Abstract
Background/Aim: The antiproliferative effects of cold atmospheric plasma (CAP) make it a promising application option in oncology. The aim of the present study was to examine whether short-term CAP treatment leads to an initial partial elimination of the treated cells or to long-term impairement and inhibition of cell growth. Materials and Methods: Cells were treated with CAP and biostatistical modelling was used to estimate growth rates over the incubation time. Four cell lines (U2-OS and MNNG osteosarcoma cells, 3T3 fibroblasts, HaCaT keratinocytes) and three CAP sources (MiniJet-R, kINPen MED, Maxium) were used. Results: The antiproliferative efficacy of CAP was due to a significant reduction in cell count during treatment and the long-lasting inhibition of growth rate in the remaining cells, detectable in all cell lines and after treatment using all three CAP devices. Conclusion: Induction of cell death and inhibition of cell growth are part of a general mechanism of biological CAP efficacy. However, data contradict the hypothesis that cancer cells respond more sensitively to CAP treatment compared to non-malignant cells.
Pulsed physical plasma under atmospheric pressure (cold atmospheric plasma: CAP) is already routinely used in dermatology and increasingly in other medical disciplines (1). The application in oncological therapy is regarded as promising. In particular the anti-proliferative, pro-apoptotic, anti-angiogenetic and cytoskeleton-stabilizing properties of CAP components including the electromagnetic radiation lead to the inactivation of cancer cells (2-4). In in vitro studies, the principle anti-cancer efficacy of CAP has already been demonstrated in various tumor entities including prostate cancer, ovarian cancer, osteosarcoma, and pancreatic cancer (5-8). The studies demonstrated a direct antiproliferative effect, but also an indirect effect when untreated cells were incubated with CAP treated medium. At the cellular level, CAP induces a redox response of the cells that involves the peroxiredoxin system, and finally induces programmed cell death.
During chemotherapy, biologically active molecules circulate in the body after the first administration of an active substance. They are thus present in the tumor for a longer period, albeit at variable concentrations and under possible enzymatic transformation, and can exert their activity (9). This is not the case with CAP treatment. Similar to radiation treatment, CAP only acts on the treated tissue for a short period of a few seconds to minutes. Nevertheless, this is sufficient to develop long-lasting anti-cancer effects that remain detectable for days to weeks.
With regard to the antiproliferative effect of CAP, two possible mechanisms of action are conceivable: (i) CAP treatment eliminates some of the cells, and therefore the cells show a reduced total growth rate compared to untreated control cells. (ii) CAP treatment could also permanently disrupt cellular mechanisms, e.g. specific metabolic processes, sustainably reducing the growth rate of each cell.
Technical parameters of CAP sources. Manufacturer, carrier gas, flow rate, power and frequency of high-frequency (HF) generators of CAP devices MiniJet, kINPen, and Maxium.
The aim of this analysis was to differentiate whether CAP treatment leads to an initial elimination and/or a persistent physiological impairment of the cells. For this purpose the growth curves of CAP treated cells were measured and biostatistically evaluated. In order to provide a general a statement, four different cell lines were included in the investigations, each of which was treated with three different CAP devices for three different treatment times.
Materials and Methods
Cell culture. The human osteosarcoma cell lines U2-OS and MNNG (both from the American Type Culture Collection, Manassas, VA, USA) 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 from PAN Biotech, Aidenbach, Germany). Murine 3T3 fibroblasts (American Type Culture Collection) were propagated in DMEM medium containing 4.5 g/l glucose, 10% fetal bovine serum, 1 mM sodium pyruvate, and 1% penicillin/streptomycin (all from PAN Biotech). Human keratinocytes HaCaT (German Cancer Research Center, Heidelberg, Germany) were cultured in DMEM supplemented with 2 mM glutamine, 1% penicillin/streptomycin, and 8% fetal bovine serum. After 3 to 4 days, cells were washed twice in phosphate-buffered saline, detached using 0.1% trypsin/0.04% ethylendiamine tetra-acetic acid (PAN Biotech), resuspended in cell culture medium, and seeded in new cell culture flasks. All cells were propagated in humidified atmosphere at 37°C and 5% CO2.
