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Introduction

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor in humans with a survival time often less than 1 year even following treatment (1–3). There remains a need for animal models that can be used to assess the efficacy of new and innovative treatments for experimental neuro-oncology (4,5). The blood-brain barrier (BBB) composed of tight junctions prevents the central nervous system (CNS) from being infiltrated by neurotoxic substances or pharmaceuticals from the peripheral circulatory system. Although the BBB of a tumor-bearing animal is severely compromised (6), some difficulties still remain in monitoring brain tumor progression or treatment response in experimental and clinical approaches. For example, since the introduction of 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG)/PET, it has been widely used in clinical settings for evaluation of several malignancies, such as lung, colon, breast, head and neck and esophageal cancers (7). 18F-FDG is metabolized as glucose but is stopped at the early step of metabolism, and can be used to detect the proliferation of tumor cells. Hence radiolabeled glucose analogs may serve as probes for molecular imaging of brain tumors. However, 18F-FDG also accumulates in benign lesions, and false-positive results may occur during infection, inflammation and other therapy-related conditions (8). 3′-Deoxy-3′-[18F]fluorothymidine (18F-FLT), a thymidine analog, often used in PET imaging for monitoring non-invasive cancer treatment, can be phosphorylated by thymidine kinase 1 (TK1) and trapped within tumor cells (9). It has been suggested that a multitracer approach using a combination of [18F]FLT and [18F]FDG may monitor early treatment response more effectively following cancer therapy (10). Other radiolabeled pyrimidine nucleoside derivatives, such as 2′-fluoro-2′-deoxy-β-D-arabinofuranosyl-5-iodouracil (FIAU) labeled with radioiodine, 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG), and 9-[(1-[18F]fluoro-3-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG), have been demonstrated to accumulate selectively in HSV1-tk transfected cells, but significant non-specific phosphorylation is still noted (11,12). On the other hand, 5-[76Br]bromo-2′-fluoro-2′-deoxyuridine ([76Br]BFU) and 1-[2-deoxy-2-[18F]fluoro-β-arabinofuranosyl-5-bromouracil] ([18F]FBAU), both uracil analogs for DNA incorporation, have been developed for the imaging of tumor proliferation with superior specificity for the HSV1-tk reporter system (13,14). However, one disadvantage of [76Br] is that patients may be exposed to a higher radiation dose due to its long half-life and high energy (16.2 h, 3.6 MeV). The shorter half-life and optimal energy of [18F] (110 min, 0.64 MeV) makes [18F]-labeled pharmaceuticals more convenient for clinical PET imaging. It has been shown that [18F]-labeled proteins provide higher availability and better imaging quality (15). The advantages of [18F]-labeled FBAU and combined with PET imaging in cellular proliferation, neuroscience and oncology have been reported (16–18).

In our previous studies, 4-borono-2-[18F]-fluoro-L-phenylalanine-fructose ([18F]FBPA-Fr) has been shown to have higher accumulation in tumors of F98 glioma-bearing rats, and can be used as a probe to mimic BPA-Fr in the treatment of brain tumors by boron neutron capture therapy (BNCT) (19). Furthermore, specific tumor accumulation with higher tumor-to-normal brain ratio of [18F]FBPA-Fr than that of [18F]FDG has also been demonstrated (20).

Establishment of a brain tumor-bearing animal model with a reporter gene-reporter probe (RGRP) system for multimodalities of molecular imaging may provide a useful platform for monitoring theranostics of tumor treatment (21–23), particularly for aggressive brain tumors such as GBM. The purpose of the present study was to investigate the potential application of [18F]FBAU as a PET reporter probe for monitoring tumor progression in an F98/tk-luc glioma-bearing rat model.

