Fig. 5
Budesonide-dependent reduction of PDAC spheroid volume is Glucocorticoid Receptor (GR)-dependent. A-B Representative pictures (left) and volume quantification (right) of spheroids from PDAC#253 A and #354 B cells ± budesonide (bude), dexamethasone (dexa) or hydrocortisone (hydro) at 1 µM. DMSO was used as a control. Data are mean ± SD (***p < 0.001; n = 3, Student’s t-test). C Schematic representation of experimental procedure. NT (control/ShEmpty) and NR3C1 KD PDAC#253 cells were plated (1.5 × 104 cells/cm2) on gelatin-coated plates at day − 1. On day 0, cells were treated ± budesonide (20 µM). After 3 days in culture, cells were dissociated and plated (1 × 105 cells/cm2) on Cy3-gelatin. D Representative confocal images (left) of ACTIN staining (green) in NT and NR3C1 KD PDAC#253 cells ± budesonide (20 µM) and quantification (right) of Cy3-gelatin degraded area. Nuclei were counterstained with DAPI. Data are mean ± SEM (*p < 0.05, n = 3, Student’s t-test), after normalization vs. the total number of nuclei. E Schematic representation of the experimental design. Control (NT) and NR3C1 KD PDAC#253 cells were seeded in ultra-low attachment plates (5 × 102 cells/well) and treated ± budesonide, dexamethasone, hydrocortisone (1 µM) or DMSO for 5 days. F Representative pictures (left) and volume quantification (right) of spheroids generated from control and NR3C1 KD PDAC#253 cells treated with budesonide (bude), dexamethasone (dexa), hydrocortisone (hydro) at 1 µM or DMSO as control. Data are mean ± SD (*p < 0.05; ***p < 0.001; n = 3, Student’s t-test). G Time course analysis of NT and NR3C1 KD PDAC#253 cell proliferation in 3D spheroids ± budesonide (left; bude: 1 µM), and in 2D cultures ± budesonide (right; bude: 20 µM). Data are mean ± SD (*p < 0.05; n = 3, Student’s t-test). H Schematic representation of the GR-independent and -dependent effects of budesonide. In 2D cultures, budesonide (> 2.5 µM) promotes epithelialization and reduces PDAC cell migration independently from the GR. In PDAC spheroids (3D), nanomolar concentrations of budesonide (≤ 10− 2µM) exert a GR-dependent anti-proliferative effect