Figure 3.

Microvessel density and structure in Wnt1- and ΔNβ-catenin-induced tumors. (A) Wnt1- and ΔNβ-catenin-induced tumors have similar microvessel density. Frozen tumor sections were stained with CD31 antibody (red), and pictures were taken from at least 5 different fields per tumor (6 Group1 Wnt1-induced tumors and 5 ΔNβ-catenin-induced tumors). Note that Wnt1- induced tumors mostly have stroma-associated vessels (see Figure 3B for a higher magnification view). Vessels in ΔNβ-catenin–induced tumors were mostly mingled with tumor cells, and some dilated structures lined with CD31⁺ cells (*) were frequently observed. Representative images are shown. Scale bars=100 μm. To determine the microvessel density (CD31 positive area per cell), the value of CD31 positive area and the number of nuclei were obtained using ImageJ software (Supplemental files; Figure 3). To quantify stroma-associated vessels, the number of stroma-associated vessels was counted and divided by total number of blood vessels (total 30 images; 3 tumor samples per genotype). Error bars=standard deviation. (B) Stroma-associated microvessels have different structures from tumor cell-associated microvessels in Wnt1-induced tumors. To assess the microvessel structure, tumor sections were co-stained with CD31 (red color) and α-smooth muscle actin (SMA, green), and examined with higher magnification (100x objective lens). In Wnt1-induced tumors, microvessels in activated stroma area had a thin and serpentine structure (arrow), while microvessels mingled with tumor cells often had a big aggregate of endothelial cells (arrow head) that is similar to vessels in ΔNβ-catenin-induced tumors. Representative images are shown. All tissue sections were counter stained with TOPRO3 (blue color). Scale bars=20 μm.