Materials and methods

Establishment of the pC1-tk-IRES-luc plasmid and the F98-tk-luc stable clone

The pC1-tk-IRES-luc plasmid was constructed according to our previous study (24). F98 rat glioma tumor cells were transfected with this plasmid using cationic polymer, jetPEI (Polyplus Transfection, Strasbourg, France). F98 cells were cultured in a 6-well plate (2×105 cells/well) after transfection. In a 1.5-ml Eppendorf vial, 3 μg of DNA plasmid was mixed with 6 μl jetPEI reagent in 100 μl of 150 mM NaCl followed by gentle vortexing, and then briefly spun down. The mixtures were incubated for 30 min at room temperature and were added to the well. Transfection was performed at 37°C for 24 h in a humidified incubator with 5% CO2. Stable clones were selected with 600 μg/ml of G418 (Calbiochem, Darmstadt, Germany).

Luciferase (luc) gene expression assayed with bioluminescent imaging (BLI)

For the luc activity assay, the cell lysate was prepared by performing a freeze-thaw cycle in reporter lysis buffer (Promega, Madison, WI, USA). The bioluminescence was measured with a luminometer (Wallac 1420 Multilabel Counter; Life Sciences, Pittsburgh, PA, USA). The protein concentration of the cell lysate in each well was measured with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) (24). F98 cells transfected with pC1-tk-IRES-luc vectors were cultured in a 6-cm diameter dish for 14 days, and 30 colonies were selected. Each colony was cultured in a 3-cm diameter dish for cell proliferation. Cells were treated with 200 μl lysis buffer, and were frozen at −80°C for 1 h. After thawing and centrifugation (1,200 rpm, Kubota Laboratory Centrifuge, model 5100; Kubota Corp., Tokyo, Japan), the supernatant was transferred to a black 96-well plate filled with D-luciferin. The bioluminescence was quantified as photons/sec/mg. For BLI imaging in vivo, male Fischer 344 rats and NOD/SCID mice were anaesthetized with 1–3% isofluorane, and then an i.p. injection with 300 μl D-luciferin (150 mg/kg in PBS) as the substrate. The IVIS 50 Imaging System (Xenogen, Alameda, CA, USA) was used for image acquisition. The time duration was 15 min for rats and 2 min for mice, followed by quantification with the Living Image software (Xenogen).

HSV1-tk gene expression assay

F98/tk-luc cells were cultured in 96-well plates (3,000 cells/200 μl/well) for 24 h, and then treated with 0–200 mM ganciclovir (GCV; F. Hoffmann-La Roche Ltd., Basel, Switzerland) for 120 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) colorimetric assay was used to determine cell survival (24).

Radiosynthesis of [131I]FIAU and [18F]FBAU

[131I]FIAU was prepared according to our previous study (25). [18F]FBAU was synthesized according to a study reported by Alauddin et al (26). The purification of [18F]FBAU was followed according to our previous study (27).

[131I]FIAU cellular uptake assay

[131I]FIAU (3.7×104 Bq/μl)was added to each well (1×106 cells/3 ml/well in 6-well culture plates), followed by incubation at 37°C for 15 and 30 min, 1, 2 and 4 h, respectively. Triplicates were performed at each time point.

Cell culture, orthotropic F98/tk-luc glioma-bearing rat model and subcutaneous mouse tumor inoculation

F98 glioma cells (a kind gift from Dr Rolf F. Barth, The Ohio State University, USA) were transfected with the pC1-tk-IRES-luc plasmid (24), and renamed as F98/tk-luc glioma cells. These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin) at 37°C in a humidified atmosphere of 5% CO2. Male Fischer 344 rats (12–14 weeks of age, 250–280 g) were anesthetized intraperitoneally (i.p.) with a mixture of 110 mg/kg ketamine and 9 mg/kg xylazine. F98/tk-luc glioma cells (1×105) in 10 μl Mg2+/Ca2+-free Hanks’ balanced salt solution (19) were then slowly (15–20 sec) injected into the left brain region of the rats. The syringe was held still for 2 min after injection before withdrawing, and the injection hole was sealed with bone wax. Finally, the wound was flushed with iodinated alcohol and sutured with a sterilized steel clip. Male NOD/SCID mice were inoculated with 5×105 F98/tk-luc cells in 200 μl serum-free medium subcutaneously in the right flank. The tumor volume was measured with a digital caliper.

