FLIPi/TRAIL inhibits tumour initialisation and metastatic potential in vivo. A, BT474 cells were transfected with c-FLIP siRNA (FLIPi) or control siRNA (SCi) and 1 × 106 cells orthotopically transplanted in the presence or absence of 100 ng/ml TRAIL into the abdominal mammary fat pad of athymic nude mice in the presence of systemic estradiol. Transplant sites were monitored and measurements of palpable tumours taken twice weekly. B, FLIPi-transfected BT474 cells were treated for 18 hours with 20 ng/ml of TRAIL, the surviving adherent cells were then harvested and re-plated either in mammosphere culture (TRAIL/FLIPi Treatment 1) or adherent conditions at a density of 20,000 cells/ml. After four weeks culture, the adherent cell cultures were re-treated with FLIPi or SCi and 20 ng/ml TRAIL (18 hours) before plating for mammosphere assay (TRAIL/FLIPi Treatment 2). Images illustrate mammospheres formed in SCi conditions. C, 1 × 106 FLIPi (FT) or SCi (SC) treated MDA-MB-231 cells were injected, +/- 100 ng/ml TRAIL (T), into the tail veins of BALB/c severe combined immuno-deficient (SCID) mice. Mice were monitored daily and sacrificed six weeks post-surgery for histological examination of lung metastases.