Imaging and quantitation of the hypoxic cell fraction of viable tumor in an animal model of intracerebral high grade glioma using [¹⁸F]fluoromisonidazole (FMISO)

Abstract

We have demonstrated that FMISO uptake is significantly higher in tumor tissue in the C6 intracerebral glioma rat model compared to normal brain, and that there is persisting hypoxia in gliomas independent of tumor size. FMISO uptake was observed homogeneously throughout viable glioma tissue in tumor sizes ranging from 2mm to almost 1cm. Quantitation of uptake of FMISO showed a tumor/brain ratio of 1.9 and a tumor/blood ratio of 2.6 at 2 hours post injection.

Introduction

Primary cerebral tumors are responsible for approximately 2% of all cancer deaths, with the majority of these deaths due to the high grade gliomas, anaplastic astrocytoma and glioma multiforme. In the early 1950’s radiation therapists made the observation that tumor oxygenation plays a role in the response to radiotherapy treatment [12], [29]. Preclinical studies [8], [11] both in vitro and in vivo have established that hypoxia protects cells from the cytotoxic effects of radiation and chemotherapeutic agents, and thereby may be a significant factor in therapeutic resistance both of glioma and other tumor types [10], [37]. Research studies have also shown abnormal vasculature developing within tumors (angiogenesis) which is poorly regulated, and associated with reduced or uneven vascular density, abnormalities of vessel orientation, and impaired blood flow [5]. Until recently, efforts to investigate hypoxia in vivo and possible remedies for it have been hampered by difficulties in quantifying the hypoxic fraction of tissue, and suitability of appropriate models to aid in the development of new therapeutic strategies. Positron emission tomography (PET) has emerged as a powerful diagnostic tool in oncology in the staging of a range of cancers and monitoring disease recurrences [36]. Glioma grade can be assessed accurately and non-invasively by FDG PET, because the rate of glucose utilization is directly proportional to the degree of malignancy [7]. More recently, FMISO and PET have been used for identifying hypoxic tissue after ischemic stroke and intracerebral hemorrhage in human [14], [34], [35].

The structure of 2-nitro-1H-imidazole (azomycin), an antibiotic substance active against infections associated with anaerobic conditions, was described by Nakamura in 1955 [30], and in 1979 Chapman and collaborators suggested that such compounds might be adapted to allow visualization of hypoxic tissue in vivo [3]. Many nitroimidazole analogues have been synthesized since then, leading to a large range of potential hypoxic cell markers when radiolabelled with iodine-123 or -125 [18], [26], [27], fluorine-18 [4], [15], [17], [19], [23], [24], [38], [40], [42], [43] and technetium-99m [25], [28]. With the exception of the iodo-labeled compound ¹²³I-IAZA [13] and of the technetium-labeled compound ^99mTc-BMS-181321 [1], which have only been reported in one clinical study each, the fluoro-labeled compound ¹⁸F-fluoromisonidazole (FMISO) [2], [21], [32], [44] represents the only marker currently reported to image hypoxia in a variety of solid tumors in humans. In addition, FMISO uptake in patients with glioma has been reported in one PET study [39]. To date, however, the study of hypoxia in animal models has been restricted to subcutaneous tumors [22], [33], which has limitations in extrapolation to gliomas where considerations such as blood brain barrier and perfusion are critical when new therapies are being developed and validated.

The C6 rat intracranial glioma model has long been accepted as a close approximation to this tumor in humans, demonstrating many of the defining characteristics of a high grade astrocytoma including a high mitotic index, palisading of cells near the necrotic regions and pyknotic nuclei. Furthermore, it has been found to be GFAP (glial fibrillary acid protein) positive and unlike the one other commonly used glioma model 9L, it contains no sarcomatous elements. Before using such a model to test therapies that reverse hypoxia, it is necessary to define the characteristics of FMISO in relation to intracranial tumor cells in this C6 rat glioma model, and the distribution of FMISO in normal brain and other tissues. In order to determine the confounding effects of tumor size on the presence and degree of hypoxia, we also sought to study FMISO uptake in rats with differing sizes of glioma. Due to the size of intracranial tumors, we employed autoradiography using phosphorus-imaging plates to determine the relationship of hypoxic cell fractions to viable tumor cells in the C6 rat glioma model.