Fig. 3
IL-10-stimulated macrophages are more efficient in stimulating cancer cell motility and migration. a Representative images of actin and tubulin stainings of AGS cells incubated, during 6 h, with RPMI (AGS + RPMI), LPS- (AGS + CM(LPSmac)) or IL-10-stimulated macrophage conditioned medium (AGS + CM(IL-10mac)). F-actin was stained with Phalloidin-FITC (green), α-tubulin with a specific monoclonal antibody following incubation with an AlexaFluor594 secondary antibody (red) while nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm. b Quantification of motility-associated actin/tubulin structures on AGS cells treated for 6 h with RPMI (-) or CM from LPS- (+CM(LPSmac)) and IL-10- (+CM(IL-10mac)) stimulated macrophages. % of cells with filopodia, lamellipodia or stress fibers was calculated relatively to total cell number on images of F-actin/α-tubulin staining. Bars represent mean values obtained with at least 100 cells in independent experiments with CM of macrophages obained from at least 3 different blood donors; flags indicate standard error mean. *, significantly different at p < 0.05. c Representative images of AGS cell trajectories followed for 13 h, using timelapse microscopy. Cells were incubated in the presence of RPMI (AGS + RPMI) or CM from LPS- (AGS + CM(LPSmac)) or IL-10-stimulated macrophages (AGS + CM(IL-10mac)). Trajectories are represented as white lines traced between initial, intermediate and final xy positions. Scale bar represents 50 μm. d Distance (μm) travelled by AGS cells in the presence of RPMI (AGS) or CM from LPS- (CM(LPSmac)) or IL-10-stimulated macrophages (CM(IL-10mac)) was quantified using the LSMib Zeiss software (Carl Zeiss, Aalen, Germany) and bars represent mean values of distance migrated. A minimum of 100 cell trajectories were measured in independent experiments, with CM from macrophages of at least 5 different donors; flags indicate standard deviations. *, significantly different from AGS or RKO in RPMI medium at p < 0.05; **, significantly different at p < 0.05