Radiolabeled Probes Targeting Hypoxia-Inducible Factor-1-Active Tumor Microenvironments

Because tumor cells grow rapidly and randomly, hypoxic regions arise from the lack of oxygen supply in solid tumors. Hypoxic regions in tumors are known to be resistant to chemotherapy and radiotherapy. Hypoxia-inducible factor-1 (HIF-1) expressed in hypoxic regions regulates the expression of genes related to tumor growth, angiogenesis, metastasis, and therapy resistance. Thus, imaging of HIF-1-active regions in tumors is of great interest. HIF-1 activity is regulated by the expression and degradation of its α subunit (HIF-1α), which is degraded in the proteasome under normoxic conditions, but escapes degradation under hypoxic conditions, allowing it to activate transcription of HIF-1-target genes. Therefore, to image HIF-1-active regions, HIF-1-dependent reporter systems and injectable probes that are degraded in a manner similar to HIF-1α have been recently developed and used in preclinical studies. However, no probe currently used in clinical practice directly assesses HIF-1 activity. Whether the accumulation of 18F-FDG or 18F-FMISO can be utilized as an index of HIF-1 activity has been investigated in clinical studies. In this review, the current status of HIF-1 imaging in preclinical and clinical studies is discussed.


Introduction
Insufficient blood supply to a rapidly growing solid tumor leads to hypoxia-oxygen tension that is below physiological levels. The median value of partial oxygen pressure (pO 2 ) is reportedly 10 mmHg in breast cancers but is 65 mmHg in normal breast tissue [1]. Hypoxic regions are not only critically important in tumor physiology and treatment, but also strongly associated with malignant progression, therapeutic resistance, and poor prognosis [2][3][4]. In such regions, the transcription factor hypoxia-inducible factor-1 (HIF-1) is overexpressed. HIF-1, one of the critical components of hypoxic responses, is a master transcriptional activator of various genes related to malignant tumor phenotypes [5][6][7]. Therefore, the development of techniques to noninvasively detect HIF-1-active hypoxic tumor cells has received considerable interest. Such techniques will provide useful information that can be applied to qualitative diagnosis of tumors and development of therapeutic strategies for cancer.
HIF-1 , leading to its degradation in the 26S proteasome [16,17]. However, proline residue hydroxylation does not occur under hypoxic conditions, allowing HIF-1 to escape degradation and move into the nucleus where it associates with HIF-1 and exerts its transcriptional activity by binding to hypoxia-responsive elements (HREs) ( Figure 1) [18,19]. Because HIF-1 is constitutively expressed in the nucleus, the transcriptional activity of HIF-1 is regulated by HIF-1 expression and degradation.
HIF-1 expression is reported to increase dramatically in conditions where pO 2 is less than 6% (40 mmHg) [20]. However, the pO 2 threshold values that lead to HIF-1 stabilization vary among different organs [21]. Because pulmonary cells are consistently exposed to relatively high pO 2 , the values that these cells perceive as abnormal are relatively high [11]. In contrast, the bone marrow is the only tissue that expresses HIF-1 protein under normal physiological conditions [22]. Therefore, HIF-1 is stably present in "biologically hypoxic regions, " where the tissues and/or cells themselves sense oxygen deficiency. These regions do not coincide completely with "physically hypoxic regions, " where pO 2 is less than 10 mmHg. Thus, HIF-1-dependent probes or reporter systems are required to directly visualize HIF-1active hypoxic regions.

