Figure 1. The oncogenic potential of RUNX3 in cancer-induced bone destruction in vivo.

hCTRL or shRUNX3 Ca9.22 OSCC cells (1 × 10⁷ cells/100 μl of HBSS) were subcutaneously injected at the mouse calvaria (N = 11). Control mice (N = 9) were injected with HBSS only. (A) RUNX3 expression level in wild type (WT), shCTRL, and shRUNX3 Ca9.22 cells was detected with a Western blot analysis with its specific primary antibody. On day 28, the tumor volumes were measured. (B) On day 28, two-dimensional (2D) images of the collected carvaria were generated from the μCT data using the NRrecon software, and 3D images were reconstructed from 2D images with the rapidform2006 software. (C) BV/TV (%), BS/TV (1/mm), and BS/BV (1/mm) served as bone morphometric parameters of the calvaria were determined using the μCT images. (D) Serum levels of the bone turnover markers Ca²⁺, ALP, and TRAP5b were estimated using kits as described in the Materials and Methods. (E, F) The calvarial tissues were fixed with 1% buffered formalin, decalcified in 10% EDTA solution and sectioned. The sections were stained with H&E (original magnification, 100×) (E) and immunostained with specific antibodies against RUNX3, CD31, and Ki67 (original magnification, 200×) (F). Scale bar = 100 μm. Proliferative index and microvessel density were evaluated by immunostaining for Ki67 and CD31, respectively. The images are representative of two independent experiments. The results are combined data from two independent experiments and expressed as the median with interquartile range of 9 or 11 mice per group. *P < 0.05, ^*P < 0.005 versus HBSS-injected control mice, ^#P < 0.05, ^##P < 0.005 versus shCTRL cell-inoculated mice.