Fig. 2.

Budesonide reduces mesenchymal markers and PDAC cell migration and invasion. A Schematic representation of experimental procedure. PDAC#253 and #354 cells were plated (1.5 × 10⁴ cells/cm²) on gelatin-coated plates at day − 1. On day 0, cells were treated ± budesonide (20 µM) or DMSO (control). After 3 days, cells were dissociated and plated (2.5 × 10⁴ cells/well) on Boyden chambers or (1 × 10⁵ cells/cm²) on Cy3-conjugated gelatin. B Representative crystal violet images (left) and quantification (right) of PDAC#253 and #354 cells ± budesonide migrating through the transwell. Cell migration was quantified at 6 h (h) after seeding. Data are shown as fold-change vs. control (DMSO) and are mean ± SEM (*p < 0.05; n = 3, Student’s t-test). C Representative confocal images of ACTIN (green) staining (left) and quantification (right) of Cy3-gelatin degraded area in PDAC#253 and #354 cells ± budesonide. Invasion was quantified 6 h after seeding. Nuclei were counterstained with DAPI. Data are mean ± SEM (**p < 0.005, ***p < 0.001; n = 3, Student’s t-test) after normalization vs. the total number of nuclei. D Schematic representation of the effects of budesonide in 2D culture. Budesonide promotes epithelialization and reduces PDAC cell migration and invasion. E Schematic representation of 3D organotypic culture procedure. PDAC (#253 and #354) and PANC1 (1 × 10³ cells/cm²) cells were plated on a layer of 100% matrigel in complete medium containing 2% matrigel ± budesonide (20 µM) for 6 days. Medium was refreshed at day 3. F-G Representative phase-contrast images (F, left), frequency of cohesive vs. not cohesive structures (F, right) and quantification of the area (G) of PDAC (#253 and #354) spheroids ± budesonide at day 6 after plating. Data are mean ± SEM (F) or mean ± SD (G) (***p < 0.001; n = 4, Student’s t-test). H Representative images of PKH26-labeled (red) spheroids at day 6 post labeling derived from PDAC#253 and #354 cells treated ± budesonide (20 µM)