Figure 1: Melanomas become differentiated after dissemination to secondary sites.

(a, top) ZMEL1-GFP zebrafish melanoma cells (mitf-BRAF^V600E; p53^−/−) were orthotopically transplanted into the ventral skin of the transparent casper strain and then imaged using brightfield and GFP over 28 days. At days 1 and 7 post-transplant, the tumours are GFP+ but devoid of pigmentation, a marker of differentiation. By day 14 post transplant, the primary tumour mass in the orthotopic site has become deeply pigmented and the animal has developed small anterior metastases that are GFP+ but unpigmented (red arrowhead). By day 28, these metastases have now enlarged and are clearly pigmented (black arrowhead), consistent with metastatic differentiation. (a, bottom) ZMEL1-GFP cells were transplanted into the vasculature of larval casper recipients to assess direct differentiation capacity at sites of metastatic colonization, bypassing the primary skin site. Similar to what is seen in the orthotopic transplantation, the cells are initially unpigmented at days 1 and 7, but become increasingly pigmented at days 14 and day 28, indicating that cells can directly differentiate after extravasation. (b) Histological analysis of a larval casper recipient transplanted with ZMEL1-GFP cells shows heterogeneous acquisition of pigmentation, with cells near muscle invasive disease showing increased evidence of melanization (N=notochord, T=tumour, M=muscle). (c) Time-lapse imaging of ZMEL1-GFP morphology in a larval casper recipient shows that cells that exit the vasculature and enter the tailfin epithelial layer gradually acquire a dendritic phenotype that is characteristic of differentiated melanocytes. (d) Enlargement of a dendritic cell in the tailfin. Images are representative of n=10–20 fish per group.