ABCC5 promotes the formation of osteolytic bone metastases induced by 4T1 murine breast cancer cells. (A) ABCC5 expression is considerably elevated in the highly metastatic mouse breast cancer cell line 4T1, which exhibits bone metastatic activity, when compared with a less-metastatic 66cl4 (lung) and nonmetastatic 67NR populations. (B) ABCC5 expression was stably silenced by using two independent short-hairpin RNAs (sh 1, sh 2) in 4T1 breast cancer cells, as demonstrated with immunoblot analysis, when compared with parental 4T1 cells (4T1p) and a scrambled shRNA control (Scr shRNA). (C) All of the animals had osteolytic lesions after breast cancer cell injections. The number of lesions per animal is indicated and was established by examining blinded μCT images. (D) Reduction in ABCC5 levels in 4T1 breast cancer cells results in the diminished formation of osteolytic bone metastases after cardiac injection (scr shRNA, n = 19; abcc5 shRNA 1, n = 16; abcc5 shRNA 2, n = 19; *P = 0.015; **P < 0.001). Controls (n = 18) refers to age-matched female mice that were not injected with breast cancer cells. The degree of osteolytic bone destruction in the hindlimbs was quantified by in vivo μCT imaging. Bone volume of defined regions of both the proximal tibia and femur is shown for animals imaged on day 21 after injection. Representative images of bone reconstructions are shown.