Multimodal hypoxia imaging in a preclinical glioma model

Introduction: The aim of our study is to analyze the relationship between tumor growth and tumor hypoxia in an orthotopic glioma murine model by using a multimodal procedure, and to compare the features of the different imaging techniques in revealing hypoxia induction. Materials and methods: Engineered U251 cells were gently provided by Dr. Giovanni Melillo, National Cancer Institute, Frederick (MD). These cells express the luciferase reporter gene under control of a constitutive promoter (U251-LUC) or under control of three copies of a Hypoxic Responsive Element (U251-HRE). Cells has been analyzed by means of three different approaches: 1) In vitro evaluation of transcriptional activation of HIF-1? at different times after deferoxamine (DFX) treatment by immunocytochemistry (ICC). 2) In vivo analysis of tumoral progression in orthotopic murine models obtained by stereotaxic injection of 10^5 glioma cells; animals were monitored weekly with BLI (CCD camera), PET ([18F]FDG,[18F]FAZA,[18F]FLT) and MRI to evaluate tumoral progression and hypoxia activation. 3)Ex vivo analysis by H&E staining and hypoxia markers (HIF-1? and CAIX) were performed. Results: In vitro studies showed no differences among the two cell lines as regards to HIF-1? activation kinetic with a peak of activation between 3 and 6h and a decrease at 20h. In vivo, U251-HRE model showed a detectable and progressive luciferase activity induction starting at 18 days from injection demonstrating that luminescence expression is dependent on HRE-mediated activation. On the other hand, in U251-LUC model luciferase activity was detectable immediately after injection and remained proportional to tumor growth. Lesions were highly proliferative ( [18F]FLT) but hypo-metabolic ([18F]FDG). In partial agreement with the results of U251-HRE, a hypoxia dependent [18F]FAZA uptake was observed at later times (30 days). MRI provided morphological characterization and diffusion studies showing hypoxic necrotic areas only at later days. Ex vivo H&E staining and immunohistochemistry (HIF-1? and CAIX) performed after 30 days confirmed in vivo results including the presence of hypoxic regions. Conclusions: This study demonstrates that the U251-HRE orthotopic murine model may be proposed as a predictive and reliable tool to evaluate hypoxia dependent processes of human glioma in preclinical studies by BLI. Differences among the three imaging techniques may be related to methods sensitivity. Further ex vivo post mortem measurements of HIF-1 and CAIX at earlier times will be performed. Additional studies will be conducted to understand the clinical meaning of early or delayed hypoxia identification in terms of response to treatment.