Tumor volume assayed by MR imaging

3T MR scanning was performed within 24 h post BLI imaging. A 40-mm diameter field-of-view (FOV) orbital surface coil was used as the receiver (28). Each rat was anesthetized with 3–4 ml 10% chloride hydrate. To obtain accurate position of the brain slice, the coronal and transverse plane scout images were scanned first. 2–4 coronal T1-weighted slices with a 1.5-mm thickness would cover the tumor. The T1-weighted images were acquired with a 256×256 matrix, FOV=89 mm, repetition time=435 msec and echo time=12 msec. For contrast-enhanced images, each animal received 0.2 mmol/kg contrast medium i.v. 5 min prior to T1-weighted sequence imaging. T2-weighted images were acquired with a 256×256 matrix, FOV=89 mm, repetition time=5,000 msec and echo time=104 msec. The DICOM images were collected, and the tumor boundary visualized in each slice was defined as the region-of-interest (ROI) using ImageJ (National Institutes of Health, Bethesda, MD, USA) for calculation. The sum of the ROI areas was processed by slice thickness to obtain the tumor volume.

Pathology examination

After examination, the rats were sacrificed with CO2. The brains were removed and fixed with 4% paraformaldehyde. The sections (5-μm) obtained from the paraffin-embedded samples were stained with hematoxylin and eosin (H&E).

Biodistribution of [18F]FBAU in the F98/tk-luc glioma-bearing rats

Biodistribution of [18F]FBAU was determined on the 13th day post tumor implantation as previously described (19). Each rat was anesthetized with 3–4 ml of 10% chloride hydrate. The F98/tk-luc glioma-bearing rats were injected with 15 MBq/0.2 ml [18F]FBAU via lateral tail veins. At 0.5, 1, 1.5 and 2 h post injection, the rats (n=5 per group) were sacrificed with CO2. Left and right brains, tumors, intestine, spleen, heart, blood, lung, liver, stomach, pancreas, kidneys, bone and muscles were removed, and the radioactivity was measured in a well-type multichannel analyzer (Raytest, Straubenhardt, Germany) (27). The uptake of [18F]FBAU in tissues or organs was expressed in counts per second (cps) corrected with the decay, and was normalized as the percentage injected dose per gram of tissue (%ID/g).

PET scanning

The PET images of F98/tk-luc glioma-bearing rats i.v. injected with 50 MBq/0.1 ml [18F]FBAU were acquired up to 160 min using the PET/CT system (GE Discovery LS PET-CT; GE Healthcare, Cleveland, OH, USA) on day 13 post tumor implantation. The imaging parameters were set as follows: 2-D brain model, FOV=15.52 cm, spatial resolution=5.46 mm, 128×128 matrix size. The reconstruction algorithm using ordered subset expectation maximization (OSEM) was similar to that of a previous study (19). The images were acquired with axial, coronal and sagittal planes, respectively. Each rat was anesthetized with 3–4 ml 10% chloride hydrate and i.v. injected with 0.1 ml of 50 MBq [18F]FBAU. Data acquisition by PET scanning was initiated at the first minute after drug injection. Dynamic coronal and sagittal images were acquired using 10 60-sec frames and 10 2-min frames, followed by 10-min frames up to 2 h. Standardized uptake values (SUVs) were calculated and normalized to body weight (BW) (29). %SUV was calculated using the following formula: %SUV = [(left brain SUV - right brain SUV)/right brain SUV] × 100%.

Ex vivo autoradiography

The ex vivo autoradiography was performed according to a previous report (20). Fischer 344 rats were anesthetized with 1–3% isofluorane. At 30, 60, 90 and 120 min post i.v. injection of [18F]FBAU (15–20 MBq/0.7 ml), the rats were sacrificed with CO2. The brains were removed and embedded with OCT on a cryostat holder (diameter 2.7 cm), and were frozen immediately at −80°C. Coronal sections (20-μm thick) were performed using a cryomicrotome (Leica CM3050; Leica Microsystems, Bensheim, Germany). The imaging plates were assayed with an FLA5000 reader (Fuji Photo Film Co., Ltd., Tokyo, Japan) with the following settings: PMT=800 V, resolution=50 μm, gradation=16 bits, dynamic range=L5 and sensitivity=5,000 to acquire the phosphor images. ROIs were circled along the tumor contour of the F98/tk-luc glioma, and ROIs of equal size in the normal brain region at corresponding brain position were used as the reference. The intensity of photo-stimulated luminescence (PSL) of the tumor and normal brain tissue was measured with Image Gauge (version 4.0, Science Lab 2001; Fuji Photo Film).

