LNCaPRANKLcell 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. LNCaPRANKL 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 LNCaPNeo 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 LNCaPRANKL cells was enhanced by 3 fold when the cells were pre-conditioned in 3-D suspension culture. Integrated distance traveled was normalized to LNCaPNeo cells and presented as the mean ± SD of 5 separate experiments. (C) Pre-conditioning in 3-D suspension culture increased the migration potential of LNCaPRANKL 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.