Figure 7. Effects of LGR5 modulation on cell-cell adhesion and migration.

Lim1899 cells were transfected with vector controls (Cy3V and pTuneV), with Cy3- siLGR5 or with pTune/LGR5. Parental, transiently transfected cells (Tr) or stably transfected cell lines (St) were cultured under the following conditions: A) Cells were seeded in 30 µl droplets on a plastic surface, and the plate inverted to create hanging drops as described in Methods. Images were taken after 8 days by re-inverting the plastic support and imaging in bright field with a Nikon 90i microscope and a 10× lens. Digital images were acquired with a Photometrics CoolSnap digital camera. Spheroid volumes (left-hand graph) were calculated from these images using the modified ellipsoid formula. Spheroid sizes differed significantly between siLGR5 or M2LGR5 transfected cells and their counterparts transfected with empty vector (p = 0.0312 and p = 0.321, respectively, by the unpaired t-test). The cellularity of the spheroids (right hand graph) was assessed as described in Methods. In both graphs data represent the mean and standard deviation of 10 individual spheroids per cell line. B) Parental cells and cells stably transfected with pTune/LGR5 (clones 6-1) were plated at high density in 24-well plates. Wounds were scratched in the adherent monolyers and the wells were imaged every two days with a Nikon90i microscope using a 10× lens (upper panels). The photomicrograph on lower right shows a higher magnification of LGR5 6-1 wound at day 6 (20× lens). Insert: Rate of wound closure over 96 hr. C) Cells were seeded in Transwell inserts (8 mm pore size) and cultured for 4 days. Filters were fixed and stained with May-Grumwald/Giemsa. Cells on the upper side of the filters were removed, and filters mounted on glass slides. Cells present on the underside of the filters (migrating cells) were counted by light microscopy as described in Methods. The graph presents average and sd of three separate samples for each cell type. Tr and St denote transient and stable LGR5 transfectants. Significance levels were determined by the unpaired t-test. *** = p<0.001; ** = p<0.005.