Monitoring HIF-1 Activity In Vivo by Using HIF-1-Dependent Reporter Gene Imaging
For the first time, nuclear medical molecular imaging of HIF-1 activity in vivo has been performed using a HIF-1-dependent reporter system. In the field of molecular biology, reporter systems are commonly used to monitor the expression of a gene of interest. To directly monitor HIF-1 activity, cell lines that stably contain a reporter gene suitable for in vivo imaging downstream of HRE repeats have been established. When implanted into animals, these cells express the reporter protein in a HIF-1-dependent manner, and the amount of reporter protein expression can be measured using a molecular probe that binds to or is metabolized by these proteins. Serganova et al. [23] and Wen et al. [24] performed positron emission tomography (PET) imaging to detect HIF-1 activity in tumors, using herpes simplex virus type 1 thymidine kinase (HSV1-TK) as a reporter gene; a PET reporter of in vivo gene expression has been widely used [25,26]. Although endogenous TKs are normally present in mammalian cells, their substrate specificity differs from that of HSV-1TK. Because HSV1-TK has broader specificity than mammalian TKs, a probe that is selectively phosphorylated by viral TKs can be used to visualize HSV1-TK expression [27]. Serganova et al. [23] constructed a retroviral vector bearing the HRE-HSV1-TK reporter gene and transfected it into C6 glioma cells. The reporter system showed that dose-dependent patterns in the temporal dynamics of HIF-1 transcriptional activity were induced by decreased pO 2 . PET imaging using 2 -[ 18 F]fluoro-2 -deoxy-1 -darabinofuranosyl-5-ethyl-uracil ( 18 F-FEAU), a radioactive probe selectively metabolized and retained in HSV1-TKexpressing cells, clearly visualized tumors. The spatial heterogeneity of HIF-1 transcriptional activity as a function The Scientific World Journal 3 of tumor size was shown in a study of reporter xenografts in mice. With increasing tumor diameter (>3 mm), HIF-1 transcriptional activity markedly increased in the tumor core regions. This marked one of the earliest successes in nuclear medical molecular imaging for HIF-1 activity in vivo. Since then, several approaches for noninvasive imaging of HIF-1 activity have been reported, including those using different combinations of reporter genes and radiolabeled probes such as sodium-iodide symporter and 99m Tc-pertechnetate [28], glycosylphosphatidylinositol-anchored avidin and 67 Ga-DOTA-biotin [29], and HSV1-TK and 9-(4-[ 18 F]fluoro-3-hydroxymethyl-butyl)guanine ( 18 F-FHBG) [30]. Brader et al. [31] constructed RH7777 Morris hepatoma cells bearing a triple reporter gene (HRE-HSV1-TK/green fluorescent protein/firefly luciferase) and prepared an orthotopic liver tumor model. They performed 18 F-FEAU-PET and luciferase bioluminescence imaging and succeeded in hypoxia-driven reporter imaging using both techniques.
HIF-1-dependent reporter systems are also useful for comparing HIF-1 activity and exogenous markers of tumor hypoxia. Wen et al. [24] generated rat prostate adenocarcinoma cells containing HRE-HSV1-TK fused to enhanced green fluorescent protein as a reporter. PET imaging was performed using 2 -fluoro-2 -deoxy-1 -darabinofuranosyl-5-[ 124 I]iodouracil ( 124 I-FIAU), and the distributions of 124 I-FIAU and the exogenous hypoxic cell marker [ 18 F]fluoromisonidazole ( 18 F-FMISO) were compared. 124 I-FIAU PET imaging of hypoxia-induced reporter gene expression was feasible, and the intratumoral distributions of 124 I-FIAU and 18 F-FMISO were similar. Similar results were obtained for human colorectal HT29 cancer cells bearing the same reporter system [32]. However, no significant correlation between the 18 F-FMISO PET and HIF-1-dependent reporter readouts has also been reported [33]. The reasons for the discrepancy between these findings remain unclear and warrant further investigation. Notably, these described reporter systems did not include the ODD in the expressed reporter proteins. The degradation of HIF-1 is reported to occur within a few minutes under normoxic conditions [34]. Thus, although reporter protein expression was regulated by HIF-1, it is necessary to acknowledge that the reporter proteins may not necessarily reflect real-time HIF-1 activity once the expression was completed.

Molecular Imaging of HIF-1-Active Tumor Microenvironments by Using Probes with Oxygen-Dependent Degradation
HIF-1-dependent reporter imaging systems are excellent tools not only for obtaining spatiotemporal information regarding HIF-1 expression, but also for evaluating the effectiveness of hypoxia-targeting therapies in vivo. However, these systems require exogenous gene transfection and are difficult to apply to humans. To overcome this limitation, a number of injectable probes containing ODD that degrade in an oxygen-dependent manner have been developed [35][36][37][38][39][40][41]. to be delivered to all tissues. In normoxic tissues, oxygen-dependent POS degradation occurs and 123 I-IBB loses its binding partner and is cleared. In contrast, in hypoxic tissues, 123 I-IPOS escapes degradation and is retained within cells owing to its molecular size. Thus, 123 I-IPOS allows for specific imaging of HIF-1-active hypoxic microenvironments.

