The formation of a vasculature in vivo requires CD44v6. (A) A spheroid-based in vivo angiogenic assay was performed as described in “In vivo angiogenesis assay.” Growth factor and peptide treatments are indicated. Two representative pictures of blood vessels in plugs are shown. Bars represent 50 μm. Staining of blood vessels was performed with CD34 antibody (green); red indicates smooth muscle actin staining (including pericytes; Sigma-Aldrich). Nuclei were stained with Hoechst dye 33258 (blue; Sigma-Aldrich). A minimum of 3 different plugs per condition were analyzed and quantified by immunologic staining (“Immunohistologic analysis”). (B) L3.6pl human pancreatic cancer cells were injected orthotopically into male nude mice as described.18 Three groups of 5 mice each were injected 7 days later with PBS or control peptide (20 μg) or CD44 v6 peptide (20 μg). Injection was repeated 3 times per week. Animals were killed 21 days after the beginning of the treatment. Tissues were stained with the endothelial marker CD31 (“Immunohistologic analysis”). A representative staining is shown. Bars represent 50 μm. Tumor volume, vessel number, and average vessel size were determined and evaluated in the graphs. All animals were handled according to German regulations for animal experimentation. The animal experiments were approved by the Regierungspäsidium Karlsruhe (35-9185.81/G-83/04). All mice were obtained from Harlan.
