Bioluminescent imaging (BLI) detects that TGF-ß activates the RANKL promoter in C4-2B cells in bone in vivo. C4-2B prostate cancer cells were stably transfected with a reporter vector of the RANKL promoter driving luciferase. The transfected C4-2B cells were injected intratibially into SCID mice tumors were allowed to develop over 6 weeks and vehicle (PBS), TGF-ß (100ng/mouse), or 1,25-dihydroxyvitamin D (VitD3) (200ng/mouse) was then administered intraperitoneally into the mice (n=6/group). The mice were then anesthetized at the indicated times pre- or post-injection and BLI was performed. Photon counts were collected over a minute period. A, representative BLI images of mice treated with either TGF-ß or VitD3. The same mouse is shown over time for each treatment. The scale bar indicates luminescence intensity from high (H) to low (L). B, luciferin was injected 10 minutes prior to the desired timepoint for BLI measurement. Luminescence emitted from the mouse was then integrated for 1 minute starting 10 minutes after injection of luciferin. Results are reported as mean±SD (n=6/group). *P<0.05, **P<0.01 compared with pre-treatment (Pre) for that compound. C, a section of the intratibial tumors were mechanically ground and then homogenized to collect total RNA which was then subjected to real-time PCR for RANKL mRNA and GAPDH mRNA. RANKL mRNA levels were normalized to GAPDH mRNA levels. Results are reported as mean±SD. *P<0.01 compared with vehicle. D, Sections of intratibal tumors were decalcified and processed for histology. Sections were stained with hematoxylin and eosin H&E or subjected to immunohistochemistry for luciferase. The top sections are 100 X magnification and the bottom sections (indicated by the squares in the top section) are 400 X magnification. The bar represents the cartilage epiphysis. Bone is represented by pink osteoid. Tumor cells are seen surrounded by bone and are indicated by the arrow. In the luciferase sections, luciferase is represented by the brown color (Zhang et al The Prostate 9999:1-10 2004).