Chimeric Fusion Protein Probes Containing Oxygen-Dependent Degradation Domains.
To design a probe that degraded in a manner similar to HIF-1 , Kudo et al. [35] selected HIF-1 548-603 , the region of ODD that is essential for oxygen-dependent degradation [42], as the core structure of the probe. An amino acid sequence that increases cell membrane permeability-the protein transduction domain (PTD) [43]-was included in the probe to facilitate its transport into cells. PTD-ODD was fused to monomeric streptavidin (SAV), generating the chimeric protein PTD-ODD-SAV (POS). POS was then labeled with the radioiodinated biotin derivative (3-[ 123 I]iodobenzoyl)norbiotinamide ( 123 I-IBB) to produce 123 I-IBB-POS ( 123 I-IPOS) [35]. The principle behind the imaging of HIF-1-active hypoxic microenvironments by using 123 I-IPOS is outlined schematically in Figure 2. 125 I-IPOS showed more than 2-fold greater accumulation in cells incubated under hypoxic conditions (0.1% O 2 ) than under normoxic conditions (20% O 2 ). Size-exclusion high-performance liquid chromatography (HPLC) analysis revealed that more than 80% of intracellular radioactivity was derived from intact 125 I-IPOS in the hypoxic cells. Reoxygenation analysis showed that the intracellular radioactivity of the reoxygenated group decreased by approximately 40%, compared to that under hypoxic conditions, and the radioactivity excreted into the medium were mostly attributable to 125 I-IBB and other small molecules [35]. These findings suggest that 125 I-IPOS is stable under hypoxic conditions but is degraded under normoxic conditions and that 125 I-IBB is excreted from cells. Biodistribution analysis showed that radioactivity accumulation in tumors 24 h after 125 I-IPOS administration was 1.4 ± 0.3% of the injected dose per gram of tissue, and the tumor-to-blood ratio, which serves as an index of favorable image contrast, was 5.1. Forty-eight hours after 125 I-IPOS administration, radioactivity in the tumor decreased, but the tumor-to-blood ratio increased [35]. Single-photon emission computed tomography (SPECT)/Xray computed tomography imaging with 125 I-IPOS clearly visualized the tumors. Autoradiographic study showed that the intratumoral distribution of 125 I-IPOS is heterogeneous and corresponds to HIF-1 -positive regions detected using immunostaining [38]. Moreover, a positive and significant correlation was observed between 125 I-IPOS accumulation and HIF-1-dependent luciferase bioluminescent signals in the identical tumor [35]. These findings indicate that 123 I-IPOS is a promising probe for imaging HIF-1-active hypoxic microenvironments in tumors.

