Targeting the Hypoxic Response in Multiple Myeloma Cells Engrafted in the Bone Marrow

This protocol will be found in our soon to be published manuscript titled “A DNA-Binding Molecule Targeting the Adaptive Hypoxic Response in Multiple Myeloma has Potent in vivo and in vitro Anti-tumor Effects.”

Cell lines and reagents:

The human MM cells line (8226) was maintained at 37^oC and 5% CO₂ (under “normoxic” condition). Our experimental molecules (HIF-PA) were synthesized by solid-phase methods on Kaiser oxime resin. For the bone marrow xenograft studies, luciferase-expressing 8226 cells (8226-LUC) were stably transfected with the pGL4.5 luciferase reporter vector followed by selection with hygromycin (350 mg/ml). The in vitro luciferase activity was confirmed and measured using the dual-luciferase reporter assay kit using a luminometer. The in vivo luciferase activity was measured using the VivoGlo luciferine substrate.

Mouse tumor model:

8226-LUC cells were injected (10 X 10⁶ cells/mouse) through the tail vein. Real time 8226-LUC engraftment in the bone marrow was measured in anesthetized mice given an IP injection of VivoGlo luciferin substrate (100 mg/kg mouse) and then monitored for luciferase activity in the skeleton using a Perkin-Elmer IVIS Lumina XRMS small animal imaging system (examples of this can be seen in our methods section). Luciferase activity in the skeleton of mice challenged with 8226-LUC was typically observed between day +15 and day +20 post challenge. Once a positive bioluminescent signal was observed, the animals were randomized into groups (6-8 animals/group) and were treated with HIF-PA or vehicle control and a total of 6 drug injections were given. The luciferase activity was measured twice/week and the change in average radiance (photons/sec/cm²/steradian) was measured and analyzed using the LivingImage version 4.4 imaging software.