In vivo study

Animal studies were performed according to the German law and with the approval by the local animal ethics committee. We used 8-week-old female CB17/Icr-Prkdc^scid/IcrCr mice (Charles River), housed in individually ventilated cages (two mice per cage), with free access to food (autoclaved pellets) and water. At the start of the experiment, mice were injected subcutaneously (s.c.) in the right side flank with 5 × 10⁶ MDA-MB435S cells in 200 µl PBS. The area of injection had been previously shaved and was maintained without hair to minimize measurement errors. Starting at 3 weeks, tumor size was controlled weekly by measuring the major axis (length) and a perpendicular diameter (width), using a caliper always by the same operator. Tumor volume was calculated with the following formula: tumor volume = length × width² × 0.52. The animals were randomized after the first measurement point in four groups: (A) control, (B) doxorubicin (freshly prepared in PBS), (C) scFv62-TRAIL, and (D) doxorubicin in combination with scFv62-TRAIL. One treatment cycle lasted 4 days. On day 1, group A and C received 200 µl PBS and group B and C 200 µl doxorubicin in PBS (0.9 mg/kg). On the following 2 days, mice received scFv62-TRAIL (0.15 mg/kg) (group C and D) or PBS (group A and B). On day 4, mice were not treated. After three cycles, the dose of scFv62-TRAIL was reduced: On day 1, group A and C received 200 µl PBS and group B and C 200 µl PBS and doxorubicin (0.9 mg/kg). The following days, mice received scFv62-TRAIL (0.15 mg/kg) (group C and D) or PBS (group A and B). On days 3 and 4, mice were not treated. Tumor growth and body weight were measured once per week. Six weeks after tumor implantation, the animals were killed and the tumors were dissected and weighted.