CAP treatment. The devices MiniJet-R (MiniJet, Heuermann HF-Technik, Aachen, Germany), kINPen MED (kINPen, neoplas tools, Greifswald, Germany) and Maxium with beam electrode (Maxium, KLS Martin, Tuttlingen, Germany) were used for CAP treatment. All three CAP devices were operated with argon as carrier gas. For experiments, 200 μl cell suspension with 2.0×104 (U2-OS, MNNG) and 1.0×104 (3T3, HaCaT) cells were treated for 5 s, 10 s, and 30 s in a 24-well cell culture plate (Sarstedt, Nümbrecht, Germany). During treatment, the handpiece of the CAP devices was guided over the cell suspension so that the CAP flame was in contact with the surface of the medium and all cells were treated equally. The feed gas pressure of the plasma sources ensured mild vortexing of the cell suspension to propagate the plasma effect evenly. The instrument specifications are given in Table I. After treatment, cells were seeded in 24-well cell culture plates (Sarstedt) with 800 μl medium for subsequent proliferation analysis.
Growth kinetics using the cell line U2-OS as an example. U2-OS cells were treated for 30 s with the CAP device MiniJet, seeded, and cell numbers were determined over 120 h. B Logarithmic plot of the same growth curve for the calculation of the initial cell number at t=0 (intersection with the Y-axis) and the growth rate (slope of the graphs).
Cell growth assay. To determine the initial cell number at time t=0 and the growth rate, cell numbers were measured over 120 h using a CASY Cell Counter and Analyzer Model TT (Roche Applied Science, Mannheim, Germany). For the measurement, cells were suspended as described in the cell culture section, diluted 1:100 with CASYton (Roche Applied Science), and 400 μl of this dilution was measured in triplicates. Cell number determination was performed using a capillary with 150 μm diameter and the following cell line-specific gate settings were used to discriminate between viable cells, dead cells, and cellular debris: 7.20 μm/13.95 μm (U2-OS, MNNG), 7.17 μm/11.90 μm (3T3), and 6.60 μm/10.95 μm (HaCaT).
Biostatistical modelling and statistical analysis. Statistical analysis was performed in R version 3.5 (R foundation for statistical computing, http://www.r-project.org). Data were log-transformed and a linear model fitted to the growth curves, corresponding to an exponential growth model on the untransformed data. Slope and intercept were subsequently compared across conditions using Student's t-test. Resulting p-values p≤0.05 (*), p≤0.01 (**), and p≤0.001 (***) were considered significant. Data are shown as mean±SD of at least four independent experiments. The doubling time was calculated according to the formula ln2/growth rate.
Growth rates of all four cell lines after treatment with different CAP devices. U2-OS (A), MNNG (B), 3T3 (C), and HaCaT (D) cells were treated with the CAP sources MiniJet, kINPen, and Maxium for 5s, 10s, and 30s, respectively, and growth rates were compared with those of the corresponding untreated control cells. Results are expressed as mean±SD values. Differences of model slopes and intercepts were assessed using Student's t-test (*p<0.05, **p<0.01, ***p<0.001).
Results
Growth kinetics of CAP-treated cells were analyzed to differentiate between CAP-dependent influences on cell number and the growth behavior of surviving cells. Four cell lines were selected for this purpose: The two osteosarcoma cell lines U2-OS and MNNG, as tumor cells, as well as the keratinocyte cell line HaCaT and the fibroblastic cell line 3T3, as non-malignant cells.
CAP treatment was performed with the devices MiniJet, kINPen, and Maxium for 5 s, 10 s, and 30 s. Subsequently cells were incubated and cell counts examined after 4 h, 24 h, 48 h, 72 h, 96 h, and 120 h. As shown in Figure 1, as an example, (cell line U2-OS; 30 s treatment; MiniJet) cell numbers of at least 4 independent runs were plotted over time (Figure 1A). After log-transformation of the data sets (Figure 1B), the growth rate was determined as the slope of the graph. Furthermore, the cell number at time t=0, which would correspond to the direct cell number reduction immediately after CAP treatment, was calculated.
