2 weeks. Leukemia progression was evaluated weekly by luciferase imaging weekly as described elsewhere [54]. For the dose-response experiments, NOD/SCID mice were inoculated one of two ALL xenografts (ALL-8 and ALL-19) [34]. PR-104 was administered i.p. at doses of 550 mg/kg, 200 mg/kg, 100 mg/kg, or 50 mg/kg once a week for 6 weeks (seven mice per treatment group). Treatment commenced when the percentage of human CD45+ cells in peripheral blood exceeded the median value of 1% for the complete cohort. The experimental endpoint for each mouse was reached when the percentage of human CD45+ cells in peripheral blood reached 25%. The study was continued until all mice from the cohort reached this leukemia-related event or were sacrificed. Mice were sacrificed if they became morbid or had a weight loss of 20% or greater. Both event free survival (EFS) and leukemia growth delay (LGD) estimations were carried out according to established methodology. Individual mice were assigned a clinical ‘‘score’’ depending on the leukemic growth characteristics observed in the 42 days following treatment initiation according to the established criteria used for evaluating single agents [34,35]. For the primary refractory FLT3-mutated AML model, NSG mice were injected i.v. with 206106 cells/mouse after irradiation (2.5 Gy). Starting on day 72 after cell injection, five mice were treated with PR-104 250 mg/kg or with PBS i.p. 3 times a week for 2 weeks. Cell engraftment and leukemia progression were measured by determining the percentage of CD45+ cells in peripheral blood by FACS. Detection of hypoxia in BM Bones were collected from mice 3 hours after they received PIM 100 mg/kg i.p. and were processed for IHC. Detection of microvessel density in BM TRITC conjugated Lectin from Ulex europaeus (SIGMA- ALDRICH) was injected into mice at 0.5 mg//mouse/IV 1 hour prior to sacrifice. Bones were collected and processed for IHC. Statistics Unless otherwise indicated, results are expressed as mean 6 SD of three independent experiments. P-values were determined by one- way ANOVA followed by F statistics or paired t test (when comparing two groups). A P-value less than 0.05 is considered significant. EFS between control and treated mice were compared by logrank test. Supporting Information Figure S1 Pimonidazole binding to Nalm6 and REH cell lines in a hypoxia dependent manner. Cells were incubated with 100 mM PIM for 3 hours at different oxygen concentrations and then washed, fixed and processed for FACS. Control, cells stained with antibody only without PIM pre-incubation. MFI ratios compared to control were as follows: Nalm6 21% O2 1.9; Nalm6 1%O2 6.9; Nalm6 0%O2 9.5; REH 21% O2 1.6; REH 1%O2 10.3; REH 0%O2 22.2. (TIF) Figure S2 BM vasculature is altered 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. Six days after cell transplantation, mice were injected with TRITC conjugated lectin 1 hr prior to sacrifice. Micro vessels were detected by fluorescence in BM from control leukemia-free mice (NBM) or bcCML bearing mice (bcCML). The lower panel shows quantification of lectin-positive vessels by CRi spectral imaging and Inform software analysis (3 mice/each, at least 10 slides per Targeting Leukemia-Associated Hypoxia with PR-104 mouse were analyzed). Original magnification is shown on the figures. ***P,0.0001. (TIF) Figure S3 A. HIF-1a is expressed at low levels in normal BM samples. Representative images from 3 normal donors are shown. Original magnification, 6500. (HIF-1a: green; nuclei: red). B. HIF-1a is expressed in stromal cells as well as in leukemic blasts in BM biopsy from ALL patients. A representative image from one ALL BM sample obtained at diagnosis (4 samples were analyzed with similar findings). HIF-1a was detected by IHC and the different cells were identified based on morphology. Original magnification 61000. (TIF) Figure S4 AKR1C3 expression in cell lines (A), primary ALL samples (B), CD34+ cells from normal donors and cord blood (C) was detected by Western Blot. Extract from SKOV3 cells was used as positive control. (TIF) Figure S5 PR-104 decreased leukemia burden of NSG mice transplanted with primary refractory FLT3-mutat- ed AML and restored tissue architecture. Treatment started on day 72 after cell injection: 250 mg/kg, i.p. 3 times a week for 2 weeks (A) PBS (upper panel) or PR-104 (lower panel) treated mice were bled periodically and leukemia progression was assessed by determining the percentage of circulating human CD45 positive cells by FACS. X axis represents time with respect to starting of the treatment. (B) PR-104 inhibited AML leukemia growth in NSG mice in the lung, liver, bone, and spleen and resulted in dramatic reduction of hypoxic expansion observed in the control mice. CD45 and H&E IHC in lung, liver, BM, and spleen from PBS-treated control and PR- 104-treated mice. (C) Pimonidazole was administered 3 hours prior to sacrifice and its adducts were detected in BM sections. Original magnification, 6 400. (TIF) Table S1 LGD summary and objective response mea- sures (ORM) for PR-104-treated ALL-8 or ALL-19 engrafted mice. (DOC) Primary patient data summary. N/A: not Table S2 available. (DOC) Acknowledgments We are indebted to all members of Dr. Michael Andreeff’s Section of Molecular Hematology & Therapy at MDACC for valuable discussions and technical suggestions. Many thanks to Wendy Schober, Jared Burks and Amy Hazen from the Flow Cytometry and Cellular Imaging Facility and personnel from the Research Animal Support Facility at MD Anderson Cancer Center. We thank Dr. Peter Ruvolo for editorial assistance. Children’s Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children’s Hospital. Author Contributions Conceived and designed the experiments: JB BS HC IB SK PAZ-M DC GB CB-R ES DAT CTJ HK WRW RL MA MK. Performed the experiments: JB YS BS HC IB HL SK WF. Analyzed the data: JB YS BS HC IB HL SK PAZ-M WRW RL MA MK. Contributed reagents/ materials/analysis tools: DC CTJ. Wrote the paper: JB RL MK. Revised manuscript for intellectual content: YS HL SK PAZ-M DC CB-R ES DAT CTJ HK WRW RL MA MK. PLoS ONE | www.plosone.org 12 August 2011 | Volume 6 | Issue 8 | e23108