Tumorigenicity assay in vivo

To evaluate the tumor promoting role of OPN in vivo, a HNC xenograft model was implemented in BALB/C nude mice (4-weeks-old). Briefly, a total of 1×10⁶ CAL-27 cells or OPN-expressing CAL-27 cells in 100 μL serum-free DMEM were subcutaneously injected into the left and right buttocks. For the anti-tumor effects of neutralized antibodies, 5×10⁵ CAL-27 cells mixed with 3×10⁵ NFs in 100 μL serum-free DMEM were subcutaneously injected into the left and right buttocks. Three days after the injections, all mice were divided randomly into 4 groups (4 mice per group) and were treated with neutralized antibodies (anti-OPN, anti-IL-6 or anti-OPN plus anti-IL-6) every 3 days. As previously noted ³³, the tumor volume was measured every 3 day by the following formula: tumor volume = length × width × width / 2. After treatment for 15 days, the animals were sacrificed and the tumor volume and tumor weight were measured. The tumor growth curve was plotted using the seeding day as the horizontal axis and the tumor volume as the vertical axis.