliferation assay with reduced FCS concentrations. The minimization of cell division is crucial for the assessment of cell motility. Therefore, a 120 h growth kinetics study was performed to determine the appropriate concentration of FCS in the cell culture medium. 2.5×104 cells per well were transferred into the wells of a 24-well plate. Full medium with various FCS concentrations (U2-OS: 10%, 0.2%, 0.1%, and 0%; A673: 10%, 1%, 0.5%, and 0.1%; CAL-78: 20%, 2%, 1%, and 0.2%) was added. The number of viable cells was determined at 0, 24, 48, 72, 96, and 120 h using the CASY Cell Counter and Analyzer (OLS OMNI Livescience, Bremen, Germany).

Cold physical plasma treatment. The kINPen MED device (neoplas MED, Greifswald, Germany) is an approved medical product in Europe and was used for CPP treatment. The gas flow of the carrier gas argon (Alphagaz 1 AIR LIQUIDE Deutschland, Düsseldorf, Germany) was adjusted to three standard liters per minute. CPP treatment of bone sarcoma cells was performed in medium cell suspension with exponential increases in application time, first for 5 s, then for 10 s, and finally for 20 s. The control treatment was performed with carrier gas argon.

Cell viability assay after CPP exposure. CellTiter-BlueCell Viability Assay (Promega, Walldorf, Germany) was used to determine the effects of CPP on bone sarcoma cells. Cells were treated for 5, 10, or 20 s with CPP or argon as a control. Preparations were performed by diluting the stock solution to the desired concentrations in DMSO. Cells were incubated with standard cell culture conditions over 0, 12, and 24 h. The cellTiter-Blue reagent was added to the media, and the fluorescence signal was measured with a multimode plate reader at 560Ex/590Em (TECAN, Männedorf, Switzerland) after 2 h of incubation. The fluorescence signals of the cells treated with CPP were then normalized to the signals of cells treated with argon (control) to determine their respective cell viability.

Radius cell migration assay. 1×106 cells were incubated on the Radius Assay (Cell Biolabs, San Diego, CA, USA) 96-well plate for 24 h. Subsequently, the gel spot on the bottom of the wells was removed according to the manufacturer’s instructions, creating a cell-free area on the well’s bottom. After three washes with PBS, 200 μl of 5 s, 10 s, or 20 s CPP-treated cell culture medium with reduced FCS concentration (A673: 0.5%, CAL-78: 1.0%, U2-OS: 0.1%) was added. Immediately after treatment, light microscopic images were taken (Zeiss Axio Observer, Zeiss Zen 2021 pro, Carl Zeiss, Jena, Germany). After 12 h of incubation at 37°C and 5% CO2, images were taken again. The cell-free area was determined using Image J (Wayne Rasband, NIH, Bethesda, MD, USA). The cell-free area at 12 h was normalized to the cell-free area at the starting time point.

Cytoskeleton. The cells were seeded onto coverslips and incubated for at least 24 h. Subsequently, the cells were incubated in 10 s CPP-treated cell culture medium for 20 min. The CPP-treated medium was then diluted with untreated cell culture medium, and the cells were further incubated for 24, 48, and 72 h. Following triple washing, permeabilization was carried out using 0.1% Triton X-100 for 15 min. The cells were stained with Deoxyribonuclease I, Alexa Flour 588 Conjugate for G-actin, and Alexa Flour 546 Phalloidin for F-actin (both from Thermo Fisher Scientific, Waltham, MA, USA) for 20 min in a humid chamber. After final DAPI staining (DAPI Fluorescence Stain 1000X, Cell Biolabs), mounting on slides was performed, followed by fluorescence microscopy.

Data analysis. For data analysis and visualization, GraphPad Prism Version 9.5.1 (GraphPad Software, La Jolla, CA, USA) was used. Differences were examined using a two-way ANOVA, followed by Bonferroni’s multiple comparison test or Fisher’s LSD test. For experiments with repeated measurements at different times (e.g., growth kinetics), repeated-measure two-way ANOVAs were used.

Results

To investigate the effects of CPP on cell motility, it was first necessary to determine a concentration of FCS that would ensure the survival of the cell population without stimulating its proliferation. To achieve this, cells were incubated in varying concentrations of FCS for 120 h, and growth kinetics were assessed. Across all cell lines, FCS concentration had a significant impact on cell proliferation (p<0.0001). The mean doubling time for the osteosarcoma cell line U2-OS was 39.46 h when using the recommended FCS concentration of 10%. However, by reducing the FCS concentration to 0.1%, the calculated doubling time increased to an average of 101.4 h. The Ewing sarcoma cell line A637 exhibited a doubling time of 16.45 h under the recommended 10% FCS concentration, and a reduction to 0.5% FCS extended the doubling time to 49.3 h. The slowly proliferating chondrosarcoma cell line CAL-78 had a doubling time of 61.46 h under the recommended 20% FCS concentration. Utilizing a 1% FCS concentration resulted in an average doubling time of 392.8 h (Figure 1).

