The role of hypoxia as a key determinant of outcome for human cancers has encouraged efforts to noninvasively detect and localize regions of poor oxygenation in tumors. In this review, we will summarize existing and developing techniques for imaging tumoral hypoxia. A brief review of the biology of tumor oxygenation and its effect on tumor cells will be provided initially. We will then describe existing methods for measurement of tissue oxygenation status. An overview of emerging molecular imaging techniques based on radiolabeled hypoxic markers such as misonidazole or hypoxia-related genes and proteins will then be given, and the usefulness of these approaches toward targeting hypoxia directly will be assessed. Finally, we will evaluate the clinical potential of oxygen- and molecular-specific techniques for imaging hypoxia, and discuss how these methods will individually and collectively advance oncology.
Clinically Relevant Approaches
Clinical studies of HIF-1 have relied upon detection and quantitation of the amount of this protein in tumors through immunohistochemical analysis of tumor biopsies using antibodies against HIF-1-alpha. Such methods are limited in their utility because (1) microscopic biopsy specimens do not offer a complete picture of the macroscopic tumor, and (2) one commonly cannot obtain serial biopsies over the course of treatment. Reporter gene techniques such as those described above have limited capacity for translation because of the difficulties in efficiently and stably transfecting an intact human tumor with a reporter gene construct. A more clinically relevant method of imaging HIF-1 would be to target it with a systemically administered imaging probe.
Figure 1 describes three potential strategies for achieving HIF-1-specific localization of such a probe. The first two approaches involve the imaging agent crossing the cell membrane to bind either HIF-1-alpha in the cytoplasm, or the composite HIF-1 transcription factor in the nucleus. Development of agents that perform these tasks is difficult because of the complexities of facilitating both passage of the agent across membranes as well as rapid clearance from cells without HIF-1-alpha and/or HIF-1. Strategy 3 examines an alternate method of detection of HIF-1 activity. Instead of targeting the imaging probe directly to HIF-1-alpha or HIF-1, it is instead directed toward CA IX, a cell membrane enzyme that is transcriptionally regulated by HIF-1 through an HRE in its promoter. As the probe can target and bind the extracellular portion of the protein, it is not hampered by the difficulty of crossing the cell membrane. In this respect, CA IX can be thought of as an endogenous reporter gene for HIF-1, one that is suitable for imaging using clinically relevant methods.
The carbonic anhydrases (CAs) are a family of enzymes that catalyze the reversible hydration of carbon dioxide to generate carbonic acid. CA IX, along with the other members of the CA enzyme family, is involved in a variety of physiologic tasks including respiration, pH and CO2 homeostasis, electrolyte secretion, and biosynthesis. An analysis of CA IX expression in mice has demonstrated the presence of mRNAs for this protein in the stomach, small intestine, and colon, as well as in skeletal muscle and kidney cells. However, the protein is expressed in moderate amounts only in the stomach and liver, suggesting tissue-specific posttranscriptional regulation. In humans, CA IX is expressed at low levels in normal tissues, with northern blot analysis of tissue extracts revealing moderate levels of CA IX mRNA in only the heart, stomach, liver, pancreas, and salivary gland.
Over the past 15 years, there has been increasing interest in CA IX because of its association with a variety of types of cancer. Expression of this cell-surface CA isoform has been correlated with prognosis in a variety of tumor types.[83-89] Overexpression of this enzyme in tumors is thought to facilitate the characteristic low pH of the tumor extracellular space, which in turn expedites tumor growth and invasion by activating extracellular matrix metalloproteinases. Experiments in CA IX-deficient cell lines transfected with the gene and with CA IX-competent cells transfected with a small interfering RNA to silence expression of this protein have demonstrated the functional importance of CA IX in tumor survival and growth.
Several antibodies to CA IX have been generated, including G250 and M75.[91,92] Further engineering of these antibodies has produced a humanized chimeric form of G250 as well as a number of variants of M75 specific to different epitopes of wild-type and mutant CA IX. The chimeric cG250 antibody against CA IX has been radiolabeled and evaluated as an agent for imaging as well as radioimmunotherapy for renal cell cancers with high expression of CA IX. Preclinical investigations of radiolabeled cG250 in rats bearing renal cell xenograft tumors have demonstrated that this agent achieves tumor-to-blood ratios of ~3 at 72 hours postinjection. A number of clinical trials of cG250 labeled with iodine-131 have been conducted and exhibited a positive therapeutic effect in the treatment of renal cancers.[96-98] These findings suggest localization of the radiolabeled antibody within the tumor and delivery of therapeutic doses of radiation while sparing other organs, conclusions that have been supported by radioimmunoscintigraphy imaging of the radiolabeled antibodies.
Engineered fragments of this antibody have also recently been developed and investigated as methods for targeting imaging and therapeutic agents, with similar pharmacokinetic profiles. Chrastina and colleagues have studied M75 antibodies labeled with iodine-125 as a potential diagnostic and therapeutic agent. The binding affinity of this antibody, which recognizes an epitope in the extracellular proteoglycan domain of CA IX, was measured to be 1.5 nM, with approximately 2.4 X 105 molecules of 125I-M75 binding to a single hypoxic HT-29 colon adenocarcinoma cell in vitro. While antibodies facilitate the targeting of CA IX in tumors with high specificity, this method of localizing drugs and/or imaging probes requires circulation times on the order of 2 to 4 days in order to achieve optimal target-to-background ratios. This limits the type of labels that may be conjugated to the antibodies to generate CA IX imaging probes to long-lived radioactive isotopes (copper-64, iodine-124) or nondecaying signal-generating groups such as fluorophores.
Small-molecule inhibitors of CA IX have also been developed that may prove useful as starting compounds for the development of imaging probes against this protein. Compounds containing a terminal sulfonamide group are potent inhibitors of the carbonic anhydrases because the sulfonamide group binds to to the zinc ion and hydrogen bonds with several amino acid residues within the enzyme's active site. Some molecules of this type have previously been applied in the clinic for other purposes, such as acetazolamide which is used to treat glaucoma and altitude sickness. Recently, several screens of sulfonamide-containing molecules have identified several promising new candidate CA inhibitors, with inhibition coefficients (Kis) in the low nanomolar range and with good specificity for CA IX relative to other CA isoforms.[102-104] These compounds therefore can be expected to possess binding affinities to CA IX similar to those of the antibodies discussed above while exhibiting more rapid clearance.
The utility of one of these small molecules as a potential imaging probe for detection of CA IX and indirectly HIF-1 activity is shown in Figure 2. The positron-emitting CA IX inhibitor 2-[18F]-3,5,6-trifluoro-3´-sulfamoylbenzanilide ([18F]-TFSB) was synthesized through an [18F]-[19F] isotope exchange reaction. Incubation of this radiotracer with cells with varying degrees of expression of CA IX demonstrated a good correlation between retention of [18F]-TFSB and the level of the protein target. Preliminary in vivo microPET imaging of this agent exhibited favorable tumor-muscle uptake ratios for CA IX-expressing HT-29 xenograft models. Background signals are evident in the area of the gut, which may be indicative of clearance pathways of TFSB and/or normal tissue expression patterns of CA IX as discussed above. Further work is currently being conducted to improve the specific activity and binding affinity and specificity of this potential clinical vehicle for imaging CA IX and HIF-1.
The authors have no significant financial interest or other relationship with the manufacturers of any products or providers of any service mentioned in this article.
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