Fig. 5

Hybrid spheroids of breast cancer cells and ln-aT/ob-aT bASCs demonstrate an increased resistance to chemotherapeutic agents. A-C BT474 spheroids (A), BT474/ln-aT bASCs (B) and BT474/ob-aT bASCs (C) hybrid spheroids, generated for 24 h, were transferred into 96-well low attachment plates, treated with DMSO, TMX (5 μM) or DTX (50 nM) for up to 96 h. Cell viability was measured at indicated time points. The results are based on three independent experiments and presented as mean ± SEM. D-G Indicated spheroids were treated with 50 nM DTX for 72 h, stained for DNA damage markers γ-H2AX (red) and 53BP1 (green), and DNA (DAPI, blue). G Representatives are shown. White arrows indicate cell nuclei with 20 ≥ foci. Scale: 25 μm. D-F Quantification of γ-H2AX and 53BP1 double positive cells (D, n = 15 fields in each group), cells with 20 ≥ foci (E, n = 15 fields in each group) and double positive foci per cell (F, n = 90 cells in each group). H-L BT474- and hybrid spheroids were stained using the live/dead viability/cytotoxicity kit. Spheroid surface area (H and I, n = 7–11 fields in each group), and the ratio between the fluorescence intensity of viable cells (calcein/green) and the dead fraction (PI/red) (J and K, n = 7–12 fields in each group) were evaluated. The results are based on three independent experiments and presented as mean ± SEM. L Representative images of spheroids treated with DTX (50 nM) for 72 h are shown. White dotted lines depict the measured area of the spheroids. Scale: 250 μm. Student’s t test was used in (A-C). Unpaired Mann-Whitney U test was used in (D-F) and (H-K). *p < 0.05, **p < 0.01, ***p < 0.001