4.13. Xenograft Model and In Vivo Antitumor Activity Assay

As previously described [4,31,32] NOD/LtSz-scid (NOD/SCID) and NOD/LtSz-scid/IL-2Rγchain null (NSG) mice were produced at the Genotoul Anexplo platform (Toulouse, France) using breeders obtained from Charles River Laboratory. Animals were used in accordance with a protocol reviewed and approved by the Institutional Animal Care and Use Committee of Région Midi-Pyrénées 9001v2008 ISO certified in June 2010 (Toulouse, France). For tumor xenograft experiments, Molm14 (2 × 10⁶) were inoculated subcutaneously into the flanks of NOD/SCID mice. When tumors were palpable, mice were treated. Tumor volume (tumor length × width² × 0.5236) was measured every 2–3 days using calipers. At the end of the experiment, or when animals show bad health condition, mice were humanely sacrificed. For orthotopic xenograft, AML cell lines were transplanted into NSG mice, as reported previously [4,7,8]. Briefly, mice were housed in sterile conditions using HEPA-filtered micro-isolators and fed with irradiated food and sterile water. Transplanted mice were treated with antibiotic (baytril) for the duration of the experiment. Mice (6–9 weeks old) were sublethally treated with busulfan (20 mg/kg) 24 h before injection of leukemic cells. Leukemia samples were thawed at room temperature, washed twice in PBS, and suspended in phosphate saline buffer at a final concentration of 1 million cells per 100 μL. A total of 200 µL of phosphate saline buffer solution containing 2 × 10⁶ of AML cells were injected in tail vein. Five days after AML cells transplantation, NSG recipients transplanted mice were injected in blood tail with 0.15 mg/kg of idarubicin every two days for 5 days and/or 20 mg/kg/day of DDA 5 days/7 during two weeks. For negative controls, NSG mice were treated daily with IP injection of vehicle, PBS 1×. Mice were monitored for toxicity and provided nutritional supplements as needed. NSG mice were humanely killed in accordance with European ethic protocols. Bone marrow (mixed from tibias and femurs) and spleen were dissected in a sterile environment and crushed in Hanks balanced salt solution with 1% FBS. Mononuclear cells from bone marrow and spleen were labeled with FITC-conjugated anti-hCD3, PE-conjugated anti-hCD33, PerCP-Cy5.5-conjugated anti-mCD45.1, APC-conjugated anti-hCD45 and PeCy7-conjugated anti-hCD44 (all antibodies from BD, except FITC-conjugated anti-hCD3 from Biolegend, San Diego, CA, to determine the fraction of human AML blasts (hCD45+hCD33+mCD45.1- cells) using flow cytometry. Analyses were performed on a Beckman coulter cytoflex flow cytometer. The number of AML cells/µL peripheral blood and number of AML cells in total cell tumor burden (in bone marrow and spleen) were determined by using CountBright beads (Invitrogen, Waltham, MA, USA) using the described protocol.