Statistical analysis

The Studen’t t-test was used to analyze the significant difference between the control and drug-treated rats. All data are shown as the means ± standard error (SE). Differences between the means were considered significant at P≤0.05. The correlation between tumor volume and BLI photon flux was performed by Pearson correlation.

Results

Selection of stable clones and luc gene expression assay

The pC1-tk-IRES-luc vectors as shown in Fig. 1A were transfected into the F98 cell line, which was renamed F98/tk-luc. The colony exhibiting luc expression of >1,500 counts per sec per mg (cps/mg) was selected for cell expansion as shown in Fig. 1B (colony no. 11). The bioluminescence of luc gene expression in the F98/tk-luc cells was 3.24 photons/sec/cell (Fig. 1C).

Figure 1 Bioluminescence assay of F98/tk-luc cells in vitro. (A) The genetic map of pCMV-tk-luc plasmid. (B) Luciferase expression assay of selected colonies was expressed in counts per second (CPS)/cell. The colony 11 with the highest value of light intensity was chosen for cell proliferation. (C) Cells (1×105) obtained from the expansion of colony 11 were serially diluted in a 96-well plate for assay of the luciferase activity. D-luciferin was added to each well and the plate was imaged for 1 min. The expression of luciferase was 3.24 photons/sec/cell.

Expression of thymidine kinase (tk)

F98/tk-luc cells were treated with various concentrations of ganciclovir (GCV). The surviving fraction (SF) of F98/tk-luc cells was 18% at 15 μM and remained ~100% up to 200 μM for parental F98 cells after GCV treatment (Fig. 2A). In addition, the uptake of [131I]FIAU in the F98/tk-luc cells was increased with time, but was not found in the parental F98 cells (Fig. 2B).

Figure 2 HSV1-tk reporter gene assay of F98/tk-luc cells in vitro. (A) The surviving fractions of F98/tk-luc cells that were decreased to 0.2 or less after treatment with various concentrations of GCV (n=6). (B) Cellular uptake was determined by γ-counter after [131I]FIAU addition. The uptake of F98/tk-luc cells increased to ~10% dose/106 cells after 240 min (n=5).

Tumor growth monitored using bioluminescent imaging (BLI) in F98/tk-luc tumor-bearing mice

F98/tk-luc cells were inoculated subcutaneously into the right flank of NOD/SCID mice. Two weeks later, tumor growth was monitored with BLI as shown in Fig. 3A. The quantification of bioluminescence (photons/sec × 105) vs. tumor volume (mm3 × 10) from representative mice is plotted in Fig. 3B with linear regression (r2=0.90).

Figure 3 Luc expression of F98/tk-luc glioma in NOD/SCID mice. (A) The increasing luminescence of F98/tk-luc tumors in mice was consistent with the increasing size of the tumors measured with a caliper over 2–3 weeks post inoculation. (B) Linear plot indicating the correlation of luminescence with increasing tumor volume (r2=0.908, P<0.05, n=4).

Bioluminescent imaging (BLI), MR imaging and histopathology

The BLI of F98/tk-luc glioma-bearing rats was obtained using the IVIS50 optical imaging system (Xenogen Corp.) on day 13 post inoculation of F98-tk-luc glioma cells into the left brain. The total photon flux rates of the tumors were 5.50×105-4.14×106 as shown in Fig. 4A (images were from representative rats). MR images of the representative rat brains were obtained from a 3T MR scanner including T1-, T2-weighted and contrast-enhanced images (Gd 0.2 mmol/kg) on day 14 (Fig. 4B).

