Cell culture REH and Nalm6 cells were purchased from American Type Tissue Collection. Nalm6-GFP-luciferase labeled cells were made at St Jude Children’s Research Hospital by transducing Nalm6 cells with a murine stem cell virus-internal ribosome entry site- GFP retroviral vector containing the firefly luciferase gene. BM or peripheral blood samples were obtained for in vitro studies from patients with AML or ALL. Samples were collected during routine diagnostic procedures after written informed consent was obtained; protocols for studies in humans were approved by the Human Subjects Committee of The University of Texas MD Anderson Cancer Center. Mononuclear cells were separated by Ficoll-Hypaque (Sigma-Aldrich) density gradient centrifugation. Cell lines and primary samples were maintained in RPMI 1640 containing 10% FCS (Gemini Bio-Products) and 1% penicillin- streptomycin (Life Technologies Laboratories). Summarized patient characteristics are shown in Table S2. Standard laboratory (normoxic) conditions comprised 21% O2 and 5% CO2 at 37uC. For experiments in a reduced oxygen environment, the hypoxic Workstation INVIVO2 400 from Ruskinn Technology was used. Cells were incubated in 1% O2 and 5% CO2 at 37uC. Immunohistochemistry Human BM or mouse organs were harvested and fixed by immersion in 4% PFA. The use of human samples was approved by IRB and mouse samples by MDACC IACUC (ACUF Protocol # 01-06-01032). Sections (5 mm thick) were stained with H&E (X5000; Sigma-Aldrich) and analyzed by light microscopy. For the different staining of mouse tissue, the sections were incubated for 1 hour in blocking solution (PBS, 0.5% Tween-20, 0.1% BSA) and 10% FBS followed by incubation overnight with primary antibody. After washing, sections were incubated for 1 hour with secondary antibody, washed in PBS three times, and coverslips were mounted with Fluoromount-G (Electron Microscope Sci- ence). For human BM staining, formalin-fixed, paraffin-embedded tissue sections of BM biopsy specimens were deparaffinized in xylene (3 times for 5 minutes) and then rehydrated through graded concentrations of alcohol. For antigen retrieval, tissue sections were heated in EDTA (Biocare Medical, Concord, Calif) for 30 minutes in a Decloaking Chamber (Biocare Medical, Concord, Calif). Tissue sections were incubated with Biocare Medical peroxidase blocking reagent for 5 minutes. After washing with buffer, tissue sections were incubated with the primary antibodies for 60 minutes at room temperature (20uC). Sections were again washed, and incubated with secondary antibodies using Mach 4 AP Universal Polymer kit (Biocare Medical) according to manufacturer’s instructions. For visualization, Vulcan Fast Red chromogen (Biocare Medical) was used for 10 minutes. All sections were counterstained with hematoxylin (Dako, Carpintera, Calif) for 3 minutes. Slides were then air dried and cover-slipped. Slides were analyzed under a 60x/1.40 PlanApo objective lens on an Olympus FV500 confocal microscope with Fluoview version 4.3 software (Olympus). For quantification of HIF-1a expression or PIM, at least 10 images/sample were aquired using the Nuance FX multispectral imaging system (CRi) which utilizes an optimized high-throughput tunable filter which has been matched to the bandwidths of common chromogens and fluorophores. Images were next quantified and scored by inForm software (CRi). The Nuance FX multispectral imaging system (CRi) was also used to generate pseudofluorescent images of DAB based IHC (PIM and HIF-1a). For assessment of bone marrow vasculature, slides were stained with DAPI and lectin was detected by TRITC fluores- cence using the CRi multispectral imaging system. To quantify Targeting Leukemia-Associated Hypoxia with PR-104 microvessel density (MVD), 10 random fields at 2006 final magnification were examined for each condition (three mice each, NBM and bcCML) and the number of lectin positive microvessels per field was counted. PR-104A in vitro studies Cells were seeded at 16106 cells/mL, treated with PR-104A at a final concentration of 5, 10, 15, or 25 mM, and incubated in normoxic (21%O2) or hypoxic conditions (1%O2) for 6 hours. The medium was then replaced with fresh medium, and cells were incubated in normoxic conditions until collection for FACS analysis. Cell death assessment After appropriate treatment, cells were washed twice in PBS and resuspended in 100 mL Annexin binding buffer containing a 1:100 dilution of Annexin V–FLUOS (Roche Applied Science). Cell numbers were quantitated after the addition of 10,000 CountBright counting beads (Invitrogen) per sample. Cells were then analyzed by a FACS Calibur flow cytometer (BD Biosciences) using 488-nm argon ion and 633-nm HeNe excitation lasers. For primary ALL samples, results were expressed as percentage of specific apoptosis calculated as: (% AnnV positive cells sample-% AnnV positive cells control)/(100-%AnnV positive cells con- trol)*100. Western blot analysis Cells were subjected to lysis in buffer supplemented with a protease inhibitor cocktail (Roche Diagnostics). Lysates were then separated on a 10% to 12% polyacrylamide gel, transferred to PVDF membranes (Life Science Research; Bio-Rad), probed with appropriate antibodies, visualized by using an enhanced chemi- luminescence plus kit (GE Healthcare), and analyzed on the Odyssey imaging system from LI-COR Biosciences. Hypoxia measurement in BM flushes BM flushes from PIM-administered mice were fixed in 4% PFA for 45 minutes at 4uC. After three washes with PBS, specimens were blocked overnight at 4uC in 0.3% Tween20, 1.5% BSA, and 5% mouse serum in PBS. Samples were stained overnight at 4uC with FITC-conjugated mouse mAb anti-pimonidazole (HPI, Inc) at 75 mg/mL. After two washes with 0.3% Tween-20 in PBS and one with PBS, specimens were resuspended in 1% PFA and analyzed by FACS. Results are expressed in terms of MFI. Animal studies All animal work was done in accordance with a protocol approved by the institutional animal care and use committee of MD Anderson Cancer Center or University of New South Wales. Three-month-old NSG mice were irradiated with 300 cGy and injected with leukemic cells from an infant with MLL-rearranged B-lineage ALL (56104 viable cells per mouse). PR-104 treatment started 10 days after injection of cells under the following regimen: 200 mg/kg i.p., 3 days per week for 2 weeks. After 3 weeks without treatment, treatment was resumed at the same dose, twice a week for 2 weeks. For the blast crisis CML model, C57Bl6/J mice were injected with 46106 murine HSC expressing the oncogenes BCR/ABL and Nup98 and labeled with GFP/YFP [30]. Mice were humanly sacrificed 20 hours, 3 days, or 6 days after cell injection. For the Nalm6 B-cell ALL model, NSG mice were injected i.v. with 16106 Nalm6-Luciferase cells/mouse. Starting on day 3, PR- 104 at 250 mg/kg or PBS was administered i.p. 3 times a week for PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e23108