C. Clifton Ling: Tumor Hypoxia Imaging

The focus of our laboratory is the study and noninvasive imaging of tumor hypoxia. One major goal is to examine the biological underpinning of tumor hypoxia imaging and to validate noninvasive nuclear imaging approaches using rodent models in laboratory studies.

To examine the biological basis of hypoxia imaging, we are using a novel model system with a hypoxia-induced molecular switch. Specifically, R3327 AT Dunning rat prostate cancer cells have been transduced with a reporter gene construct tkeGFP (the herpes simplex virus 1 thymidine kinase HSV1:tk fused with the enhanced green fluorescent protein eGFP) under the regulation of the hypoxia responsive element (HRE). In the derived clonal cell lines, the stabilization and upregulation of the hypoxia inducible factor 1 alpha (HIF-1a) by tumor hypoxia and the transactivation of the HRE by HIF-1a, provide the molecular switch for activating the downstream genes.

In transplanted tumors/xenografts using these cell lines, the “switching-on” of the reporter gene will be visualized by nuclear imaging techniques, noninvasively with PET and invasively with digital autoradiography of tumor sections, using the positron-emitting tracers ¹²⁴I-FIAU or ¹⁸F-FEAU. Fluorescence microscopy of tumor sections can be performed to image the distribution of endogenous and exogenous hypoxia markers, e.g., HIF-1α, Ca9, VEGF, pimonidazole, and EF5. With this approach, the initial molecular events and its spatial distribution, induced by hypoxia, can be determined experimentally and compared with similar determination of phenomena associated with tumor hypoxia, i.e., expression of downstream genes and trapping of exogenous markers.

These tumor models serve as valuable tools for evaluating the use of promising PET tracers for noninvasive imaging hypoxia; and we are studying ¹⁸F-FMISO, ¹²⁴I-IAZG and ¹⁸F-EF5. For such evaluation, microPET images of the endogeneous reporter system are obtained with either ¹⁸F-FEAU or ¹²⁴I-FIAU and compared with similar images obtained with each of the 3 exogenous tumor hypoxia imaging agents mentioned above.

As a reference, pO₂ probe measurements will be performed with the Oxylite and Eppendorf Hypoximeter systems. These measurements will be image-guided, and the data will therefore be spatially correlated with the microPET images, using a stereotactic marker template system that permits multimodality image registration and image-guided placement of probes and needles. In pilot studies, we have successfully used this system for image-guided pO₂ measurement using the Oxylite probe.

The xenograft models with the hypoxia-inducible reporter gene and microPET imaging provide unique opportunities to study changes in tumor hypoxia. For example, we shall study the dynamics of tumor hypoxia in xenograft models using repeated ¹⁸F-FEAU / microPET imaging. In addition, the alteration of tumor hypoxia, due to intervention such as carbogen or hyperbaric oxygen, breathing will be similarly evaluated. Also, we plan to assess radiosensitivity in regions of high- and low-tumor hypoxia, based on the radiation response assay of histone cluster γH2AX analysis. Radioresponse assessment will be compared with microPET or digital autoradiographic images of the same sections.

In collaboration with Dr. Jason Koutcher and his nuclear magnetic resonance (NMR) laboratory, we shall perform multimodality tumor hypoxia imaging to compare NMR with nuclear imaging approaches in yielding information on tumor hypoxia, using the tumor model described above. The NMR methods will include dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), blood oxygen level dependent (BOLD) and FLOOD T2* imaging, and the measurement of lactate. Although the images obtained are not of hypoxia per se, they are surrogates of it and may provide valuable information of the pO₂ status of the tumor. To facilitate registration between the NMR and μPET images, NMR imaging will be carried out using the stereotactic marker template previously described.