Uptake of [18F] EF5 as a Tracer for Hypoxic and Aggressive Phenotype in Experimental Head and Neck Squamous Cell Carcinoma1

PURPOSE: This study aims to investigate whether the uptake of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide ([18F]EF5) and 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) is associated with a hypoxia-driven adverse phenotype in head and neck squamous cell carcinoma cell lines and tumor xenografts. METHODS: Xenografts were imaged in vivo, and tumor sections were stained for hypoxia-inducible factor 1α (Hif-1α), carbonic anhydrase IX (CA IX), and glucose transporter 1 (Glut-1). Tracer uptakes and the expression of Hif-1α were determined in cell lines under 1% hypoxia. RESULTS: High [18F]EF5 uptake was seen in xenografts expressing high levels of CA IX, Glut-1, and Hif-1α, whereas low [18F]EF5 uptake was detected in xenografts expressing low amounts of CA IX and Hif-1α. The uptake of [18F]EF5 between cell lines varied extensively under normoxic conditions. A clear correlation was found between the expression of Hif-1α and the uptake of [18F]FDG during hypoxia. CONCLUSIONS: The UT-SCC cell lines studied differed with respect to their hypoxic phenotypes, and these variations were detectable with [18F]EF5. Acute hypoxia increases [18F]FDG uptake in vitro, whereas a high [18F]EF5 uptake reflects a more complex phenotype associated with hypoxia and an aggressive growth pattern.


Introduction
Imaging of tumor hypoxia using 2-nitroimidazoles has increased during recent years. For a number of cancers, including head and neck squamous cell carcinomas (HNSCCs), radiotherapy (RT) may fail due to the presence of tumor hypoxia [1]. However, before imaging of hypoxic tumors can be employed for hypoxia-directed treatment strategies, the biologic significance of imaging data needs to be validated [2]. This molecular information may be useful for planning RT, as well as in drug development. Image-guided radiotherapy is routinely implemented to reduce safety margins associated with delineation of clinical target volume, but it is also necessary to irradiate biologically relevant subvolumes within the tumor [3]. In view of the heterogeneity of tumor tissue, it is hoped that this targeted irradiation can improve the survival prospects of patients with cancer. lactate, and H + ions, develop at the microregional level [4]. Hence, tumor cells must survive in this hypoxic environment and the acidic surroundings, both of which are currently considered as hallmarks of cancer [5]. Hypoxic cells are able to adapt to the demanding environments by activating hypoxia-inducible factor 1 (Hif-1), a heterodimer consisting of α and β-subunits [6,7]. Hif-1 activates the transcription of many genes, for example, those involved in angiogenesis, glycolysis [e.g., glucose transporters (GLUTs)], pH maintenance [e.g., carbonic anhydrases (CAs)], and proliferation [8,9]. In summary, the activation of Hif-1 helps cells to adapt to an environment with a low-oxygen level. CAs are a family of proteins that catalyze reversibly the hydration of the carbon dioxide to carbonic acid, and thus help cells to survive in an acidic environment [10]. CA isoform 9 (CA IX) is found in many aggressive tumors, including HNSCC, and has been associated with poor treatment outcomes [11,12]. The acidic microenvironment can also trigger nonhypoxic cells to use glycolysis as their primary energy source [13]. Glucose is transported into cells by GLUTs, which are overexpressed in many cancers, including HNSCC [14]. Higher Glut-1 expression has been shown to correlate with a poorer survival in many cancers [14,15], although contradictory results on the correlation between Glut-1 expression and the uptake of 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG) have been reported [16].
Hypoxia imaging with positron emission tomography (PET) is usually based on 18 F-labeled 2-nitroimidazole compounds [17]. We have earlier evaluated the hypoxia tracer 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide ([ 18 F]EF5) in patients with HNSCC [18]. In this study, the uptake of [ 18 F]EF5 and [ 18 F]FDG into primary tumors and cervical lymph node metastases was found to be heterogeneous. Previous studies using unlabeled EF5 have described a correlation between hypoxia and tumor aggressiveness [19,20]. Understanding the relationship between oxygen and glucose metabolism is crucial for the planning of hypoxia-directed therapies, such as biologically guided RT. Therefore, these findings encouraged us to investigate in detail whether a certain molecular profile might affect [ 18 F]EF5 and [ 18 F]FDG uptake in HNSCC.
We studied the in vivo uptake of [ 18 F]EF5 and [ 18 F]FDG in xenografts from UT-SCC cell lines. We also evaluated the in vitro accumulation of these tracers in the same cell lines. Finally, we investigated the association between these PET tracers and biologic markers commonly considered to be related to [ 18 [21]. The specific activity of [ 18 F]EF5, decay corrected to the end of synthesis, exceeded 3.7 GBq/ μmol. Radiochemical purity was higher than 98.5% in every production batch.
[ 18 F]FDG was synthesized from mannosyl triflate using a nucleophilic method. The radiochemical purity exceeded 95% in every production batch.

