Figure 1

LNCaP^RANKLcell adhesion and migration are enhanced in 3-D suspension culture. (A) In the left panel, RANKL expression is shown to enhance the adhesion of PCa cells to ColI. For each condition, 5,000 single cells from 2-D monolayer or 3-D suspension culture were seeded on 96-well plates pre-coated with ColI, ColIV or FN. After 30 minutes of incubation, the number of adherent cells was determined using alamarBlue assay. LNCaP^RANKL cells preferentially adhered to ColI, especially when the cells were pre-conditioned in 3-D suspension culture. In the right panel, a similar assay was conducted with cells pre-conditioned in 3-D suspension culture, where antagonizing antibody to α₂β₁ integrin was introduced to interfere with adhesion. The results are presented along with ratios to the control LNCaP^Neo cells for each condition. Each data point is the mean ± SD of 6 measurements from 2 independent experiments. (B) Time lapse fluorescence microscopy was used to determine cell motility. Compared to control, motility of LNCaP^RANKL cells was enhanced by 3 fold when the cells were pre-conditioned in 3-D suspension culture. Integrated distance traveled was normalized to LNCaP^Neo cells and presented as the mean ± SD of 5 separate experiments. (C) Pre-conditioning in 3-D suspension culture increased the migration potential of LNCaP^RANKL cells. In the upper left panels, cells in 2-D monolayer culture on ColI-coated plates were subjected to a wound healing assay. In the upper right panels, migration of cells pre-conditioned in 3-D suspension culture was assessed. In the lower panels, changes in cell migration were quantified based on the results of 3 separate experiments.