6. Functional testing after PEF-5 exposure
6.1. Clonogenic survival assay and IR combined treatment
To assess possible influence of PEF-5 on GBM clonogenic capacity, the survival clonogenic assay was performed on both first neurospheres derived from U87 ML (U87 NS1) and second generation neurospheres, derived from U87 NS cells (U87 NS2). As shown in Figure 5, exposure to PEF-5 efficiently decreased the clonogenic capacity in both neurosphere generations (about 2-fold lower than sham-exposed cells; p < 0.0001 in U87 ML and p < 0.001 in U87 NS). These results again suggested a strong ability of PEF-5 to modify the stem phenotype of CSCs decreasing their self-renewal capacity (Figure 5a-d). As radiosensitivity increases with cell differentiation status, researchers tested the effectiveness of a combined protocol, irradiating U87 ML and U87 NS cells with increasing IR doses (2, 5, 8 Gy), 3 hours after PEF-5 exposure, as suggested in the previous work. As shown in Figure 5a-d, the combined treatment of PEF-5 and IR was able to reduce clone formation as function of delivered doses, suggesting an additive effect between these two physical agents.
To assess possible influence of PEF-5 on GBM clonogenic capacity, the survival clonogenic assay was performed on both first neurospheres derived from U87 ML (U87 NS1) and second generation neurospheres, derived from U87 NS cells (U87 NS2). As shown in Figure 5, exposure to PEF-5 efficiently decreased the clonogenic capacity in both neurosphere generations (about 2-fold lower than sham-exposed cells; p<0.0001 in U87 ML and p<0.001 in U87 NS). These results again suggested a strong ability of PEF-5 to modify the stem phenotype of CSCs decreasing their self-renewal capacity (Figure 5 a-d). As radiosensitivity increases with cell differentiation status, we tested the effectiveness of a combined protocol, irradiating U87 ML and U87 NS cells with increasing IR doses (2, 5, 8 Gy), 3 hours after PEF-5 exposure, as suggested in our previous work [34]. As shown in Figure 5 a-d, the combined treatment of PEF-5 and IR was able to reduce clone formation as function of delivered doses, suggesting an additive effect between these two physical agents.
Figure 5. Survival clonogenic assay. (a-b) Representative images of primary and secondary neurospheres after PEF-5 exposure and IR treatments. (c-d) Quantitative analysis revealed a significant decrease in the clonogenic capacity between sham- and PEF-5 exposed cells, regardless of culture conditions. The combined treatment (PEF-5 + IR) reduced clone formation as function of delivered radiation doses.
Figure 5. Survival clonogenic assay. (a-b) Representative images of primary and secondary neurospheres after PEF-5 exposure and IR treatments. (c-d) Quantitative analysis revealed a significant decrease in the clonogenic capacity between sham- and PEF-5 exposed cells, regardless of culture conditions. The combined treatment (PEF-5 + IR) reduced clone formation as function of delivered radiation doses.
6.23. Invasion assay after PEF-5 exposure
As invasiveness is another pathophysiological feature of human malignant gliomas, the effects of PEF-5 on the invasiveness and migration of GBM cells were checked
in vitro by Transwell Matrigel invasion assay. As shown in Figure 6(a-d), the invasiveness of U87 ML and NS cells exposed to PEF-5 was strongly reduced compared with the control groups. U87 ML PEF-5 exposed cells showed a corresponding decrease of the invasion of 34% and U87 NS cells of 47%. This finding indicates the capacity of PEF-5 alone to inhibit the invasive ability of GBM cells
by Transwell Matrigel invasion assay. As shown in Figure 6 (a-d), the invasiveness of U87 ML and NS cells exposed to PEF-5 was strongly reduced compared with the control groups. U87 ML PEF-5 exposed cells showed a corresponding decrease of the invasion of 34% and U87 NS cells of 47%. This finding indicates the capacity of PEF-5 alone to inhibit the invasive ability of GBM cells
in vitro
. Also, for this assay, a combined protocol, PEF-5 + IR, was carried out using increasing IR doses (2, 5, 8 Gy) delivered 3 h after PEF-5 treatment. No further decrease in invasion ability was observed in only irradiated U87 ML samples (Figure 6c), instead, a decrease of 28% (
p = 0.006) and of 29% (
=0.006) and of 29% (
p = 0.009) was observed in U87 NS cells irradiated with 5 and 8 Gy, respectively (Figure 6d). This result may point out a migration response mediated by the cell types, and the specific delivered radiation dose. After the combined protocol of exposure, no significant changes were observed in migration and invasion abilities of these two cell types compared to those observed in the PEF-5 only treated groups. This result highlights the radio-resistance of GBM cells in both culture conditions. Notably, PEF-5 treatment alone was as effective as the combined treatment with radiations at all the delivered doses in both U87 ML (PEF vs 2Gy, -41%
=0.009) was observed in U87 NS cells irradiated with 5 and 8 Gy, respectively (Figure 6d). This result may point out a migration response mediated by the cell types, and the specific delivered radiation dose. After the combined protocol of exposure, no significant changes were observed in migration and invasion abilities of these two cell types compared to those observed in the PEF-5 only treated groups. This result highlights the radio-resistance of GBM cells in both culture conditions. Notably, PEF-5 treatment alone was as effective as the combined treatment with radiations at all the delivered doses in both U87 ML (PEF vs 2Gy, -41%
p = 0.04; PEF vs 5Gy, -32%
=0.04; PEF vs 5Gy, -32%
p
=0.0096; PEF vs 8Gy, -25%
p
=0.016), and U87 NS cells (PEF vs 2Gy, -41%
p
=0.04).
Figure 6. Invasion and migration assay. Representative images of transmigrated cells for (a) U87 ML and (b) U87 NS cells. (c-d) Percentage of invasiveness in U87 ML and NS cells evaluated 24 hours after PEF-5 exposure and IR combined treatment.
7. Conclusions
Our data set was obtained by analyzing different endpoints, i.e., cell viability, cell permeabilization, ROS generation, cell cycle perturbation, as well as functional testing. The evidence suggests that PEF-5 treatment on homogeneous and more heterogeneous U87 GBM cell populations has a significant effect. PEF-5 exposure inhibited clonogenic and invasion potentials in both U87 ML and NS cells, reducing the ability to form new neurospheres and transmigrate in
= 0.04).
vitro. This exposure substantially influenced CSCs fate and specifically affected their proliferation, by differentially regulating cell cycle checkpoints by ROS signal transduction processes. This physical stimulus is easily modifiable in terms of the number of pulses delivered, the degree of intensity and repetition frequency, and this technique could be adapted to target specific tumor cells to optimize personalized therapy, alongside conventional oncologic therapies. PEF therefore represents a promising therapeutic approach to pre-treat CSCs and cancer cells, reducing radio or chemotherapy doses and helping to prevent tumor relapse.
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Figure 6. Invasion and migration assay. Representative images of transmigrated cells for (a) U87 ML and (b) U87 NS cells. (c-d) Percentage of invasiveness in U87 ML and NS cells evaluated 24 hours after PEF-5 exposure and IR combined treatment.
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