Animals and Tumor Models
Male nude mice (Athymic nu/nu; Harlan Laboratories, Horst, The Netherlands), weighing 33.1 (29.0-37.1) g, were used to establish xenografts from UT-SCC-8, UT-SCC-25, UT-SCC-34, and UT-SCC-74A HNSCC cell lines. Mice were irradiated with 4 Gy (3 Gy/min) 1 day before tumor transplantation to reduce their immunity. Depending on the cell line, 1 to 10 × 10 6 cells were subcutaneously injected into the flank of each mouse. Throughout this study, cell line passage numbers were kept as low as possible (p6-p40). Mice were maintained in individually ventilated animal cages under controlled pathogen-free environmental conditions (21°C, humidity = 55 ± 5%, and lights on from 6:00 A.M. to 6:00 P.M.) with free access to water and standard food. Animals were observed on daily basis, and tumor sizes were measured weekly (V = (π/6) × a × c × b). The experimental procedures were reviewed by the local Ethics Committee on Animal Experimentation at the University of Turku and approved by the Provincial State Office of Western Finland.

In vivo Imaging of Xenografts
Mice (n = 3 per cell line) bearing tumors with an average size of 406 (105-685) mm 3 were anesthetized with 2.5% isoflurane, and body temperature was maintained using a heating pad. Following a transmission scan for attenuation correction using the computer thomography (CT) modality, an emission scan was acquired in three-dimensional list mode (Inveon; Siemens Medical Solutions, Knoxville, TN, USA).
Mice were injected with 11.6 (9. Dynamic 80-minute-long scans were acquired. Sinograms were framed into 25 frames (6 × 10 seconds, 4 × 15 seconds, 2 × 30 seconds, 2 × 120 seconds, 1 × 180 seconds, 6 × 300 seconds, and 4 × 600 seconds) and reconstructed with an ordered subset expectation maximation (OSEM) two-dimensional iterative algorithm. Images were summed from 60 to 80 minutes, and volumes of interest were drawn over whole tumors with Inveon Research Workplace image analysis software 4.1 (Siemens Medical Solutions), using the CT template as an anatomic reference. Radioactivity uptake was calculated as the percentage of injected dose per gram tissue (%ID/g) in whole tumor.

Cell Line
Primary Tumor as described previously [11]. The expressions of CA IX, Glut-1, and Hif-1α were visually analyzed from 10 different areas in each tumor section using a × 20 microscope objective. The percentage of positively stained tumor cells was counted, and staining intensity was described as weak (1), moderate (2), or strong (3). Each tumor was scored (range = 0-300) by multiplying the average intensity value by the average percentage of positively stained cells. Analyses were performed independently by two investigators (J.S. and K.K.).

In vitro Uptake of Tracers in Cell Lines
Digital autoradiography was used to measure the uptake of [ 18 F]EF5 and [ 18 F]FDG in cell lines grown on eight-well chamber slides (Nunc™, Thermo Scientific, Waltham, MA, USA) under normoxia and different time periods of hypoxia (1% O 2 ). Four days before tracer incubation, cells were plated onto chamber slides in duplicate (two wells per cell line) and cultured under normal culturing conditions. Medium was changed on the third day. Cells were seeded at various densities according to their growth rates to ensure that there would be equal amount of cells at the time of tracer incubation. On day 4, one chamber slide per time point was transferred to a hypoxia workstation (Invivo2; Ruskinn Technology Ltd, Pencoed, United Kingdom) at 24, 12, 6, 3, and 1 hour before the end of tracer incubation time. Chambers were removed, and slides were washed with phosphate-buffered saline before being incubated with [  The workstation and all solutions used were stabilized in 1% O 2 before the experiment. Control cells were incubated with tracers under normal culture conditions (normoxia, 0 hour). Cells were then washed with phosphate-buffered saline and fixed in 4% paraformaldehyde for 10 minutes at room temperature. Air-dried slides were exposed with an imaging plate (BAS-TR2025; Fuji Photo Film Co, Tokyo, Japan) for 4 hours before scanning with the Fuji Analyzer FLA-5100.
Images were analyzed for count density [photostimulated luminescence (PSL) per unit area] with a computerized image analysis program (AIDA Image Analyzer version 4.22; Raytest Isotopenmessgeräte, Straubenhardt, Germany). A square-sized region of interest was drawn over each chamber containing cultured cells, and the background was subtracted from the image data. Data were normalized to the total amount of radioactivity used in each experiment.

