Molecular imaging applied on hypoxia evaluation: comparison of BLI, FLI and PET imaging

Introduction: Hypoxia is implicated in many aspects of tumor growth and progression, indeed it is considered the major driving force of glioma angiogenesis, and it is involved in the intracellular stabilization of the hypoxia master-regulator gene HIF-1?. To this aim, we have used a cell imaging tool based on the U251-HRE cell line, to study simultaneously glioma features and hypoxia by means of optical and nuclear based imaging techniques. Methods: We performed the in vivo analysis of tumoral progression in an orthotopic murine model obtained by stereotaxic injection of 105 glioma U251-HRE cells. Mice were analyzed by means of different approaches:1) comparison of 3D bioluminescence and fluorescence (Hypoxisense680 fluorescent probe), both supplemented by CT anatomical data; 2) comparison of fluorescence imaging (Hypoxisense680 probe) and nuclear imaging ([18F]FAZA) in hypoxia detection; 3) correlation between in vivo [18F]FLT uptake and ex vivo Ki67 proliferation marker; 4) ex vivo validation of bioluminescence signal through IHC luciferase staining. Results: 3D bioluminescent signals provided information about tumor growth and hypoxia presence, underlining the volumetric dimension and the depth of the bioluminescent signal, by the overlay with the CT anatomical data. Fluorescence acquisition with the HypoxiSense680 probe showed a wide hypoxic area overlaying with the bioluminescent signal, demonstrating that also the observed bioluminescence was due to hypoxia induction. HypoxiSense680 fluorescent probe analysis and [18F]FAZA PET analysis showed similar results, even if fluorescent signal detected hypoxia presence at earlier time point than PET imaging. IHC for Ki67 confirmed the data obtained by [18F]FLT PET, showing a high rate of proliferation within the lesions. Finally, IHC for luciferase antibody supported results observed in vivo by bioluminescent analysis. Conclusions: Our preclinical model, and the powerful imaging strategy proposed herein, would be useful for the early identification of hypoxic regions and for the validation of specific treatments, accelerating the translation of preclinical data into clinical applications. Targeting HIF-1? activity in hypoxia microenvironment could be extremely important to improve therapeutic approaches and, maybe in the future, to stratify glioma patients basing on intratumoral hypoxia level and to maximize toxic effects of the antineoplastic treatment. References: Cancer Research 62, 4316-4324, 2002