Figure 1

Endothelial cells induce branching in colonies of VA10 epithelial cells in reconstituted basement membrane matrix. A-E. Phase contrast images of cells cultured in rBM. A. VA10 cells form spherical colonies when cultured in rBM. B. HUVEC cells cultured in rBM do not form colonies but occur as single non-proliferative cells. C. Complex epithelial branching structures form when VA10 cells are co-cultured with endothelial cells. Secondary bifurcations (asterisks) are seen at the ends of elongated branches (arrow). D. A549 cells form colonies that do not branch when co-cultured with HUVEC. E. Co-culture of VA10 and HUVEC cells, showing extensive branching network formation after 19 days in culture. F-I. Confocal images of branching structures. F. Cryosection stained with a nuclear marker (TO-PRO-3, blue) and CD31 (red) to label endothelial cells, see arrowheads. G, H. Details of terminal buds, TO-PRO-3, blue. G. β4-Integrin expression (green) at the interphase between the structures and the matrix. H. E-Cadherin (green) outlines the epithelial cell junctions in branching structures. Scale bars 100 μm (A-E)/50 μm (F-I) I. Confocal sections and orthogonal sections showing gaps in the nuclear pattern (TO-PRO-3, white) in a tube (upper half) and terminal buds (lower half). Dotted lines indicate the location of the orthogonal sections (-z-).