Figure 3.

CR-CSCs #21 response to MHT alone (TR-Cubes) or combined with chemotherapy (TR-DOXO). (a) Schematic representation of the experimental protocols followed to study the effect of TR-Cubes and TR-DOXO upon exposure to MHT on CR tumor cells containing quiescent PKH^pos qCR-CSCs. The first step consists of identifying the qCR-CSCs followed by the use of TR-Cubes or TR-DOXO for MHT (f = 182 kHz with a fixed temperature of 45 °C) and finally reseeding the cells in culture to evaluate their response to the treatment in vitro. (b) Optical microscope representative pictures of control cells, cells exposed to MHT for 90 min (three cycles of 30 min), and cells treated with only DOXO 1 and 16 days post-treatment showing no spheroid formation for the cells treated with TR-DOXO. Scale bars: 200 μm. Viability profiles, analyzed by trypan blue assay, of the CR-CSCs treated with TR-Cubes (green bars) or TR-DOXO (red bars) after different exposure times of MHT: (c) 30 min, (d) two cycles of 30 min (2 × 30 min) for a maximum duration of 60 min, and (e) three cycles of 30 min (3 × 30 min) for a maximum duration of 90 min. The blue bars indicate the control untreated cells. (f) Viability profiles, analyzed by trypan blue assay, of the CR-CSCs treated with TR-DOXO in the presence (red bars) or absence (black bars) of MHT. Values are presented as mean with error bars indicating the standard deviation (SD) for n = 3 independent experiments. Statistical analysis was performed using ANOVA with a Tukey post hoc test. *p < 0.05, * *p < 0.01, ***p < 0.001, and *** *p < 0.0001.