Rapid Detection of HIF-1-Active Tumor Microenvironments by Using POS: A Pretargeting Approach.
Although PET is used less widely than SPECT in nuclear medical molecular imaging, it allows for higher sensitivity and resolution, as well as high-quality quantitative imaging. Thus, PET imaging of HIF-1-active tumors can provide precise information for determining appropriate therapeutic strategies and predicting prognoses of patients. Fluorine-18 is one of the most widely used radionuclides for PET imaging because of its ease of production, low positron energy, and adequate half-life (110 min). However, the half-life is too short to obtain signals 24 h after administration, at which time clear images can be obtained using 123 I-IPOS. Therefore, development of an imaging procedure that can be used to obtain highcontrast images during the early period after administration is required for successful PET imaging using 18 F.
A pretargeting approach uses a combination of tumortargeting molecules and the prompt clearance of small radioactive compounds from the blood. One advantage of this method is that it can provide a high target: nontarget organ ratios shortly after injection. Therefore, we utilized a pretargeting approach based on the high-affinity and specific interaction between SAV and biotin for the PET imaging of HIF-1-active tumor microenvironments. The underlying principle of pretargeted imaging of HIF-1active hypoxic microenvironments by using POS and (4-[ 18 F]fluorobenzoyl)norbiotinamide ( 18 F-FBB) is outlined schematically in Figure 3.
Conjugation of N-succinimidyl-4-[ 18 F]fluorobenzoate and norbiotinamine yielded 18 F-FBB, with a total synthesis time of 150 min, radiochemical yield of 23%, and radiochemical purity of >95%. An in vitro binding assay confirmed that 18 F-FBB bound to the SAV moiety of POS. Biodistribution analysis showed that 18 F-FBB was rapidly cleared from the body without POS pretargeting. The tumor-to-blood and tumor-to-muscle ratios were <1 at all investigated time points, indicating that 18 F-FBB itself does not show tumor accumulation. In contrast, 18 F-FBB accumulated in tumors in POS-pretargeted mice. 18 F-FBB was able to enter the tumor cells by passive diffusion owing to its lipophilicity  Figure 3: Principle underlying the imaging of HIF-1-active tumor microenvironments, using pretargeted POS and 18 F-FBB. The PTD enables the delivery of POS to all tissues. In normoxic tissues, POS degrades in a manner similar to HIF-1 . In contrast, in hypoxic tissues, POS escapes degradation and is retained inside the cells. After allowing sufficient time for POS to degrade in normal tissues, 18 F-FBB is administered. 18 F-FBB enters cells by passive diffusion and binds to the SAV moiety of the POS retained in hypoxic cells; this does not occur in normoxic tissues. Therefore, pretargeting POS followed by 18 F-FBB administration enables specific imaging of HIF-1-active hypoxic microenvironments. and bind to POS retained in tumor cells. Both tumor-toblood and tumor-to-muscle radioactivity ratios increased over time [37]. Tumor-to-blood ratios comparable to those at 24 h after 123 I-IPOS administration can be obtained within 3 h of 18 F-FBB administration by using the pretargeting method. Pretreatment with excessive d-biotin significantly inhibited 18 F-FBB accumulation in POS-pretargeted tumors. Size-exclusion HPLC analyses revealed that 80% of intratumoral radioactivity is attributable to macromolecules. Taken together, these findings show that 18 F-FBB binds to POS in vivo, and PET imaging clearly delineated tumors within 3 h after injecting 18 F-FBB into POS-pretargeted mice. Therefore, the pretargeting approach makes it possible to reduce the time taken from probe administration to image acquisition by 8fold, allowing imaging to be performed within the half-life of 18 F. In mice with tumors that had been transfected with a HIF-1-dependent luciferase reporter gene, 18 F-FBB accumulation positively correlated with HIF-1-dependent luciferase bioluminescent signals. The 18 F-FBB-distributed areas were consistent with HIF-1 -positive areas in tumors pretargeted with POS [37]. Thus, these findings demonstrate that 18 F-FBB accumulation in the POS-pretargeted tumors reflects HIF-1activity and the pretargeting approach with POS and 18 F-FBB is suitable for rapid imaging of HIF-1-active tumors.

Peptide Probes Containing Essential Amino Acids for
Oxygen-Dependent Degradation. POS is a 34 kDa protein The Scientific World Journal 5 generated in Escherichia coli. Although promising results for the imaging of HIF-1-active tumor microenvironments were obtained, as described above, clinical applications are limited by the possibility of immunogenicity. Peptide probes or small molecules are favorable for clinical applications because they can be easily synthesized with high purity and show no immunogenicity. Our research group succeeded in developing a peptide-based imaging probe that is degraded in a manner similar to HIF-1 [41].
Based on the degradation mechanism of HIF-1 , we selected HIF-1 547-574 as the basic structure for the oxygensensitive peptide probe. For site-specific radiolabeling, a glycyl cysteine was introduced into the C-terminal of the HIF-1 547-574 scaffold, yielding a peptide named OP30. OP30 was degraded when incubated with HeLa cell lysates but was unable to penetrate cell membranes. Thus, to increase its membrane permeability, the nine -terminal amino acids of OP30 were replaced by l-lysine or d-lysine, producing peptides KOP30 and DKOP30, respectively. As expected, these modified peptides were also degraded in HeLa cell lysates and accumulated in the tumor cells. Degradation was inhibited by the addition of a proteasome inhibitor. Furthermore, by replacing the proline residues in KOP30 and DKOP30 that are essential for oxygen-dependent degradation with L-alanine, the resulting peptides (mKOP30 and mDKOP30, resp.,) were not degraded in HeLa cell lysates. Taken together, these results indicate that the proline residues and proteasome function are required for the degradation of KOP30 and DKOP30, similar to HIF-1 . Biodistribution analysis showed that 125 I-DKOP30 had better tumor accumulation and tumor-to-blood ratio than 125 I-KOP30. Therefore, we performed an additional study using 123/125 I-DKOP30. Tumors were visualized clearly by planar imaging using 123 I-DKOP30, and autoradiographic studies showed that the intratumoral distribution of 125 I-DKOP30 was consistent with the HIF-1 -positive areas detected using immunohistochemical staining. Moreover, a positive and significant correlation was observed between 125 I-DKOP30 accumulation and HIF-1-dependent luciferase bioluminescent signals within the same tumor, whereas no correlation between 125 I-mDKOP30 accumulation and bioluminescent signals was observed. These findings indicate that 123 I-DKOP30 is a useful peptide probe for imaging HIF-1-active tumors.
Because many peptide probes have already been used in PET/SPECT imaging in humans [44][45][46], 123 I-DKOP30 has the most potential probe for imaging HIF-1-active tumors in clinical settings so far. Using planar imaging, the accumulation of 123 I-DKOP30 in tumors was found to be higher than the background radioactivity in the cervicofacial and thoracic regions; therefore, it is possible to detect HIF-1 activity in the head and neck, breast, and lung cancers. However, the high background radioactivity in the abdominal region would hamper the imaging of HIF-1-active abdominal tumors. To overcome this issue, a probe that demonstrates greater accumulation in tumors and/or faster clearance from blood and normal abdominal organs should be developed.

