Non-malignant migration of B16 mouse melanoma cells in the neural crest and invasive growth in the eye cup of the chick embryo

Abstract

Melanocytes originate from the neural crest. In a previous study, we observed that human SK-Mel 28 human melanoma cells resumed neural crest cell migration after transplantation into the chick embryo neural tube. Here, we used transgenic mouse B16-F1 melanoma cells transfected with green fluorescent protein-vasodilator-stimulated phosphoprotein construct to extend these observations. After the injection of a cell suspension into the trunk neural tube of E2 chick embryos, the migration of melanoma cells was followed by live fluorescence microscopy. Within 12 h, the melanoma cells formed clusters in the neural tube at the levels of the intersegmental clefts between somites. After 24 h, a segmental pattern of emigration was visible. Emigrated melanoma cells were identified in serial paraffin sections by immunohistochemistry with ab732 as a marker for melanoma cells and by in-situ hybridization of mouse-specific repetitive genomic sequence mL1. After 24 h, melanoma cells were found along the medial neural crest pathway and in the sympathetic trunk ganglia and, after 48 h, also in the lateral melanocytic pathway. During migration along the neural crest pathways, mouse melanoma cells underwent apoptosis, which was assessed by anti-caspase 3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining. To prove the ablation of malignant behavior after back-transplantation into the original embryonic neural crest environment, we injected the same cell suspension into the eye cup of the E3 embryo. In this location, invasive melanomas formed.