Reduction of Human Glioblastoma Spheroids Using Cold Atmospheric Plasma: The Combined Effect of Short- and Long-Lived Reactive Species

Abstract

Cold atmospheric plasma (CAP) is a promising technology against multiple types of cancer. However, the current findings on the effect of CAP on two-dimensional glioblastoma cultures do not consider the role of the tumour microenvironment. The aim of this study was to determine the ability of CAP to reduce and control glioblastoma spheroid tumours in vitro. Three-dimensional glioblastoma spheroid tumours (U87-Red, U251-Red) were consecutively treated directly and indirectly with a CAP using dry He, He + 5% H₂O or He + 20% H₂O. The cytotoxicity and spheroid shrinkage were monitored using live imaging. The reactive oxygen and nitrogen species produced in phosphate buffered saline (PBS) were measured by electron paramagnetic resonance (EPR) and colourimetry. Cell migration was also assessed. Our results demonstrate that consecutive CAP treatments (He + 20% H₂O) substantially shrank U87-Red spheroids and to a lesser degree, U251-Red spheroids. The cytotoxic effect was due to the short- and long-lived species delivered by CAP: they inhibited spheroid growth, reduced cell migration and decreased proliferation in CAP-treated spheroids. Direct treatments were more effective than indirect treatments, suggesting the importance of CAP-generated, short-lived species for the growth inhibition and cell cytotoxicity of solid glioblastoma tumours. We concluded that CAP treatment can effectively reduce glioblastoma tumour size and restrict cell migration, thus demonstrating the potential of CAP therapies for glioblastoma.

Keywords: cancer; cell migration; cold atmospheric plasma (CAP); cytotoxicity; glioblastoma; proliferation; short-lived reactive species; spheroid shrinkage; tumour reduction.

Figure 1

Consecutive plasma treatment inhibited growth in glioblastoma spheroids. (a) The area of the spheroid core (viable cells) was reduced by 2× direct plasma treatment generated with dry He, He + 5% H₂O or He + 20% H₂O vapour saturation. The most effective inhibition of growth in both U87-Red and U251-Red was achieved using plasma generated with He + 20% H₂O (p ≤ 0.0001). (b) Similar values of the total spheroid area (comprising living spheroid core and dead cells) were obtained for all the conditions in both cell lines, except for U87-Red, He + 20% H₂O, where the total spheroid area was smaller. (c) The addition of H₂O to the gas feed resulted in increased cytotoxicity for both cell lines. Cell death is expressed as the ratio of Cytotox Green⁺ cells in treated spheroids/untreated controls at each time point (a.u.). Dead cells confluence = confluence percentage of the image area occupied by dead cells. Results corresponding to U87-Red (left) and U251-Red spheroids (right). Data representative of two independent experiments, 4–6 spheroids per condition. Mean ± SD; ** = p ≤ 0.01; **** = p ≤ 0.0001.

Figure 2

Spheroid growth/shrinkage upon the different treatments applied. Representative images of spheroid growth/shrinkage and cytotoxic effect in U87-Red spheroids on day 1 and 7 days after exposure to the different treatments tested. Living cells in red, Cytotox Green⁺ cells in green. Scale bar = 300 µm.

Figure 3

Short- and long-lived RONS present in plasma-treated PBS. (a) H₂O₂; (b) DMPO-OH and DEPMPO-OOH adducts; (c) NO₂⁻, NO₃⁻ and NO₂⁻ + NO₃⁻ were detected in plasma-treated PBS by EPR and colourimetry (see Mat. and Met.). Experiments were carried out in 200 μL pPBS, using dry He, He + 5% or 20% H₂O vapour saturation. Data represent mean values (n ≥ 2).

Figure 4

Short- and long-lived species delivered by plasma are needed to inhibit spheroid growth. (a) A statistically significant difference was observed in the area of the spheroid core (viable cells) in spheroids exposed to 2× direct plasma treatment compared to those treated with pPBS (indirect) and the RONS mix (U87-Red, p ≤ 0.0001; U251-Red, p ≤ 0.001). (b) U87-Red spheroids treated with pPBS and the RONS mix showed higher total spheroid area than spheroids treated 2× directly with plasma (p ≤ 0.0001). In U251-Red spheroids, higher total spheroid area was observed only in spheroids treated with the RONS mix. (c) The amount of cell death induced in spheroids by pPBS and RONS mix were comparable. Cell death is expressed as the ratio of Cytotox Green⁺ cells in treated spheroids/untreated controls at each time point (a.u.). Dead cells confluence = confluence percentage of the image area occupied by dead cells. Results corresponding to U87-Red (left) and U251-Red (right) spheroids. Data representative of two independent experiments, 4–6 spheroids per condition. Mean ± SD; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.

Figure 5

The migratory ability of U87-Red and U251-Red cells is affected by the 2× direct plasma treatment. The 2× direct plasma treatment inhibited cell migration for (a) up to 48 h in U87-Red and (b) up to 72 h in U251-Red. 2× pPBS and RONS mix had a significant but less inhibitory effect, reaching similar migration areas than the untreated PBS controls after 48 h (U87-Red) and 72 h (U251-Red). (c) Representative images of U87-Red and U251-Red cell migration (0 and 48 h after consecutive treatments). % Migration area = 100*(area T_x-T₀/area T₀). Data representative of two independent experiments, 4–6 spheroids per condition. Mean ± SD; *** = p ≤ 0.001; **** = p ≤ 0.0001. Scale bar = 1000 µm.

Figure 6

The 2× direct plasma treatments reduced the expression of the proliferative marker ki67 in glioblastoma spheroids. The 2× direct plasma treatment decreased the % ki67⁺ cells in (a) U87-Red spheroids (p < 0.05) and was lower than in those untreated or treated with pPBS and RONS mix (p < 0.05). (b) The same trend was observed in U251-Red spheroids (p > 0.05). The staining was scored using IHC Profiler in ImageJ. (c) Representative images of ki67 staining of U87-Red and U251-Red spheroids exposed to untreated PBS or He + 20% H₂O direct treatment. Enlarged image demonstrates variable ki67 staining. No ki67 staining control on the right. Data representative of two independent experiments, 4–6 spheroids per condition. Mean ± SD; * = p < 0.05. Scale bar = 500 µm.