Figure 4 Brain images of Fischer 344 rats obtained by bioluminescent imaging (BLI), MR and histology. (A) BLI. (B) MR images by T1- and T2-weighed and post gadolinium (0.2 mmol/kg) injection compared to BLI (left panel) in a representative mouse. (C) Histopathology of a brain section stained with H&E (scale bar, 1.01 mm). (D) Tumor volume estimated from photon flux rates of ROIs by BLI are correlated with those calculated from MR images (r=0.95).

In the present study, the tumor volume and its extent were not able to be demonstrated well by T1-weighted images but were greatly enhanced post contrast injection, which showed a clear boundary between the tumor and normal brain tissue. On the other hand, the tumor location could be shown by T2-weighted images whereas the boundary between the tumor and normal tissue was still indistinguishable. Fischer 344 rats after MR scanning were sacrificed, and the brain tissues were removed and preserved in 4% paraformaldehyde. H&E staining was subsequently performed. The results are shown in Fig. 4C, in which the tumor location and tumor volume were the same as those of the MR images shown in Fig. 4B.

The total photon fluxes of F98-tk-luc tumors obtained from BLI vs. tumor volumes calculated by 3T MR contrast-enhanced images are tabulated in Table I and plotted in Fig. 4D. The Pearson correlation coefficient (r) is 0.95.

Table I The tumor volumes assayed by 3T MR and photon flux rates of regions-of-interest (ROI) obtained from BLI of the rat brains were compared.

Table I

The tumor volumes assayed by 3T MR and photon flux rates of regions-of-interest (ROI) obtained from BLI of the rat brains were compared.

Rat ID	Tumor volume (mm3) assayed by MR	Total photon flux rate (photons/sec, × 105) obtained from BLI
1	1.93	4.38
2	5.07	4.56
3	8.62	9.78
4	8.94	5.51
5	52.77	41.40
6	126.90	41.00
7	213.0	58.90

Biodistribution of 18F-FBAU in the F98/tk-luc tumor-bearing rats

The accumulation of [18F]FBAU in tissues of the F98/tk-luc tumor-bearing Fischer 344 rats (n=5) was measured at different time points after administration of 15 MBq/0.2 ml. The highest uptake of [18F]FBAU was found in kidneys, suggesting that the elimination route of [18F]FBAU is via renal clearance. The uptake of [18F]FBAU in the left and right normal brain tissues was very low in the range of 0.03–0.09 %ID/g (Fig. 5A and Table II). Notably, the uptake of [18F]FBAU in the F98/tk-luc glioma tumors reached up to 0.51±0.06 at 90 min post injection (Fig. 5B). The uptake of [18F]FBAU in the tumors was significantly higher than those of normal brain tissues (P<0.01). In addition, the uptake of [18F]FBAU in tumors in the 60 and 90 min post-injection groups was significantly higher than that of the 30 min post-injection group (P<0.05).

Figure 5 Biodistribution of [18F]FBAU in F98/tk-luc tumor-bearing rats (n=5). (A) Specific radioactivities (%ID/g) in the left and right brain, intestine, spleen, heart, kidney, lung, liver, stomach, pancreas, bone, muscle, blood and tumor at various time points are shown. (B) The ratios calculated from %ID/g of [18F]FBAU in F98/tk-luc glioma-to-normal right brain at 30, 60, 90 and 120 min post injection of [18F]FBAU were 9.16, 14.24, 5.70 and 13.70, respectively. The %ID/g of [18F]FBAU in the F98/tk-luc glioma was significantly higher than that of the right brain (**P<0.005). In addition, %ID/g values for the 60 and 90 min groups were also significantly higher than that of the 30 min post injection group (#P<0.05).

Table II Biodistribution of [18F]FBAU in various tissues/organs of the F98/tk-luc glioma-bearing Fischer 344 rats after intravenous injection (n=5; mean ± SE).

Table II

Biodistribution of [18F]FBAU in various tissues/organs of the F98/tk-luc glioma-bearing Fischer 344 rats after intravenous injection (n=5; mean ± SE).