Analysis of the tumor cells U2-OS (Figure 2A) showed a significant reduction of their growth rate after 10 s, with the lowest after 30 s CAP treatment with the 3 CAP devices compared to the controls. For comparison purposes, the x-fold changes in growth rates are given in Table II. These results were confirmed by studies with the cancer cell line MNNG (Figure 2B). Although both cell lines have human osteosarcoma origin, a slightly stronger effect on the cellular growth rate was observed in MNNG cells. In non-malignant cells, the reduction in growth rate was even more pronounced. Both the fibroblastic 3T3 cells and the keratinocytes HaCaT showed in part considerably stronger differences between control and CAP treated cells (Figure 2C and D).
Growth rate changes of U2-OS, MNNG, 3T3, and HaCaT cells treated with the CAP devices MiniJet, kINPen, and Maxium.
Analysis of the calculated doubling times clarified the growth inhibitory effect of the CAP devices (Table III). With increasing treatment time, the doubling time increased significantly, whereby the 30 s treatment with all 3 devices resulted in a pronounced extension of the doubling time. In contrast to treatment times, the effect of the different devices on all cell lines was quite similar. Remarkably, the cell number of the non-malignant fibroblasts and keratinocytes doubled significantly faster than that of malignant osteosarcoma cells.
As a further parameter influencing kinetics of cell growth, the extent to which CAP treatment led to an immediate reduction of the initial cell number during the treatment procedure was investigated (x-fold changes in the initial cell number see Table IV). Again, there were significant effects with all CAP devices and all cell lines that were increased with extended treatment times. The two cancer cell lines U2-OS and MNNG responded similarly to treatment with CAP sources (Figure 3A and B). Also the two non-malignant cell lines 3T3 and HaCaT exhibited approximately the same significant reduction in the initial cell numbers due to the influence of CAP treatment (Figure 3C and D).
Discussion
In all four cell lines and after treatment with all three CAP devices, the anti-proliferative effects increased with prolonged treatment time. The biostatistical analysis of the growth kinetics showed that both the initial cell numbers at time t=0 and the growth rates of the cell populations were reduced by CAP treatment. Thus, both rapid cell death during CAP treatment and long-term inhibition of the growth of the surviving cells seem to contribute to reduced cell proliferation.
Doubling rates of U2-OS, MNNG, 3T3, and HaCaT cells treatet with the CAP devices MiniJet, kINPen, and Maxium.
Initial cell number changes of U2-OS, MNNG, 3T3, and HaCaT cells treated with the CAP devices MiniJet, kINPen, and Maxium.
The impact of CAP on the physiology of cells and in particular on cell growth has been known for over a decade (10-12). The antibacterial effect in particular has long been used for sterilization of sensitive materials, but also in the treatment of skin diseases (13, 14). Eukaryotic cells can also be inactivated by CAP treatment, which could be particularly beneficial in oncological therapy. The effect on eukaryotic cells has been attributed to CAP-induced changes in membrane permeability (15, 16). This leads, among others, to metabolic dyshomeostasis, which is one of the reasons for the long-lasting effect of CAP treatment. The direct reduction of the cell number during the treatment itself has not yet been described. Reactive species probably play a decisive role in this process, and depending on their composition and local concentration, they may also have toxic effects.
Initial cell number of all four cell lines after treatment with different CAP devices. U2-OS (A), MNNG (B), 3T3 (C), and HaCaT (D) cells were treated with the CAP sources MiniJet, kINPen, and Maxium for 5s, 10s, and 30s, respectively, and growth rates were compared with the corresponding untreated control cells. Results are expressed as mean±SD values. Differences in model slopes and intercepts were assessed using Student's t-test (*p<0.05, **p<0.01, ***p<0.001).
In addition, it could be shown that the surviving cells showed significantly reduced growth rates at least after the longer CAP treatment times of 10 s and 30 s up to 120 h. The cellular causes for this could be manifold (e.g. changes in membrane functionality, inhibition of metabolic processes, persistent redox stress) and require functional analyses. Since only living cells were measured during cell count determination, it is also conceivable that prolonged CAP-induced apoptosis leads to the ongoing death of individual cells, which would reduce the cell count over time. The induction of apoptotic processes by CAP has been extensively described (5, 17, 18). Interestingly, the presented data also suggests a CAP effect, which is of major significance for wound healing. Short treatment times with CAP (‘low-level treatment’) have stimulating and proliferative effects, as can be seen with U2-OS (5s MiniJet, 5s kINPen), MNNG (5s MiniJet), and HaCaT (5s Minijet). This effect has been reported before in endothelial cells, monocytes and also in keratinocytes (19-21) and is not desired in the clinical setup of cancer therapy. However, since the stimulation of cell growth could no longer be observed in any cell line or CAP device using treatment times above 10 s, this effect is unlikely to play a negative role in oncological practice.