Statistics
Descriptive statistics for the data are presented as arithmetic means and range. Mean levels of [ 18 F]EF5 and [ 18 F]FDG (tumor uptake), membranous CA IX, membranous Glut-1, and nuclear Hif-1α scores were compared between groups (UT-SCC-8, UT-SCC-34, and UT-SCC-74A) with one-way analysis of variance (ANOVA). Likewise, mean levels of [ 18 F]EF5 (in vitro uptake) were compared between UT-    Table 2. One-way ANOVA revealed differences between the cell lines for CA IX and Hif-1α expression (P = .046 and P = .014, respectively), whereas there were no significant differences between the cell lines in the levels of Glut-1 expression (P = .147).
All xenografts exhibited membranous CA IX expression. In UT-SCC-8 xenografts, the mean CA IX-positive tumor cells was below 10% ( Table 2). The highest CA IX scores were observed in UT-SCC-34 tumors ( Figure 2B), which exhibited weak to strong staining in more than 50% of tumor cells ( Table 2). In UT-SCC-74A xenografts, below 30% of tumor cells were positive for membranous CA IX (Table 2) All xenografts were positive for membranous Glut-1. More than 60% of cells in UT-SCC-8 xenografts were Glut-1 positive, with mostly moderate to strong staining intensities. In UT-SCC-34 xenografts, 65% of the cancer cells were Glut-1 positive, exhibiting divergent staining intensities but equal distribution within the UT-SCC-34 xenografts exhibited strongest nuclear Hif-1α expression (Figure 2A). In these xenografts, more than 40% of tumor cells were positive for nuclear Hif-1α, compared with only 5% of positive tumor cells in UT-SCC-74A xenografts and 20% in UT-SCC-8 xenografts (Table 2)

Cell Line-Specific Uptake of [ 18 F]EF5
UT-SCC-34 and UT-SCC-74A cells exhibited the highest [ 18 F] EF5 uptake, whereas low uptake was seen in UT-SCC-25 cells (Figure 3). After 1 hour of exposure to hypoxia, [ 18 F]EF5 uptake increased slightly in all UT-SCC cell lines. However, this uptake declined over time toward the levels detected in the normoxic conditions ( Figure 3). One exception to this pattern was detected in UT-SCC-74A cells in which a higher [ 18 F]EF5 uptake was seen at 24 hours after exposure to hypoxia in comparison to normoxic conditions. However, significantly different (P b .0001) [ 18 F]EF5 uptake was already seen between the cell lines under normoxia, except between UT-SCC-34 and UT-SCC-74A, which showed similar high uptake.

