Figure 1.
Egg preparation and fenestration.
(A) Incubator containing eggs. (B) The top of each egg is marked to indicate the location of the embryo (blastoderm). (C) Tools required for fenestration of the eggs are depicted: (from left to right) paper towels, tungsten needle and forceps, 2 egg holding devices, syringe with needle, 80% ethanol, plastic petri dish for egg shell waste, egg piercer, hacksaw, adhesive tape, scissors. (D) A pre-determined breaking point is generated after removal of 2 ml albumen to lower the level of the blastoderm. (E) After removal of the shell, the embryo is discernible on the blastoderm (arrow). (F) The egg is sealed with adhesive tape and re-incubated. (G) Capillaries are pulled before transplantation. (H) Working place (stereo-microscope with epi-illumination, diluted black ink, PBS, pipette) and (I) tools (mouth pipette, forceps, tungsten needle) required for transplantation of the cells.
Figure 2.
Transplantation of melanoma cells into three distinct niches of the chick embryo.
(A) Chick embryo stage 12–13 HH before and (B) directly after transplantation of B16-F1 melanoma cells into the neural tube. The entering site of the micro-pipette is marked (asterisk in A). Note the dilated neural tube (frame in B) due to the transplanted cells when compared to (A). (C) B16-F1 cells can be detected via GFP epifluorescence in the lumen of the neural tube directly after transplantation. (D) 48 h after transplantation ventrally emigrating B16-F1 cells are clearly discernible (arrows) in lateral view; the borders of the neural tube are outlined in green. (E) Chick embryo stage 19 HH directly after transplantation of B16-F1 melanoma cell aggregates into the optic cup. (F) Aggregates were stained with nile blue sulphate before transplantation for better visibility. Higher magnification shows the aggregates behind the embryonic lens (arrow). A temporary capillary bleeding can be discerned at the injection spot at the choroid fissure in (F). (G) Macroscopically no tumor growth is visible 72 h after transplantation. (H) The former entering site of the micro-pipette (choroid fissure) is marked (arrow). (I) Chick embryo stage 12–13 HH before and (J) directly after transplantation of human melanoma cells into the ventricle of the hindbrain (rhombencephalon, frame in I). The entering site of the micro-pipette is marked with the asterisk in (J). Note the melanoma cell-filled brain ventricle (frame in J). (K) 48 h after transplantation a growing tumor is already visible in the hindbrain (frame). (L) After 96 h a single condensed tumor is visible in the dorsal midline of the neural epithelium (arrow). Scale bars in A, B and E–L: 1 mm; scale bars in C and D: 0.5 mm.
Table 1.
Evaluation of melanocyte invasion in the optic cup.
Figure 3.
Histology, immunohistochemistry and in situ hybridization of the chick embryos.
(A) Schematic drawing depicting ventral and medial neural crest migration pathways. n.c. neural crest; n.t. neural tube; s.t. sympathetic trunk. (B) Chick embryo 24 h after transplantation of SKMel28 melanoma cells into the neural tube. Melanoma cells (visualized by HMB45 immunoreactivity) spontaneously resuming neural crest migration have a stretched, mesenchymal-like morphology (arrows). (C) At the site of destination along the ventral migration pathway (para-aortic sympathetic ganglia) melanoma cells undergo apoptosis, visualized by TUNEL staining. (D,E) Chick embryo 24 h after transplantation of benign primary human melanocytes into the neural tube. Melanocytes (showing a compact, epithelial-like morphology) are encountered only in the lumen of the neural tube and, in part, integrated into the roof plate with no neural crest migration. (F) Melan A immunoreactivity confirms the melanocytic origin of the cells. (G) Schematic drawing of chick embryo 72 h after transplantation of B16-F1 melanoma cells into the optic cup. (H) Histological correlate of schematic drawing. Already in H&E staining the transplanted, invasively migrating melanoma cells are visible (arrows). (I) Single melanoma cells (identified by HMB45 immunoreactivity) form a tumor, and single melanoma cells invade the choroid of the optic cup (arrows). (J) Chick embryo 96 h after transplantation of human metastatic melanoma cells into the brain vesicle at the hindbrain (rhombencephalon). The cells form a large tumor in the dorsal neuroepithelium with (K) single HMB45 positive cells infiltrating the surrounding brain tissues. (L) MIB1 immunohistochemistry (proliferation marker not cross-reacting with chick cells) identifies melanoma cells during haematogenous spreading in blood vessels among host erythrocytes and lymphocytes, and in the surrounding neural tissue.
Figure 4.
Pre-treatment with the TGFbeta family members BMP-2 or nodal induces invasive migration of human melanocytes in the optic cup.
Untreated, BMP-2 or nodal pre-treated melanocytes were injected into the optic cup of the chick embryo (stage 20 HH). After 72 h of further incubation, the embryos were analyzed for tumor growth and invasion. Untreated melanocytes formed loosely aggregated tumors adjacent to the hyaloid vessels (left image in upper row), in the developing vitreous body and behind the lens (right image in upper row) without invasion. The BMP-2 and nodal groups formed tumors in similar locations. In the BMP-2 group single melanocytes invaded the lens epithelium (insert in left image in middle row), the retina, the hyaloid vessels, and the choroid (right image in middle row; arrows pointing at melanocytes). In the nodal group single melanocytes invaded the choroid (lower row, arrows in right image) and the hyaloid vessels.
Figure 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 30–50% of the MCF7 cells proliferate in the chicken environment.