ABCC5 promotes the formation of osteolytic bone metastases in MDA-MB-231 human breast cancer cells. (A) ABCC5 expression is appreciably higher in the bone metastatic 1833-BM1 population compared with parental MDA-MB-231 cells and the lung-metastatic 4175-LM2 population. (B) Immunoblot analysis reveals that ABCC5 expression was efficiently silenced via short-hairpin RNA (shRNA) in the 1833-BM1 breast cancer cell line. An immunoblot for α-tubulin served as a loading control in panels (A) and (B). (C) Diminished ABCC5 expression in 1833-BM1 cells resulted in reduced formation of skeletal metastases after cardiac injection, as determined by in vivo bioluminescent imaging. The data and representative images are shown for day 21 after tumor-cell injection (scr shRNA, n = 10; ABCC5 shRNA, n = 8; *P = 0.024). (D) All of the animals developed osteolytic lesions after injection of the breast cancer cells. The number of lesions per animal was determined by analyzing blinded μCT images. (E) The degree of osteolytic bone destruction was quantified by in vivo μCT imaging. Bone volume was calculated from reconstructions of defined regions of both the proximal tibia and femur from mice imaged on day 21 after injection (scr shRNA, n = 20; ABCC5 shRNA, n = 16; **P = 0.041). Controls (n = 10) refers to age-matched females mice that were not injected with breast cancer cells. Representative images of bone reconstructions are shown.