No cross-reactivity in the chick embryo [21]. AntiMIB-1 (Dako, Hamburg, Germany) immunohistochemistry (marking cells that are in the state of DNA synthesis) specifically stained nuclei of human melanoma cells without cross-reactivity with the chick host 22948146 (Fig. 3L). Finally, we used live epi-fluorescence with murine GFP-VASP-transfected B16-F1 cells [19]. GFPVASP labels lamellipodia and filopodia of migrating B16-F1 cells. The technique allowed the in ovo visualization of the melanomacells emigrating from the neural crest or colonizing the optic cup [15].Aggregate Formation and Specific TreatmentThe advantage of using aggregates instead of cell suspensions was better reproducibility of the site-specific transplantation of melanoma cells into the lumen of the neural tube, a small embryonic compartment. While melanoma cell suspensions spread out within the entire lumen of the neural tube, the aggregates remained at the site of transplantation (between the 14th and the 20th pairs of somites). For aggregate formation we used a roller culture procedure in gas permeable biofoil bags developed for reproducible generation of small organ cultures [22]. Cell suspensions of 106 cells in 1 ml transA-196 web formed into melanoma cell aggregates after rotation for 24 h [17]. During aggregation, the melanocytes were treated with BMP-2 (20 ng/ml) or nodal (30 ng/ml, both from R D Systems, Wiesbaden, Germany).The Chick Embryo in Melanoma ResearchFigure 5. Transplantation of MCF7 breast cancer cells into the rhombencephalon of the chick embryo. (A,C) Two different examples of chick BIBS39 chemical information embryos 96 h after transplantation of MCF7 breast cancer cells into the rhombencephalon, H E stainings. The cells have formed stretched, compact epithelial tumors in the roof plate and adjacent mesenchyme. (B,C) Higher magnification reveals that the MCF7 cells invade the chick host in small aggregated clusters (arrows). (B,D) MIB1 immunohistochemistry shows that 30?0 
of the MCF7 cells proliferate in the chicken environment. doi:10.1371/journal.pone.0053970.gResults and Discussion Neural Crest and EMTIn the embryo, emigration of neural crest cells from the neural tube is designated as epithelial mesenchymal transition (EMT). EMT represents a complex change in cell morphology and migratory potential of embryonic cells and is induced in the embryonic neural crest by BMP and inhibited by sonic hedgehog and noggin signaling [23]. EMT comprises two consecutive steps [24]: First, the neural crest compartment is 15755315 induced in the epithelium of the neural tube. This step is morphologically characterized by the disintegration of the basal lamina in the region of the lateral roof plate. Second, neural crest cells start migration from the dorsal edges of the neural tube along their medial and lateral pathways. Neural crest cells following the medial pathway form spinal ganglia and the autonomic ganglia of the sympathetic chain. The melanocyte precursors emigrate at a later stage and follow the lateral pathway. The melanoblasts then colonize the epidermis and are the source of melanocytes in the adult individual [25]. Melanomas arise from melanoblasts or melanocytes in the epidermis. Due to the neural crest lineage of melanocytic cells, the neural tube was chosen as transplantation site.Spontaneous Neural Crest Cell Migration of Melanoma CellsUpon transplantation into the neural tube of the early chick embryo, melanoma cells integrated into the neural crest, performed EMT, and migrated along both pathw.No cross-reactivity in the chick embryo [21]. AntiMIB-1 (Dako, Hamburg, Germany) immunohistochemistry (marking cells that are in the state of DNA synthesis) specifically stained nuclei of human melanoma cells without cross-reactivity with the chick host 22948146 (Fig. 3L). Finally, we used live epi-fluorescence with murine GFP-VASP-transfected B16-F1 cells [19]. GFPVASP labels lamellipodia and filopodia of migrating B16-F1 cells. The technique allowed the in ovo visualization of the melanomacells emigrating from the neural crest or colonizing the optic cup [15].Aggregate Formation and Specific TreatmentThe advantage of using aggregates instead of cell suspensions was better reproducibility of the site-specific transplantation of melanoma cells into the lumen of the neural tube, a small embryonic compartment. While melanoma cell suspensions spread out within the entire lumen of the neural tube, the aggregates remained at the site of transplantation (between the 14th and the 20th pairs of somites). For aggregate formation we used a roller culture procedure in gas permeable biofoil bags developed for reproducible generation of small organ cultures [22]. Cell suspensions of 106 cells in 1 ml transformed into melanoma cell aggregates after rotation for 24 h [17]. During aggregation, the melanocytes were treated with BMP-2 (20 ng/ml) or nodal (30 ng/ml, both from R D Systems, Wiesbaden, Germany).The Chick Embryo in Melanoma ResearchFigure 5. Transplantation of MCF7 breast cancer cells into the rhombencephalon of the chick embryo. (A,C) Two different examples of chick embryos 96 h after transplantation of MCF7 breast cancer cells into the rhombencephalon, H E stainings. The cells have formed stretched, compact epithelial tumors in the roof plate and adjacent mesenchyme. (B,C) Higher magnification reveals that the MCF7 cells invade the chick host in small aggregated clusters (arrows). (B,D) MIB1 immunohistochemistry shows that 30?0 of the MCF7 cells proliferate in 
the chicken environment. doi:10.1371/journal.pone.0053970.gResults and Discussion Neural Crest and EMTIn the embryo, emigration of neural crest cells from the neural tube is designated as epithelial mesenchymal transition (EMT). EMT represents a complex change in cell morphology and migratory potential of embryonic cells and is induced in the embryonic neural crest by BMP and inhibited by sonic hedgehog and noggin signaling [23]. EMT comprises two consecutive steps [24]: First, the neural crest compartment is 15755315 induced in the epithelium of the neural tube. This step is morphologically characterized by the disintegration of the basal lamina in the region of the lateral roof plate. Second, neural crest cells start migration from the dorsal edges of the neural tube along their medial and lateral pathways. Neural crest cells following the medial pathway form spinal ganglia and the autonomic ganglia of the sympathetic chain. The melanocyte precursors emigrate at a later stage and follow the lateral pathway. The melanoblasts then colonize the epidermis and are the source of melanocytes in the adult individual [25]. Melanomas arise from melanoblasts or melanocytes in the epidermis. Due to the neural crest lineage of melanocytic cells, the neural tube was chosen as transplantation site.Spontaneous Neural Crest Cell Migration of Melanoma CellsUpon transplantation into the neural tube of the early chick embryo, melanoma cells integrated into the neural crest, performed EMT, and migrated along both pathw.