Figure 5. Long-term imaging of the tongue cancer.

(A-F) Hela-O3 cells expressing either venus (A, B) or H2B-GFP (C-F) were transplanted into the tongue submucosa, as described in Material and Methods. A. After 7 days from the injection, the edges of the tumor were imaged on a daily basis by using two-photon microscopy (excitation wavelength of 930 nm) to reveal collagen fibers (red) and the tumor mass (green). Z-stacks were acquired at day 7 (D7), 8 (D8), and 9 (D9) by using a 25X water lens (N.A. 1.05, Olympus). 3D reconstructions (upper panels, yz view) and maximal projections of the xy view were performed by using Imaris (Bitplane) (middle and lower panels). The extracellular matrix at the surface of the tongue was used as a reference point (middle panels, arrows). Individual tumor cells were observed migrating from the tumor mass (arrowheads). Bar 50 μm. B After 14 days, the edge of the tumor was imaged as described above by performing Z-stacks for two hours at 5 min interval. Individual cells were observed leaving the tumor mass (insets, red arrowheads). Bar, 20 μm. C, D. Texas Red-dextran (70 kDa) was injected in the tongue in order to map the lymphatic vessels, and Z-scan were performed, as described above. Maximal projections show tumor cells expressing H2B-GFP (green) either in proximity (C) or inside lymphatic vessels (D). Note collagen fibers in C (cyan). E, F. Dextran (70 kDa) was intravenously injected to label blood vessels and stromal cells, and cervical lymph nodes were exposed and imaged as described above. E. Tumor cells (green) colonized a cervical lymph node: stromal cells (red) and collagen fibers (cyan). F. Tumor cells (green) are in close proximity to a blood vessel (red). Bars, 50 μm