3D spheroids for morphological analysis of prostate carcinoma cells with different EMT-related phenotype: tips and tricks

3D-spheroids maintain a 3D cell arrangement to mimic the tissue architecture, thus retaining most of the biological characteristics that are lost when culturing cells in 2D and represent an excellent experimental setting to study cancer cells biology. Since previous studies demonstrated that 3D spheroids allow the characterization of carcinoma and melanoma cell phenotype, in this study we aimed at comparing different 3D experimental settings for the morphological analysis of prostate cancer cells having a different EMT-related phenotype. For this purpose, 3D spheroids containing DU145 and PC3 cells were obtained using three different technical approaches: agarose-coated wells (agarose 1%) in 24-multiwell plates, Apricell 3-in-1 plates and Ibidi µ-Slide Spheroid Perfusion. Our results show that the more epithelial DU145 cells formed 3D spheroids containing tightly and densely packed cells when cultured in agarose-coated wells. By contrast, the more mesenchymal PC3 cells formed loosely aggregates that do not maintain their integrity when manipulated for morphological analysis processing. When DU145 and PC3 cells are cultured in Apricell 3-in-1 plates, densely packed 3D spheroids exhibiting regular morphology are rapidly formed, so that spheroid integrity is preserved for both cell types during the procedures to prepare the samples for morphological analysis. Ibidi µ-Slides offer advantages similar to Apricell 3-in-1 plates to facilitate the rapid formation of 3D spheroids that preserve their integrity during the manipulation, but the small amount of cell culture medium in each well does not allow to culture the spheroids for long periods without the perfusion. Overall, our results show that different methods to grow prostate cancer cells in 3D spheroids offer different advantages during the manipulation of spheroids depending on cell phenotype. However, each of the considered experimental settings confirms that 3D spheroids represent a pre-clinical model for identifying and validating tumor markers and to studying new therapeutic tools.