Discussion
Our aim in this study was to investigate whether a certain molecular profile might affect [ 18 F]EF5 and [ 18 F]FDG uptake in HNSCC. The main finding of our study is that [ 18 F]EF5 uptake appears to be related to a hypoxia-driven adverse phenotype. The highest [ 18 F]EF5 uptake was seen in UT-SCC-34 xenografts (Figure 1), which also expressed high amounts of CA IX, Glut-1, and Hif-1α ( Figure 2 and Table 2). Moreover, much lower levels of [ 18 F]EF5 uptake and CA IX and Hif-1α expression were detected in UT-SCC-8 xenografts. We consider this difference (P = .091) in [ 18 F]EF5 uptake as a trend toward significance in this limited sample number study. Compared to UT-SCC-8 xenografts, a higher, although not statistically significantly (P = .194), uptake of [ 18 F]EF5 was also detected in UT-SCC-74A xenografts. The expression of CA IX was similarly higher in UT-SCC-74A xenografts, whereas the expression of Hif-1α was significantly (P = .012) lower compared with UT-SCC-34 xenografts.
In comparison to UT-SCC-8 xenografts, the uptake of [ 18 F]FDG was also higher, although not statistically significantly, in UT-SCC-34 and UT-SCC-74A xenografts (Figure 1). This finding further supports our conclusion that the phenotype of UT-SCC-34 and UT-SCC-74A xenografts is more aggressive. Membranous Glut-1 expression was detected in all three UT-SCC xenografts. The expression of Glut-1 did not relate to the uptake of [ 18 F]FDG or [ 18 F]EF5. The highest expression was seen in UT-SCC-8 and UT-SCC-34 xenografts, whereas the lowest expression was detected in UT-SCC-74A xenografts ( Figure 2 and Table 2). Although tumors frequently overexpress Glut-1, the cellular uptake of [ 18 F]FDG is not exclusively attributable to Glut-1 [16], which probably explains the previous contradictory studies on the relationship between Glut-1 and [ 18 F]FDG.
To further evaluate phenotype differences detected in the xenografts in vivo, we determined the uptake of [ 18 F]EF5 and [ 18 F]FDG in the cell lines in vitro. Cells were grown under normoxia and for 1, 3, 6, 12, and 24 hours of 1% of oxygen (Figures 3 and 4). These experiments confirmed the existence of phenotypic differences detected in xenografts. The lowest uptake of both tracers was found in UT-SCC-25 cells, which did not form xenografts in nude mice. The greatest uptake was detected in UT-SCC-34 and UT-SCC-74A cells. The uptake of [ 18 F]EF5 increased after exposure to 1% of oxygen, but interestingly, we observed differences between the cell lines in the [ 18 F]EF5 uptake already at normoxic conditions ( Figure 3). This finding indicates that the studied cell lines express different hypoxiadriven adverse phenotypes that might influence their behavior, even without the presence of a hypoxic environment. There might also be variation in the activity of one-electron reductases required for activation of 2-nitroimidazoles in cells. Whether there is a relationship between these reductases and hypoxia-driven adverse phenotypes remains to be clarified. Recently, [ 18 F]EF5 was shown to be activated by the same reductases as CEN-209 (a tirapazamine analog) in human tumor cell lines and thus to function as a dual reporter for both hypoxia and reductase expression in tumors [29]. The impact of hypoxia as a function of time affected the uptake of [ 18 F]FDG to a greater extent than [ 18 F]EF5. The uptake of [ 18 F]FDG clearly increased after 1 hour of hypoxia exposure, typically being highest at 3 to 6 hours. UT-SCC-74A cells differed in this respect by displaying the highest uptake after 24 hours of hypoxia ( Figure 4).
To evaluate the ability of cell lines to adapt to a stressful hypoxic environment, we also determined the expression of Hif-1α as a function of time ( Figure 4B). We found an extensive variation in its expression level among the four cell lines, which furthermore correlated with the uptake of [ 18 F]FDG. This correlation most probably reflects the metabolic adaptation of cells to hypoxia in vitro, which one could speculate is regulated by the activation of Hif-1. The fact that hypoxia induces anaerobic glycolysis and therefore the increases in [ 18 F]FDG uptake have been shown previously [30,31], although there are also contradictory results as reported by Busk et al. [32]. The lack of response reported in this study might be a result of contact-inhibited cells. We observed that it is important to seed cells at correct densities to achieve proliferative active cells during the time of tracer incubation. Contactinhibited cells, or cells grown at a low density, did not increase their [ 18 F]FDG uptake in 1% O 2 (data not shown).
To summarize our observations, we found low uptake of [ 18 F]EF5 and [ 18 F]FDG in the UT-SCC-25 cell line, which was unable to form xenografts. Low tracer uptakes were also detected in UT-SCC-8 cells and corresponding xenografts that expressed low amounts of CA IX and Hif-1α. In contrast, UT-SCC-34 cells and xenografts exhibited high levels of [ 18 F]EF5 and [ 18 F]FDG uptake in addition to intense expression of CA IX, Glut-1, and Hif-1α. A more heterogeneous pattern of both uptake of tracers and expression of CA IX and Glut-1 was detected in UT-SCC-74A cells and xenografts.

Conclusion
In conclusion, our results demonstrate that the low-passage UT-SCC cell lines evaluated in this study differ in their glycolytic and hypoxic phenotypes. Importantly, these in vitro phenotypic differences can be imaged in vivo and may thus be clinically evaluable using PET. Overall, our results suggest that [ 18 F]EF5 accumulation in HNSCC not only reflects hypoxia but also is related to an adverse phenotype. [ 18 F]FDG uptake, in turn, may be sensitive to acute changes in oxygenation as suggested by rapid response of expression of HIF-1α to hypoxia in vitro. The hypoxia tracer [ 18 F]EF5 might be useful for the detection of hypoxic and more aggressive HNSCC tumors, and thus, it could assist in planning of hypoxia-directed therapies. The biologic genotype behind the phenotypes reported in this study will need to be evaluated in greater detail.