Leukemic cells engraft onto extramedullary bones. (A) MSC-ECFC-Matrigel mixture was injected into the flanks of mice 6 weeks before the leukemic-cell transplantation. (i) Images captured by the IVIS system (using ICR filters) show the osteoblastic activity of the extramedullary bones (white arrow). Two weeks after MOLM13/Luc/GFP cell transplantation, the mice were imaged in the IVIS system after injection of the luciferase substrate. (ii) A positive luciferase signal was observed in both mouse skeletons and extramedullary bones (white arrows indicate the extramedullary bones). (B) Histologic sections of spleen, liver, femur, and extramedullary bones were stained with H&E or an anti-GFP antibody. (C) Staining with the hypoxia probe pimonidazole and HIF1α also was performed to determine the presence of hypoxic conditions after leukemia engraftment. Pimonidazole (PIM) staining was converted to fluorescent (green) signal using the CRi multispectral system. The results shown are representative of at least 5 separate experiments.