Figure 6.

MMP-1 is able to rescue defects in spheroid outgrowth resulting from TWIST1 ablation. (A) WM793TR cells were engineered to express either control shRNA or TWIST1 shRNA #3 as well as a LacZ gene cassette or a MMP-1 gene cassette upon doxycycline addition. Cells were left untreated (basal TWIST1 & MMP-1 expression) or induced with doxycycline for 96 hours prior to spheroid formation. After treatment, lysates were harvested and western blots were performed. In addition, induction was performed for the final 24 hours in serum-free media for detection of secreted MMP-1. (B) WM793TR cells described above (+/− doxycycline) were allowed to form spheroids which were then implanted into 3D collagen. Bright field images were taken at 24 hour increments with careful consideration to image the same spheroids at each time point. Percent outgrowth was calculated by comparing the surface area of the spheroid (set at 100%) to that of the total outgrowth at each time point. Columns, average of three independent experiments; bars, SE; *p<0.05, **p<0.01, ***p<0.001. (C) At 96 hours, embedded spheroids were stained with both calcein AM (green = live cells) and ethidium bromide (red = dead cells) to determine apoptosis. One representative field of view is shown; scale, 100µm.