	Uptake (%ID/g)
Tissues/organs	30 min	60 min	90 min	120 min
Left brain	0.048±0.004	0.047±0.008	0.094±0.023	0.081±0.028
Right brain	0.030±0.001	0.029±0.004	0.090±0.019	0.030±0.005
Tumor	0.275±0.034	0.413±0.023	0.513±0.066	0.411±0.061
Large intestine	0.212±0.051	0.325±0.060	0.826±0.282	0.311±0.040
Small intestine	0.231±0.039	0.368±0.175	0.759±0.248	0.362±0.071
Spleen	0.315±0.025	0.311±0.030	0.650±0.158	0.936±0.375
Heart	0.296±0.054	0.252±0.044	0.648±0.212	0.176±0.059
Blood	0.296±0.032	0.207±0.027	0.699±0.217	0.178±0.058
Lung	0.203±0.030	0.146±0.012	0.562±0.158	0.172±0.066
Liver	0.248±0.031	0.189±0.033	0.476±0.240	0.191±0.053
Stomach	0.120±0.017	0.186±0.028	0.448±0.127	0.136±0.031
Pancreas	0.179±0.027	0.117±0.026	0.227±0.112	0.120±0.046
Kidney	2.151±0.544	1.822±0.480	4.183±1.604	1.868±0.550
Bone	0.170±0.026	0.159±0.046	0.657±0.160	0.479±0.116
Muscle	0.165±0.019	0.161±0.026	0.484±0.143	0.195±0.031
Tumor/right brain	9.16	14.24	5.70	13.70

PET images of 18F-FBAU in the F98/tk-luc tumor-bearing rat model

The coronal and sagittal PET images of the F98/tk-luc glioma-bearing rats were imaged with a clinical PET. The result showed that accumulation of [18F]FBAU was noted in F98/tk-luc glioma in the brain (arrows in Fig. 6A). The [18F]FBAU/PET images at 30–90 min post injection showed significantly higher %SUV values than that acquired at the initial time point (P<0.005; Fig. 6B).

Figure 6 The PET images and %SUV of F98/tk-luc glioma-bearing Fischer 344 male rats at various time points post intravenous injection of [18F]FBAU (50 MBq/0.2 ml). (A) PET image of F98/tk-luc glioma is indicated by an arrow in the image of the left brain. (B) The %SUVs of [18F]FBAU at 30, 60 and 90 min post injection of [18F]FBAU were significantly higher than that of the on-set time point post injection by 9–20% (*P<0.05; data are expressed as mean ± SE, n=5).

Ex vivo autoradiography

Biodistribution of [18F]FBAU in the rat brain was also examined by ex vivo autoradiography. The result showed that the uptake of [18F]FBAU in the F98/tk-luc gliomas reached the maximum value at 90 min post injection (Fig. 7A). Autoradiogram of [18F]FBAU in the F98/tk-luc gliomas showed a similar result as that of the biodistribution study with the highest uptake at 90 min post injection. The quantification of the autoradiogram is depicted in Fig. 7B. The uptake of [18F]FBAU assayed by BLI in units of photo-stimulated luminescence (PSL)/mm2 in the F98/tk-luc gliomas was significantly higher at each time point from 30 to 120 min compared to that of the contralateral normal brain tissue (Fig. 7B).

Figure 7 Ex vivo autoradiography. (A) Brain autoradiograms of the F98/tk-luc glioma-bearing Fischer 344 male rats after intravenous injection of 20 MBq/0.2 ml [18F]FBAU. (B) Relative optical intensity (PSL/mm2) of tumors and normal brain derived from autoradiograms (*P<0.05, **P<0.005; data were expressed as mean ± SE, n=5).

