Figure 1. Co-culture of melanoma cells and LECs reveals melanoma invasion into the LEC 3D structures and increases the metastatic potential of WM852 cells in vivo.

(a) Schematic of the experimental pipeline. (b) Confocal images of LEC spheroids (PECAM-1, red) in 3D fibrin matrix (left panel), LEC spheroids co-cultured with WM852 (green, middle panel) or Bowes (green, left panel). The area enclosed in the white square is shown enlarged below each panel. Melanoma cells were stained with GFP (green), and nuclei were counterstained with Hoechst 33342. Maximum intensity Z-projections of confocal stacks are shown. (c,d) Growth rates of the 3D LEC primed WM852* (c) and Bowes* (d) derived tumors (n = 8 for both cell types) compared to control WM852 (n = 7) and Bowes (n = 8) tumors, respectively. (e, f) Distant organ metastasis, detected by bioluminescence imaging of luciferase signal, in liver (e) and lung (f) of SCID mice subcutaneously injected with WM852 alone or co-cultured with LECs (WM852*). Upper panels: representative images of the indicated organs, each box represents an organ from one mouse. Bottom panel: quantification of luciferase signal, each dot represents the luciferase value in one sample. Horizontal line indicates the average, vertical bars represent SEM. *: p<0.05. n.s., non-significant.

Figure 1—figure supplement 1. Analysis of mouse xenografts and distant organ metastasis.

(a) mRNA expression of a panel of LEC markers (CD34, PROX1, FLT4) in 3D LEC primed Bowes and WM852 cells (*) before the cells were used for the in vivo xenograft assay. Expression in monotypic-cultured LEC was used as a control and set to one. (b) End point analysis of the volume (left panel) and weight (right panel) of the Bowes/Bowes* and WM852/WM852* tumors. A dot represents one mouse. Error bars indicate s.e.m. *: p<0.05. n.s., non-significant (c) Immunohistochemistry of xenograft sections from the LEC co-cultured WM852* (left panels) or Bowes* (right panels) stained for mouse Lyve-1 (red) to detect mouse lymphatic vessels and human MMP14 (green) as a marker of the human melanoma cells. Nuclei were counterstained with Hoechst 33342. Scale bar = 100 µm. (d, e) Luciferase signal from lymph nodes isolated from mice bearing WM852 (n = 7) and WM852*(n = 8) (d), or Bowes and Bowes* (n = 8) (e); each line shows lymph nodes from one mouse derived tumors. (f) Quantitative-PCR (q-PCR) for human Alu sequences from lung genomic DNA isolated from mice bearing WM852 and WM852* derived tumors. Each dot in the graph represents the luciferase signal intensity obtained from one isolated organ per mouse, and the red line marks the median of the samples. (g) Luciferase signal from liver and lungs isolated from mice bearing Bowes or Bowes* derived tumors (n = 8); each box represents an organ from one mouse.