Nod rag1null il2rgnull nrg mice

NOD-Rag1null IL2rgnull (NRG) mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). NRG pups (24–72 h old) were irradiated twice with 3 cGy 3 h apart and then engrafted intrahepatically with 10⁵–10⁶ CD34⁺ hematopoietic stem cells (HSC) isolated from human umbilical cord blood. At 12 weeks post engraftment, blood was collected to quantify human immune cell reconstitution using flow cytometry. Erythrocytes were lysed using an ACK lysis buffer and the remaining cells were treated with both anti-human Fc Receptor Binding Inhibitor and anti-mouse CD16/CD32 antibodies (eBiosciences). Cells were then stained with an antibody cocktail (mCD45-AlexaFluor 700 (1:50), hCD45-Pacific Blue (1:20), hCD3e-Qdot 605 (1:100), hCD4-PerCP-Cy5.5 (1:20), hCD8a-PE-Cy7 (1:20)), followed by fixable viability dye (APC-eFluor 780; eBiosciences). Samples were run on the Cytoflex LX flow cytometer equipped with a flow rate calibrator and analyzed using FlowJo software version 10 (Tree Star, Ashland, OR, USA). Mice with at least 10% or 50,000 per mL hCD45+ leukocytes in the blood were selected for subsequent experiments.

Immunodeficient NOD-Rag1^nullIL2rg^null (NRG) mice were purchased from Jackson Laboratory (Bar Harbor, ME), bred and housed at the University of Maryland Baltimore. Similar to our previous publication (20 (link)). mice were transplanted IV with luc-labeled human AML cell lines or cryopreserved AML patient cells on day -10, and baseline total body luminescence was quantified on day 0 by Xenogen bioluminescence imaging (Xenogen IVIS Spectrum; PerkinElmer, Waltham, MA). Mice were allocated to treatment groups so that each group had similar average day 0 luminescence, then groups were administered drugs per oral (PO; gavage). Luminescence of each mouse was assessed over time and compared with that mouse’s day 0 luminescence (fold-change AML burden). Clinical behavior, appearance, weight, and survival were also monitored.

All experiments utilized NOD-Rag1^null IL2rg^null (NRG) mice (The Jackson Laboratory, 4-8 weeks old, and 18-26 g at time of tumor implantation) (33 (link)). For survival assays, human prostate adenocarcinoma LNCaP cells (ATCC) xenografts were implanted 4 ± 1 weeks before the experiment by injecting 200 µL of a 1:1 matrigel (Fisher Scientific)/phoshate-buffered saline (Wisent) mixture containing 8 × 10⁶ LNCaP cells subcutaneously on mice shoulders. LNCaP PSMA expression was previously verified (31 (link)). Protocols were approved by the Animal Ethics Committee of the Université de Sherbrooke according to the Canadian Council on Animal Care guidelines.