Figure 1.
Figure 1.

Growth kinetics with different fetal calf serum (FCS) concentrations. Human Ewing’s sarcoma cells A673 (A), chondrosarcoma cells CAL-78 (B), and osteosarcoma cells U2-OS (C) were incubated with the recommended FCS concentration as well as reduced FCS concentrations. The number of viable cells was determined with a CASY Cell counter and an analyzer after 24, 48, 72, 96, and 120 h. Mean values±SD are presented. Significant differences were assessed using a two-way repeated measure ANOVA followed by Bonferroni’s multiple comparisons test. The significance level of the multiple comparisons within the lowest and highest FCS concentration is depicted (*p<0.05, **p<0.01, ***p<0.001).

To determine the effects of CPP on cell viability within 24 h of treatment, cells were exposed to CPP and incubated for 24 h. Cell viability was assessed immediately after treatment as well as at 12 h and 24 h post-treatment. All cell lines showed a trend of reduction in cell viability after CPP treatment. However, within the observed 24 h timeframe, the effects were only statistically significant for the Ewing’s sarcoma cell line A673 (0 h: p<0.05, 12 h and 24 h: p<0.001). In the case of the chondrosarcoma cell line CAL-78, a significant reduction in cell viability was observed only after a 20 s treatment following a 24 h incubation period (p<0.01) (Figure 2).

Figure 2.
Figure 2.

Cell viability after cold physical plasma treatment of human bone sarcoma cells. Ewing’s sarcoma (A673), chondrosarcoma (CAL-78), and osteosarcoma (U2-OS) cells were treated for 5 s, 10 s, and 20 s with CPP and incubated over 24 h. Cell viability was determined with Cell Titer blue assay at the indicated time points. The graphs show the mean values±SD and were tested for statistically significant differences using two-way ANOVA followed by Bonferroni’s multiple comparison tests (*p<0.05, ***p<0.001, ****p<0.0001).

Radius cell migration assays were performed to investigate cell motility. The tested cell lines exhibited markedly different migration behaviors. In the control treatment, Ewing’s sarcoma cells reduced the cell-free area to 40% of its original size after 12 h of incubation. For the CS cell line, the cell-free area was reduced to approximately 50% of the initial size after 12 h of incubation. The osteosarcoma cells U2-OS demonstrated significantly faster migration, nearly closing the cell-free area within the 12 h incubation (Figure 3).

Figure 3.
Figure 3.

Inhibition of cell motility after cold physical plasma exposure. Human Ewing’s sarcoma (A673), chondrosarcoma (CAL-78), and osteosarcoma (U2-OS) were preincubated in radius assay plates. After 24 h, the gel spot was removed, and cells were treated with CPP-exposed media. The cell-free area was measured immediately after treatment and after 12 h incubation. The cell-free area after 12 h was normalized to the cell-free area immediately after treatment. The graphs show the mean values±SD and were tested for statistically significant differences using two-way ANOVA followed by Bonferroni’s multiple comparison tests (**p<0.01, ****p<0.0001).

Furthermore, CPP treatment tended to decrease cell motility in each examined cell line. Even a 5 s treatment of the A673 cell line resulted in reduced closure of the cell-free area, with values of 48.44±3.56% compared to 41.60±2.4% in the control. While increasing the duration of the control treatment had no significant impact, prolonged CPP treatment led to more pronounced effects (10 s: 59.46±6.21%, p<0.0001; 20 s: 69.96±3.42%, p<0.0001).

In the CS cell line CAL-78, a similar trend was observed, although the effects were not significant. A 20 s CPP treatment in this experimental setup resulted in such pronounced cell death that an evaluation of cell migration became impractical.

The motility of U2-OS osteosarcoma cells, exhibiting the fastest migration behavior, was also significantly inhibited by CPP (p<0.0001). A 5 s treatment led to closure of the cell-free area comparable to the control treatment (10.17±3.3% vs. 10.34±4.9%). The effects were more pronounced with a 10 s (16.85±2.2%, p<0.01) and a 20 s (28.12±6.03%, p<0.0001) treatments.

For further investigation of migration behavior, the actin cytoskeleton was examined. Specific staining of G- and F-actin was performed for this purpose (Figure 4). In all cell lines, a clear tendency to shift from F-actin to G-actin was observed following CPP treatment. In the A673 cell line, the effect was most pronounced immediately after treatment, with the ratio of G- to F-actin more than doubled by CPP treatment (p<0.001). This effect was also observed, albeit to a lesser extent, at the observation time points of 24, 48, and 72 h post-treatment. In CAL-87 cells, a slight shift from F- to G-actin was observed at each of the observed time points, except at 24 h, due to CPP treatment. In the U2-OS cell line, the effect was observed at each of the observed time points. At 0 h and 24 h, the ratio of G- to F-actin was slightly increased compared to the control treatment (0 h and 24 h: p<0.05; 48 and 72 h: p<0.01).

