H rankl

Dissociated tumor cells mixed 1:1 with Matrigel Basement Membrane (BD Biosciences) were transplanted orthotopically in the inguinal mammary gland of 10–/12‐week‐old NSG mice and when tumors reached 5 mm of diameter mice were randomized for mock, h‐RANKL (0.75 mg/kg, 4–6 doses, twice per week; Amgen Inc.), h‐RANK‐Fc treatment (10 mg/kg, three times per week; Amgen Inc.) or denosumab (10 mg/kg, three times per week; XGEVA^®). Docetaxel (Hospira/Actavis, 20 mg/kg) was administered once per week together with dexamethasone (0.132 mg/kg, Merck), to reduce the chemotherapy‐induced inflammation. All the drugs were administered intraperitoneally. Tumor growth was monitored and measured with a caliper once per week. Tumor volume was then calculated as follows: π × length × width²/6 in cm³. Treatment was interrupted when tumors regress below 3 mm of diameter. Mice were sacrificed and tumors were surgically removed 24 h after treatment completion. In combination treatments with Docetaxel, mice were sacrificed once relapsing tumors reached 10 mm of diameter.

PDX single tumor cells were embedded in Corning™ Matrigel™ Growth Factor Reduced Basement Membrane Matrix (Corning™ 356,238), plated in DMEM/F‐12 with B‐27™ Supplement, EGF 10 ng/ml, hydrocortisone 0.5 μg/ml, insulin 5 μg/ml, cholera toxin 100 ng/ml, and penicillin/streptomycin. Cells were stimulated, or not, with h‐RANKL (500 ng/ml; Amgen Inc.) during 24 h prior to gene expression analyses.