FIGURE 4.

FGF9 imparts hierarchy to the tumor vasculature. A and B, ultraviolet fluorescence intravital microscopy images of red blood cells flowing within the vasculature of GFP-expressing (A) and FGF9-expressing (B) renal tumors 14 days after injection of Renca cells. GFP-expressing tumors (A) show a densely packed network of highly branched vessels. The network in FGF9-expressing tumors (B) is less dense and less branched. There is also a “smooth” appearance to some vessels in FGF9-expressing tumors, consistent with faster flow in an arterialized vessel (A), and a more irregular appearance in other vessels, with cell-free spaces in the lumen, consistent with slower flow in a venous structure (V). C, graphs depicting vascular length density (*, p = 0.034), lumen diameter (*, p < 0.001), and branch point density (*, p = 0.019). At least 10 fields of view were averaged for each mouse, with 3 GFP-expressing tumors and 4 FGF9-expressing tumors. D–F, higher magnification intravital images illustrating chaotic and serpentine flow routes (D, yellow arrows) and orphaned capillaries (E, white arrows) in GFP-expressing tumors, but ordered flow through an arterial-capillary-venous microcirculatory unit in an FGF9-expressing tumor (F). Arrows depict flow routes. C, capillary. G, graphs depicting the density of simple and complex microcirculatory units of arterial to capillary to venous flow (*, p < 0.001; †, p = 0.007), capillary density defined as vessels with single file red blood cell flow (p = 0.233), and percentage of capillaries positioned within a microcirculatory unit (*, p = 0.028). Movies corresponding to images in panels D, E, and F can be found in supplemental Videos S1, S3, and S4, respectively. Supplemental Video S2 depicts another example of bizarre step changes in lumen diameter and absence of flow hierarchy.