Fig. 3

Domatinostat affects PDAC stem cells by modulating oxidative stress. A The effect of domatinostat (0,5 μM) on PANC1 and ASPC1 spheroid cultures. Cells (1000/mL) seeded in a matrigel drop and sphere medium, were treated with and without domatinostat and collected 7 days after treatment. Images of one spheroid for each condition in a representative experiment is shown (white scale bar: 50 μm, magnification 20X). On the right, bar graphs show the numbers of spheroids for well (mean ±} SD of 2 or more separate experiments each one with technical triplicate). B PANC1 and ASPC1 spheroids viability treated with and without domatinostat (0.5 μM and 1 μM) was assessed by cell titer luminescence assay (see Materials and Methods) (mean ±} SD of 2 or more separate experiments each one with technical triplicate). C Flow cytometry assay shows CD133 protein expression decrease after domatinostat (0.5 and 1 μM) treatment for 16 h in PANC1 and ASPC1 cells. D qRT-PCR analysis shows Oct-4 levels drop when PANC1, and ASPC1 spheroids are treated with domatinostat (0.5 μM) for 16 h. E Cellular ROS production is visualized by Hydroethidine (HE) staining. PANC1 and ASPC1 spheroids were treated with domatinostat (0.5 μM) alone and in combination with N-acetylcysteine (NAC, 5 mM), as ROS scavenger, at the indicated timing. Cells were stained for HE as described in Material and Methods section and visualized by flow cytometry. F Mitochondrial ROS amount is analyzed by mitosox staining. PANC1 and ASPC1 spheroids treated with or without domatinostat (0.5 μM) alone at the indicated timing were fixed, stained for mitosox (red) and measured by Opera Phenix confocal microscopy. The mitosox positive cells are counted by Harmony software as described in Material and Methods section. Representative images (20X magnification) show stained cells (red) and mitosox counted positive cells (green). G-H The observed increase in ROS amount is related to an increase of apoptotic cancer stem cells upon domatinostat treatment. PANC1 and ASPC1 spheroids, treated as previously, were stained for AnnexinV-FITC and CD133-APC as described in Material and Methods section and visualized by flow cytometry. In G. PANC1 and ASPC1 spheroids were treated with domatinostat (0.5 μM) alone and in combination with NAC, 5 mM, as ROS scavenger, at the indicated timing. In H PANC1 and ASPC1 spheroids were treated with domatinostat (0.5 μM) alone and in combination with Mitoquinone mesylate (MitoQ) 100 nM, as mitochondrial ROS scavenger, at the indicated timing. (Statistically significant results by ANOVA test are reported *** indicates P < 0.0001, ** indicates P < 0.005 and * indicates P < 0.05)