Controls for GLUT-1 and CA9 staining consisted of sections where primary antibody was omitted. Proliferation marker bromodeoxyuridine staining was performed on adjacent sections that had been previously imaged for pimonidazole, GLUT-1, or CA9. Sections were treated with Bafilomycin A1 manufacturer 2 N HCl for 10 minutes at room temperature followed by 0.1 M Borax for 10 minutes at room temperature. Sections were then exposed to Alexa Fluor 594–conjugated anti-bromodeoxyuridine antibody (1:20 dilution; Molecular Probes) for 1 hour at room temperature and washed. Images were acquired using a Nikon Eclipse E800 fluorescence microscope (Nikon America Inc, Melville, NY) equipped with a

motorized stage (Ludi Electronic Products Ltd, Hawthorne, NY). Pimonidazole and Hoechst 33342 were imaged using green and blue filters, respectively. CA9 and bromodeoxyuridine were imaged using a red filter. GLUT-1 was imaged using either a red or a green filter dependent on secondary antibody.

Digital autoradiography (DAR) was obtained by placing the tumor sections in a film cassette against an imaging plate as described previously [9], [13], [16] and [17]. The same plate was used throughout the experiments; the Z-VAD-FMK cost plate was exposed for ~ 20 hours and read by a Cyclone Plus imaging system (PerkinElmer, Inc, Waltham, MA) that generated digital images with pixel dimensions of 42 × 42 μm. DAR PLEK2 images were quantified by the OptiQuant software (PerkinElmer Inc), and tracer uptake was measured as digital light unit per square millimeter (DLU/mm2), which was converted to MBq/g, based on the known section thickness (7 μm) and the system calibration factor, allowing the results to be expressed as percentage injected dose per gram tumor tissue (%ID/g). Ascites fluid pO2 was expressed as median ± SEM, and 18F-FDG uptake was expressed as mean ± SD. Statistical significance was examined by two-tailed Student’s t-test. A P value less than .05 was considered as statistically significant

difference. Ascites fluid pO2 measured by OxyLite systems was as low as 0.90 ± 0.53 mm Hg (0.12 ± 0.07% O2, median ± SEM, n = 63 measurements) in three HT29 ascites carcinoma mice ( Figure 1). Ascites pO2 was 0.97 ± 0.68 mm Hg and 1.01 ± 0.55 mm Hg in A549 and MDA-MB-231 ascites carcinoma models, respectively. For A549, MDA-MB-231, and HT29 cell lines, all single cancer cells and ascites tumors (clusters of pure cancer cells) harvested from ascites fluid were stained positive for both pimonidazole and GLUT-1 (Figure 2A), indicating uniform significant hypoxia. In contrast, larger serosal tumors contained normoxic (both pimonidazole and GLUT-1 were negative) and hypoxic (stained positive for pimonidazole and GLUT-1) cancer cells. Representative images from A549 and MDA-MB-231 serosal tumors were presented in Figure 2B. Similar pattern was observed in HT29 serosal tumors [14].