Fig.5.

Confocal immunofluorescence analyses of C8161 and DNC8161 cells grown on different matrices and for variable culture periods demonstrate distinct spatial distribution of Nodal. (A) Both C8161 (a, c), and DNC8161 cells (e, g) grown on glass substrate display an intracellular distribution of Nodal. On Matrigel, C8161 cells form vascular-like networks with Nodal mostly detected at the outer edge of the network structures, often in association with VE-cadherin or TGFβRII (yellow arrows, b & d). DN C8161 only forms unstructured clumps on Matrigel with no specific distribution of Nodal (f &h). (B) Long term cultures (16-20 days) of C8161 on 3D Collagen I matrices resulted in the formation of more developed hollow-appearing tubular structures (as shown previously in Fig. 4e by phase contrast microscopy). Elongated tumor cells (yellow arrows) delineate the tubular structures. These endothelial-like melanoma cells appear polarized based on their peri-tubular expression of Nodal and luminal expression of VE-cadherin. DNC8161 placed on Collagen I initially formed clumps; however, the clumps slowly spread and at the end of 2.5 weeks in culture, minor structures could be observed without the alignment of the tumor cells (for quadrant display of the colors in (B), see Sup. Fig. 5). Nuclei are stained with DAPI (blue). Original magnification, 63X.