Metastatic 344SQ cells form polarized epithelial spheres in 3D Matrigel culture. (A) Morphology of day 12 spheres by contrast microscopy. (B) Sphere growth versus time from plating onto Matrigel (MG); diameter measured in microns. (C) Ki-67 (green) and Topro-3 (blue) staining of developing sphere, visualized by confocal microscopy. (D) Cleaved caspase 3 staining (green) and Topro-3 (blue) of developing sphere. (E) β-catenin (green) and Topro-3 (blue) staining of developing sphere. (F) E-cadherin (green) and Topro-3 (blue) staining of developing sphere. (G) Sphere stained for ZO-1 (green), α6-integrin (red), and Topro-3 (blue). (H) Sphere stained for ParD6B (green), α6-integrin (red), and Topro-3 (blue). (I) Sphere stained for GM130 (green) and Topro-3 (blue). Bar in confocal images represents 20 μm.