Targeting Leukemia-Associated Hypoxia with PR-104 Figure 2. Expansion of the hypoxic BM niche in a syngenic model of blastic phase CML. 16107 GFP/YFP labeled cells expressing the oncogenes BCR/ABL and Nup98 were FACS-sorted and transplanted into irradiated (4.5GY) C57B16/J mice. At the indicated time-points, mice were injected with pimonidazole 3 hrs prior to sacrifice. Areas of hypoxia were detected by PIM, and leukemic cells by anti-GFP in BM (A) or spleen (B). (C) and (D) Quantification of PIM positive cells in BM and spleen, respectively by CRi spectral imaging and Inform software analysis (at least 3 mice per group were analyzed). Original magnification, 6100. *P,0.05; **P,0.01. doi:10.1371/journal.pone.0023108.g002 leukemia progression was monitored by luciferase imaging. While control animals showed luciferase positivity indicating detectable tumor burden on day 14 after injection, none of the PR-104- treated mice had a positive signal (Figure 6A). Twenty two days after injection, all control mice were strongly positive for luciferase activity while the treated animals showed much smaller positive areas, indicating that PR-104 inhibited the growth of Nalm6 cells in vivo. Remarkably, on day 28 (when mice were sacrificed) one mouse from the PR-104 treated group remained leukemia-free while all the mice in the control group exhibited high levels of luciferase activity. Before the mice were sacrificed, PIM was injected to evaluate BM hypoxia. As shown in Figure 6C, the BM of control animals were predominantly infiltrated by GFP+ leukemia cells, which colocalized with PIM adducts, whereas the PR-104-treated mice had very few GFP+ cells and only small areas of PIM staining. Expression of the HIF-1a targets CAIX and CXCR4 correlated very closely with that of PIM, confirming the expansion of hypoxic zones in the untreated leukemic BM and inhibition of this process upon PR-104 treatment. No significant differences were seen in the expression levels of stroma-derived factor 1a (SDF-1a). Although the microvessel density assessed by CD31 staining did not differ significantly between control and treated mice, vessels appeared tortured and compressed in leukemic as compared to normal BM. To examine the efficacy of PR-104 in killing primary ALL cells in vivo, NSG mice were injected with leukemic cells from an infant with MLL-rearranged B-lineage ALL. Leukemia burden was determined by the percentage of human CD45+ cells circulating in peripheral blood. In control mice, the percentage of circulating CD45+ cells increased steadily, reaching 9362.3% on day 52 (Figure 7A). In contrast, CD45+ cells remained undetectable in treated mice while they received the drug and up to a week later. Percentage of circulating CD45+ cells then increased but decreased significantly on re-initiation of treatment (from 45615% on day 38 to 10610.2% on day 52). PIM was administered to the control and treated mice before they were PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23108