OCA impairs cell migration of mixed iCCA cells.

a) Wound healing assay of mixed iCCA. A scratch was created on the confluent cell mono-layers and cells were monitored at 24 h intervals for 7 days. Cell migration was evaluated by calculating the percentage of covered area (% closure rate) at different time points vs time 0. Mixed control iCCA cells started to cover scratches after 2 days, and the wound healing was completed after 10 days. In contrast, in OCA (0.5 μM) treated cells, the scratches remained virtually uncovered until 7 days, indicating that migration of Mixed iCCA cells was almost completely impaired by OCA. Photographs (4x magnification) are representative of N = 6 independent experiments in which cells are highlighted in green and the cell-free area in grey. Data represent means ± SD of N = 6 independent experiments. ** p < 0.01 vs controls without OCA (0 μM OCA). b) In mucinous iCCA cells, in contrast, OCA showed no significant effect on cell migration; a slight inhibitory effect was seen only at days 3 and 4. Photographs (4x magnification) are representative of N = 6 independent experiments. Data represent means ± SD. * p < 0.05 vs controls without OCA (0 μM OCA). c) Cell migration was also evaluated by the Matrigel invasion assay, where the ability of tumor cells to migrate through the synthetic extracellular matrix was measured. After 96 h of culture, cells migrated through Matrigel were fixed with 0.1% crystal violet and their number reported as percentage respect to controls considered equal to 100 (see bar graph). OCA (0.5 μM for 4 days) impaired the ability to invade the Matrigel mainly in mixed iCCA and with minor effects in mucinous iCCA cells. 10x photographs were representative of N = 6 independent experiments. Means ± SD of N 6 independent experiments. ** p < 0.01 vs controls without OCA (0 μM OCA); * p < 0.05 vs controls without OCA (0 μM OCA).