In vivo colocalization of 2-nitroimidazole EF5 fluorescence intensity and electron paramagnetic resonance oximetry in mouse tumors

Background and purpose: The primary objective of this study was to establish in vivo the relationship between 2-2-nitro-1H-imidazol-1yl-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5) adduct formation and intratumoral oxygen concentrations measured by electron paramagnetic resonance (EPR) in a tumor model mimicking a clinical situation. The secondary objective was an attempt to calibrate in situ the immunofluorescence (IF) signal with EPR oximetry. Materials and methods: IM syngeneic fibrosarcoma (NFSA) bearing C3H mice were used. Three days after injection of a paramagnetic charcoal into the tumor, the mice were anesthetized, injected with the hypoxic marker EF5, and monitored every 20 min for 3 h with a low-frequency EPR spectrometer. Animals were allowed to breath either under 21 or 100% O-2. Tumors were then harvested, frozen, cut into sections including the charcoal and processed for EF5 adducts detection using monoclonal antibodies. Slices were viewed with a fluorescence microscope and 190 X 140 mum areas surrounding the charcoal were digitized and analyzed with the NIH-Image and Adobe Photoshop(TM) software. The fluorescence intensity (FI) was measured in the whole pictures and in strips of 10 mum around the charcoal. Results: EF5 binding increased with decreasing pO(2), most substantially at pO(2) below 5 mm Hg. Baseline (ambient air) pO(2) reached 3.2 +/- 2.1 mm Hg in NFSA tumors. It increased to 9.8 +/- 3.2 mm Hg under 100% O-2. A statistically significant correlation was observed on an individual tumor basis between the FI in the first 10 mum strip around the charcoal and the pO(2) determined by EPR oximetry (Wilcoxon signed rank test: P < 0.001). Conclusions: The present study confirms the intrinsic relationship between EF5 adduct binding and intraturnoral pO(2) in an in vivo environment under biologically-relevant pO(2) values of less than 10 mm Hg. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.