In vivo xenografts

Experiments were performed in SCID/beige female mice from Charles River Laboratories, Massachusetts, USA. Animals, approximately 7-8 weeks of age at the start of the treatment were allowed to acclimate in animal facility with access to food and water ad libitum for 3 days prior to manipulation. Animals were handled in accordance with IACUC regulations and guidelines for experiments performed in the USA and with European and French regulation for the protection of vertebrate animals for experiments performed in France. Animal well-being and behavior, including grooming and ambulation were monitored at least once a day. General health of mice was monitored and mortality recorded daily. Any moribund animals were sacrificed.

RS4;11 Acute Lymphoblastic Leukemia cell lines was obtained from ATCC. Toledo (Diffuse Large B-Cell Lymphoma) human cell lines were obtained internally through Bioresources and the Novartis-Cancer Cell Line Encyclopedia [43]. Cells were free of Mycoplasma and murine viral contamination in the IMPACT VIII PCR assay panel (IDEXX RADIL, IDEXX laboratories INC, Westbrook, ME, USA). Cells used for subcutaneous implantation were cultured in RPMI plus 10% FBS at 37° C in a humidified atmosphere containing 5% carbon dioxide). For RS4;11 culture, the medium was also supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM Hepes and 4.5 g/L glucose. Cells were resuspended in a 1:1 mixture of cold DPBS (Dulbecco's Phosphate-Buffered Saline) and Matrigel^™ (Becton-Dickinson #354234) at a concentration of 3 × 10⁷ cells/ml for Toledo cells, and of 1 × 10⁷ cells/ml for RS4;11 cells.

For each experiment, female SCID/beige mice were implanted subcutaneously (right axillary region) with 3 × 10⁶ Toledo or RS4;11 (1 × 10⁶ cells for efficacy studies and 1 × 10⁷ cells for pharmacodynamics studies) suspended in 1:1 mixture of cold DPBS and Matrigel^™ in a total volume of 100 μl. Body weights were recorded and tumors were measured with digital calipers twice to three times a week. Tumor volume was calculated using the formula: length × width²/2. Percent changes in body weights was calculated as (BWcurrent - BWinitial)/(BWinitial) × 100. Data is presented as percent body weight change from the day of treatment initiation.

When tumors reached approximately 200 mm³ for efficacy studies or 300 mm³ for pharmacodynamic studies, mice were randomized. {"type":"entrez-nucleotide","attrs":{"text":"S55746","term_id":"266073","term_text":"S55746"}}S55746 was formulated in PEG300/EtOH/water (40/10/50). ABT-199 was formulated in PEG300/EtOH/Phosal (30/10/60). Mice were treated via oral gavage at 10 ml/kg with the doses and schedules described in the figure.

For pharmacodynamic studies, blood and tumors were removed 16 h post dosing and immediately snap-frozen. Total proteins were extracted from the tumors and caspase-3 activity was assessed in triplicate using CaspACE^® Assay System (Promega). Platelet cell counts were determined 16 hours post dosing using Coulter AcT diff (Beckman).

For efficacy studies, percent treatment/control (T/C) values were calculated using the following formula: %T/C = 100 × ΔT/ΔC if ΔT > 0. Regression = 100 × ΔT/T_initial if ΔT < 0 where: T = mean tumor burden of the drug-treated group on the final day of the study; ΔT = mean tumor burden of the drug-treated group on the final day of the study – mean tumor burden of the drug-treated group on initial day of dosing; T_initial = mean tumor burden of the drug-treated group on initial day of dosing; C = mean tumor burden of the control group on the final day of the study; and ΔC = mean tumor burden of the control group on the final day of the study – mean tumor burden of the control group on initial day of dosing. If the tumor volume was less than 14 mm³ for more than 3 consecutive measurements, animals were considered in complete regression (CR).