Figure 4.
Figure 4.

Remodeling of actin fibers of human bone sarcoma cells after cold physical plasma. Ewing’s sarcoma (A673), chondrosarcoma (CAL-78), and osteosarcoma (U2-OS) cells were treated with cold physical plasma (CPP) and incubated for 0 h, 24 h, 48 h, and 72 h. Cells were stained for F-actin and G-actin. Ratios were calculated. Data are shown as mean with standard deviation. The graphs show the mean values±SD, and two-way ANOVA and Fisher’s LSD test were used to determine significant differences (*p<0.05, **p<0.01, ***p<0.001).

Discussion

As a promising therapy option in oncology, CPP and its effects have been investigated in various cancer types (22, 24, 31-34). Through the production of large amounts of RONS, CPP led to a reduction in cell viability, cell proliferation, cell motility, and the induction of apoptosis when applied to cancer cells (1, 2). These effects have also been demonstrated in the most common types of bone sarcoma, CS (8, 11), ES (12), and OS (10, 12, 15).

The aim of this study was to investigate the effect of CPP on cell motility in chondrosarcoma (CAL-78), Ewing’s sarcoma (A673), and osteosarcoma (U2-OS) cell lines. Since FCS was found to have a significant effect on cell proliferation, the identification of an optimal concentration of FCS was necessary for the correct performance of the migration assay.

Furthermore, applying CPP to CAL-78, A673, and U2-OS cell lines resulted in decreased cell viability, with Ewing’s sarcoma showing the most pronounced response. In the current study, the viability was measured after CPP treatment for 5 s, 10 s, and 20 s after 24 h. In their study on glioblastomas, Cheng et al. (35) suggested that CPP induces a reduction in cell viability after 72 h, particularly when applied for over 30 s. This aligns with previous studies on those cell lines (11, 12), which suggested that the anti-proliferative effects of CPP manifested after more than 24 h. Accordingly, we aimed to focus on examining the effects of CPP on cell motility by conducting our assessments after this time frame. A decrease in tumor cell motility was observed after application of CPP, with ES cells showing the most significant reduction. Although CS cells did not show a significant decrease in cell migration, our previous study showed that CPP did indeed reduce their motility (11). This aligns with the findings of several studies, which also suggested that CPP application negatively affects the migration rates of cancer cells (36-38). The reduction in cell migration is thought to be due to the modulation of the cytoskeleton, particularly of the actin fibers, that occurs in the cells after the application of CPP (20, 39). These modulations cause cancer cells to lose their polarization and undergo morphological changes, preventing them from migrating (40). The current study confirmed that CPP led to notable changes in the actin fibers in all three cell lines, with the most significant changes observed in U2-OS cells.

Conclusion

In conclusion, this study highlights the promising role of CPP in cancer treatment, especially in chondrosarcoma, Ewing’s sarcoma, and osteosarcoma. The mobility of bone sarcoma cells is effectively reduced by CPP treatment. This effect is likely due to changes in the actin cytoskeleton. The current study helps to understand how CPP affects the behavior of bone sarcoma cells and highlights the need for further research in this area to explore its clinical applications in cancer therapy.

Acknowledgements

The Authors acknowledge the support for the Article Processing Charge from the DFG (German Research Foundation, 393148499) and the Open Access Publication Fund of the University of Greifswald. A.N received the Domagk Master Class (DMC) scholarship funded by the University Medicine Greifswald. S.Q. received the Domagk scholarship funded by the University Medicine Greifswald.

Footnotes

  • Authors’ Contributions

    Conceptualization and methodology, L.H., A.N. and. P.M.; investigation and formal analysis, P.M, S.B., M.B. and S.Q.; software: P.M. and M.B. resources, G.I.W., S.B.; data curation, L.H. and G.I.W.; writing—original draft preparation, A.N., S.Q. and L.H. All Authors have read and agreed to the published version of the manuscript.

  • Conflicts of Interest

    All Authors declare no conflicts of interest in relation to this study.

  • Received February 22, 2024.
  • Revision received March 25, 2024.
  • Accepted April 8, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Cold Physical Plasma Reduces Motility of Various Bone Sarcoma Cells While Remodeling the Cytoskeleton
ANDREAS NITSCH, PAULINE MARTHALER, SARA QARQASH, MAXIMILIAN BEMMANN, SANDER BEKESCHUS, GEORGI I. WASSILEW, LYUBOMIR HARALAMBIEV
In Vivo Jul 2024, 38 (4) 1571-1578; DOI: 10.21873/invivo.13607
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