Figure 5.

TR-DOXO dual-therapy effect on qCR-CSCs #21. (a) Confocal microscope analysis of CR-CSCs treated with TR-DOXO and exposed to MHT (90 min; three cycles of 30 min). PKH^pos cell (green signal), Sytox blue (blue signal) for dead cells, and uptake of DOXO (red signal). The colocalization of green, blue, and red signals at day 5 confirms the death of quiescent PKH^pos cells that have also internalized the released DOXO following MHT. (b) Spheroid growth monitoring after MHT (f = 182 kHz reaching a fixed temperature of 45 °C for 90 min; three cycles of 30 min) using TR-DOXO compared to control cells. The lack of spheroid formation on the figures on the right panel, at day 5, confirms the dual cytotoxic effects of the treatment. All scale bars are 50 μm. (c) Estimation of PKH^pos cell death based on confocal images acquired using Sytox blue for 4 weeks after MHT and TR-DOXO administration. (d) Estimation of PKH^pos cell death based on confocal images acquired using Sytox blue for 4 weeks after treatment with DOXO. For panels (c) and (d), at least 500 PKH^pos cells were counted per each time point and per each experimental condition in n = 3 independent experiments. The mean data are reported, and error bars represent SD. Statistical analysis was done using ANOVA followed by Dunn’s multiple comparison test. The statistical difference ***p < 0.001 was calculated for each time point.