Both antiproliferative effects, reduction of the initial cell count and inhibition of the growth rate, were quite differently in the different cell lines. While the non-malignant 3T3 and HaCaT cells responded very strongly to CAP treatment, the malignant U2-OS and MNNG cells showed significantly lower sensitivity to CAP treatment. These data contradict the hypothesis that cancer cells respond more sensitively to CAP treatment and that CAP therapy therefore has a specific effect on tumor tissue and hardly any on adjacent non-malignant tissue. This assumption was primarily based on the assumption of severe growth-related (oxidative) stress in tumor cells, which can lead to cell death due to additional redox-active noxae of CAP (22-24). The present analysis cannot confirm this; conversely, tumor cells appear to be more resistant to the effects of CAP treatment than non-malignant cells. The CAP effect has also been shown in other studies (10, 21, 25). However, it has to be said that the comparison of different cell lines is only possible to a very limited extent. To answer this question, it would be better to analyse malignant and non-malignant primary tissue or cells.
In addition to cell type-specific differences, device-specific differences in cell response to CAP exposure were also observed. In most cases the effect after MiniJet treatment was not as pronounced as for the other two CAP sources. This is particularly evident in the MiniJet treatments over 5 s, which showed almost no significant differences compared to the control-treated cells. These observations are certainly an indication that the composition and thus also the biological effect of CAP is largely dictated by the technical properties of the CAP source. Since environmental parameters (e.g. humidity, temperature) also affect the formation of CAP (26, 27), it is complicated to compare different CAP devices in various studies concerning their anti-proliferative effects.
However, such comparisons must also be carried out with caution for experimental reasons. In most cases, parameters such as physiological capacity (e.g. MTT assay) and stainability of cells (e.g. DAPI staining) are determined for cell growth. Changes in signal intensity are then used as indirect indication of a change in biomass. However, it must be considered that the treatment itself can also lead to changes in cell physiology, and changes in the measured parameters are then at least partially not accompanied by changes in biomass. Although in our study cell growth was measured directly by determining the live cell count, which resulted in a high reproducibility of the measurements, in vitro data with cell lines must not be overinterpreted. However, this reproducibility was a prerequisite for the biostatistical analysis of the data. The heterogeneity of cell systems can already be seen in the two tumor cell lines U2-OS and MNNG. U2-OS cells reacted less sensitively to CAP treatments than MNNG, although both lines originate from osteosarcoma. The comparison between malignant and non-malignant cells has to be questioned even more critically, since fibroblasts (3T3) and epithelial keratinocytes (HaCaT) comprise very different cell types in this group.
Conclusion
The antiproliferative effect of CAP is based on the combination of a rapid reduction of the initial cell number with an inhibition of the growth rate of the remaining living cells. These effects were observed applying three CAP devices from different manufacturers as well as using different cell types of both malignant and non-malignant origin. Consequently, this appears to be a general mechanism of biological CAP efficacy. Nevertheless, we observed apparent sensitivity differences in the response of different cell types to CAP exposure as well as a dependence of the effect on the duration of treatment and the CAP source.
Acknowledgements
AB and LK acknowledge funding from the European Social Fund ESF via the Excellence Program of the State of Mecklenburg-Vorpommern (KoInfekt, ESF/14-BM-A55-0014/16). SB acknowledges funding from the German Federal Ministry of Education and Research (BMBF), grant number 03Z22DN11.
Footnotes
Authors' Contributions
Conceptualization, A.M, L.K. and M.B.S.; methodology, L.H. and A.B.; software, A.B. and L.K.; formal analysis, A.B., L.K. and M.B.S.; investigation, L.H., A.B., B.S, M.W. A.K., S.B.; writing – original draft preparation, L.H., S.B. and M.B.S.; writing – review and editing, A.K., A.E. and M.B.S.; visualization, L.K. and M.B.S.; supervision L.K. and M.B.S.; funding acquisition, A.B., S.B. and L.K.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received May 26, 2020.
- Revision received June 15, 2020.
- Accepted June 16, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
