Fig. 3. Invasion of cell-cell adhesion-deficient II-4 cells was preceded by their attachment and localization at the epithelial-stromal interface in 3D in vitro tissues. In vitro 3D cultures were constructed as 4:1 mixtures of HEK with either pBabe-, H-2k^d-EcadC25- or H-2k^d-Ecad-II-4 cells and were grown at an air-liquid interface for 7 days. Tissues were stained by double immunofluorescence for the basement membrane component Type IV collagen (red) and ß-gal (green). Constructs harboring mixtures of HEK with either pBabe- (A, arrows) or H-2k^d-EcadC25-expressing II-4 cells (B, arrows) demonstrated tumor cells that were limited to the suprabasal layers of the epithelium. By contrast, tissues harboring H-2k^d-Ecad-II-4 cells demonstrated ß-gal-positive cells in the basal layer of the epithelium, adjacent to the Type IV collagen present at the epithelial-stromal interface (C, arrows). Bars, 10 µm.