Figure 3
Effects of TGF-β and TGF-β antagonists on cell growth and cell-motility and -invasion of MDA-MB-231 subclones. A. Cells were plated at 2 × 104/well in 24-well plates and incubated in the presence of vehicle (Blue bars), TGF-β (100 pM) (Green bars), 1D11 (10 μg/ml) or LY2109761 (2 μM) (Red bars) or a combination of TGF-β and an inhibitor (Yellow bars) for 72 h and cell numbers determined. Treatment with exogenous TGF-β failed to significantly inhibit growth of any of the subclones, with the exception of SCP2TR cells. Neither 1D11 nor LY2109761 stimulated tumor cell growth of any of the MDA-MB-231 subclones. Means ± SD of at least three independent experiments. Unpaired 2-sided t-test ± Welch correction was used to compare treatment with or without TGF-β antagonist. For cell-motility (B) and -invasion (C) assays, MDA-MB-231 sublines were cultured in uncoated and Matrigel®-coated PET inserts, respectively. Cells were treated with TGF-β (100 pM) and either LY2109761 (2 μM) or 1D11 (10 μg/ml) for 24 h. MDA-MB-231 subclones SCP2TR and 4175TR displayed the greatest motility and invasion, which were further stimulated by exogenous TGF-β. Moreover, both TGF-β pathway antagonists significantly inhibited TGF-β-induced motility and invasion in these two cell lines. Means ± SD of at least three independent experiments. Unpaired 2-sided t-test ± Welch correction was used to compare treatment with TGF-β alone with TGF-β plus inhibitor. D. Lung-tropic 4173 cells were plated onto a layer of growth factor-reduced Matrigel® matrix, followed by treatment with either vehicle or varying concentrations of LY2109761 for 9 days. Phase contrast microscopy images of 9 day-old 3D cultures labeled with Alexa-488-phalloidin were obtained. Numbers refer to the four representative colonies from each culture shown. As can be seen, treatment with LY2109761 inhibited invasion into surrounding Matrigel® in a dose-dependent manner, resulting in a reversal from a stellate to mass-like phenotype.