Discussion

[124I]FIAU has been reported to be the first PET probe used in the HSV1-tk reporter gene expression system (11,30). Although FIAU is preferentially phosphorylated by HSV1-thymidine kinase (TK) compared to mammalian TK, a certain amount of FIAU is accumulated in cells which do not express HSV1-TK resulting in non-specific signals (31). It has been reported that no difference in biodistribution exists between [14C]FIAU and [76Br]FBAU; the uptake of the latter, however, is constantly higher than that of [14C]FIAU in RG2-TK rat glioma cells. Furthermore, the uptake of [76Br]FBAU is not affected significantly by endogenous mammalian thymidine kinase (14). 2′-Deoxy-2′-[18F]fluoro-5-ethyl-1-β-D-arabinofuranosyluracil ([18F]FEAU), another pyrimidine nucleoside analog, has been shown to have higher selectivity and affinity for HSV-tk in subcutaneous RG2-TK+ tumors and C6-tk cells compared to other radiotracers (32,33), but thorough evaluation by other modalities is lacking. In the present study, we demonstrated the tumor-specific radiotracing activity of [18F]FBAU in an F98/tk-luc glioma-bearing animal model using HSV1-tk reporter gene/[18F]FBAU reporter probe system combined with multimodalities of molecular imaging with PET, BLI, MRI and ex vivo autoradiography. [18F]FBAU showed better differentiation with higher uptake in the tumors compared to the contralateral normal brain in the F98/tk-luc glioma-bearing animal model between 30 to 120 min post injection (Figs. 5B and 6B). Both PET imaging and ex vivo autoradiography also showed the persistent accumulation of [18F]FBAU in the F98/tk-luc glioma-bearing animal model from 90 to 120 min post injection (Figs. 6A and 7B), which was ~5–14 times higher than the other radiotracers, such as [76Br]FBAU, at 120 min post injection as reported in other studies (14). The results suggest that [18F]FBAU is a suitable reporter probe for the imaging of HSV1-tk expression.

For the purpose of tracing tumor growth progression, molecular imaging techniques other than traditional caliper measurement have been developed. A statistical correlation was developed to correlate the relationship between anatomical size and bioluminescent intensity collected from ROI of the tumors in a brain-tumor bearing animal model (23). Notably, the brain tumor volume assayed by bioluminescent imaging (BLI) has been reported to poorly correlate with images obtained from MRI (34,35). Nevertheless, metastatic breast cancer in a rat model monitored using MRI at 3 tesla was found to correlate well with that by BLI (36). Although [18F]FDG/PET imaging has been used for diagnosis of tumors as well as the malfunction of the brain, the uptake of [18F]FDG remains high in normal brain tissues, causing difficulties in differentiating normal brain from malignancy (20,37). In order to monitor the progression of brain tumor growth in the glioma-bearing animal model, we found a good correlation for the tumor volume acquired from MRI vs. that obtained from BLI in the F98/tk-luc glioma-bearing rat model (Fig. 4). The SUV of [18F]FBAU in F98/tk-luc glioma was 20% higher than that of contralateral normal brain tissue (Fig. 6). Hence [18F]FBAU may provide another advantage in brain tumor diagnosis and disease monitoring. As a result, this approach may have potential for using [18F]FBAU as a molecular probe combined with a reporter gene system, such as HSV1-tk, to monitor tumor growth progression in preclinical or clinical applications.

In conclusion, the uptake of [18F]FBAU and SUV of [18F]FBAU in brain tumors of an F98/tk-luc glioma-bearing rat model was significantly higher than that of normal brain tissues determined by biodistribution and PET, respectively. Both the volume and progression of tumors determined with MRI and BLI were well correlated in the F98/tk-luc glioma-bearing rat model using [18F]FBAU as a reporter probe. Radiolabeled FBAU combined with multimodalities of molecular imaging may also provide potential benefits for the evaluation of therapeutic efficacy of newly developed drugs against other brain diseases.

Acknowledgements

The present study was supported by grant NSC92-2745-P-010-002 from the National Science Council, Taipei, Taiwan. We thank the staff of the Department of Radiopharmaceutical Production, Buddhist Tzu Chi General Hospital, Hualien, Taiwan for providing PET/CT imaging and advice for the synthesis of [18F]FBAU. We also thank the Taiwan Mouse Clinic which is funded by the National Research Program for Biopharmaceuticals (NRPB) at the National Science Council (NSC) of Taiwan for technical support with the bioluminescent imaging experiment.

References