Fig. 6.

Metastatic melanoma brain colonization requires Cx26. (A,B) b16 metastatic melanoma cells expressing the empty lentiviral vector (A; b16 control) or b16 cells stably expressing lentiviral shRNAs to Cx26 (B; b16KNcx26) along with GFP were injected into the chicken circulation and allowed to form microtumors in the brain for the indicated days. Top views are representative 3D confocal reconstructions of the brain vasculature (red, Rhodamine–lectin stained) and tumor cells (green) (60×). Insets show lower magnification (10×) of boxed areas. Note that 3D rendering shows that b16 control cells co-opt brain blood vessels whereas b16KNcx26 cells show little vessel association. Side view shows confocal z-stacks. Middle view shows representative optical slices from the middle of the z-stacks (slice positions indicated by the dashed lines in the side view). (C) Representative whole chicken brain images 4 days after intravenous injection of either b16 or b16KNCx26 cells, showing the level of brain colonization of GFP-labeled tumor cells. (D) Average volume and number of brain microtumors induced by b16 mock or b16KNCx26 cells 4 days after injection. Scale bars: 200 µm (A,B), 15 µm (A,B, inset), 0.5 cm (C).