Molecular Imaging of HIF-1-Active Tumor Microenvironments in Clinical Practice
No probes currently used in clinical practice can directly detect HIF-1 activity. As described in Section 2, pO 2 values at which HIF-1 is stable differ between organs. Thus, in principle, probes targeting physically hypoxic regions (pO 2 of ≤10 mmHg) are not suitable for the detection of HIF-1 expression or activity because the mechanisms underlying their hypoxic accumulation are independent of HIF-1 expression. However, HIF-1 -positive regions reportedly overlap with pimonidazole-adducted regions in tumors. Although microscopic observation has shown that unmatched regions also exist [47,48], the spatial resolution of nuclear medical imaging is not sufficiently high to distinguish these unmatched regions in vivo. Therefore, HIF-1 activity could potentially be evaluated indirectly by using probes targeting physically hypoxic regions.  [49], HIF-1 expression in the tumors did not correlate with 18 F-FMISO uptake in either newly diagnosed or recurrent glioma patients. A significant but weak correlation between VEGF expression and 18 F-FMISO uptake was observed in newly diagnosed glioma patients, but not in those with recurrent glioma. Hu et al. [51] investigated the relationship between [ 18 F]fluoroerythronitroimidazole ( 18 F-FETNIM) uptake and expression of HIF-1 , VEGF, and glucose transporter 1 (GLUT-1) in nonsmall cell lung cancer. A positive correlation existed between the tumor-to-muscle ratio of 18 F-FETNIM and the expression of each of these HIF-1 targets.

18 F-FDG.
Sato et al. [49] examined the relationship between the SUVmax of 18 F-FDG and HIF-1 expression in OSCC, and no significant correlation was observed. This finding is in accordance with that reported by Cheng et al. [52] who found no correlation between 18 F-FDG uptake 6 The Scientific World Journal and HIF-1 expression in breast cancer patients. Conversely, Takebayashi et al. [53] investigated the relationship between the SUVmax of 18 F-FDG, HIF-1 expression, and GLUT-1 expression in gastric cancer patients. The SUVmax was correlated with HIF-1 expression, but, interestingly, not with GLUT-1 expression. In this case, 18 F-FDG accumulation could indicate tissue hypoxia rather than glucose transport activity in aggressive cancer growths.

Conclusion
In this paper, the current status of nuclear medical molecular imaging strategies for HIF-1-active tumor microenvironments was discussed. At the preclinical level, HIF-1 activity has been directly evaluated by using HIF-1-dependent reporter systems and injectable probes that degrade in a manner similar to HIF-1 . However, these techniques have not yet been applied clinically. At the clinical level, whether the accumulation of 18 F-FDG or 18 F-FMISO can be utilized as an index of HIF-1 activity has been investigated but results obtained in these studies varied substantially. To address this issue, the development of radiolabeled probes that directly evaluate HIF-1 activity in humans is required.