Fig. 6
MMP activity influences LPS- and IL-10-stimulated macrophage-mediated cancer cell invasion and angiogenesis. a AGS cells were incubated in BD BioCoatTM MatrigelTM Invasion Chambers for 24 h with RPMI medium (-) or macrophages stimulated for 72 h with 10 ng/ml LPS (LPSmac)) or with 10 ng/ml IL-10 (IL-10mac) and supplied or not with a pharmacological inhibitor of matrix metalloproteases, Galardin (10 μM). Invasive cells were determined as described in Materials and Methods. Bars represent mean values of independent experiments performed with, at least, 4 different donors; flags indicate standard deviations. *, significantly different from AGS in RPMI medium at p < 0.05; **, significantly different at p < 0.05. b Quantification of the number of new blood vessels grown in the CAM towards each inoculation area (<20 μm diameter). A control with AGS and CM from IL-10-treated macrophages without Galardin (AGS + CM(IL-10mac)) was always included in each egg next to the inoculation area of AGS and CM of IL-10-stimulated macrophages supplemented with Galardin (30 μM) (AGS + CM(IL-10mac) + Galardin). Bars represent mean values (ratio between the vessel number in the test condition and the vessel number in the control condition, per animal) obtained from a total of 35 eggs and CM of macrophages derived from 3 different donors. Flags indicate standard error mean; *, significantly different at p <0.05. c Conditioned media from MatrigelTM invasion assays containing AGS (CMMat(AGS)), unstimulated (CMMat(mac)), LPS- (CMMat(LPSmac)), IL-10-stimulated macrophages (CMMat(IL-10mac)), AGS and unstimulated (CMMat(mac)), LPS- (CMMat(AGS + LPSmac)) and IL-10-stimulated macrophages (CMMat(AGS + IL-10mac)) were run on gelatin zymograms. Proteolytic activity bands were revealed in white on a blue background stained with Coomassie. d and e Densitometry analysis using QuantityOne® software (BioRad) allowed quantification of pro-MMP-9 and MMP-9 (d) and pro-MMP-2 and MMP-2 (e) activities. Proteolytic activity was expressed as percentage of the proteolytic activity of unstimulated macrophages. Data correspond to mean values of independent experiments performed with macrophages derived from at least 5 different blood donors. Flags indicate standard error mean; *, significantly different from AGS at p < 0.05; **, significantly different at p < 0.05