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Advances in Brief |
Department of Medical Oncology [G. A. P. H., A. C., N. H. M.] and Positron Emission Tomography Center [A. v. W., P. D., W. V., E. F. J. d. V.], Groningen University Hospital, 9700 RB Groningen, the Netherlands
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ABSTRACT |
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Introduction |
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A method to monitor the activity of HSV-tk in vivo would be extremely useful to optimize clinical gene therapy protocols.
Scintigraphic imaging, like PET and single photon emission computed tomography, can offer information about both the extent of gene expression and its distribution, provided that an appropriate reporter gene is included in the therapeutic cassette (5) . As compared with single photon emission computed tomography, PET imaging offers a higher resolution and sensitivity and allows noninvasive quantification of biological processes (6) .
Several radiolabeled thymidine and ganciclovir derivatives have been proposed as probes for imaging of HSV-tk enzyme activity with PET, including [124I]FIAU (6) , 8-[18F]fluoroganciclovir (7) , and [18F]FHPG (8 , 9) . These radiopharmaceuticals are better substrates for the HSV-tk enzyme than for human thymidine kinases (10) . After phosphorylation by HSV-tk, the tracer is trapped in HSV-tk-expressing cells only and can be visualized externally with a PET camera.
Preliminary studies showed [18F]FHPG to be a potential tracer for HSV-tk imaging with PET. In vitro, high and selective accumulation of [18F]FHPG in several HSV-tk-expressing cell lines was observed (8 , 11 , 12) . The enantiomers of [18F]FHPG did not exhibit any significant differences in cellular accumulation kinetics (13) . The first in vivo data showed that tumor uptake was 3- and 6-fold higher in mice bearing a HSV-tk-expressing HT-29 human colon cancer than in mice with a control tumor at 2 and 5 h after injection of [18F]FHPG, respectively (8) . Despite these promising results, no in vivo [18F]FHPG PET studies have yet been reported. In this study, we have performed biodistribution and PET studies on rats bearing both a HSV-tk-expressing tumor and a control tumor in their flanks to further substantiate the feasibility of monitoring HSV-tk enzyme activity using [18F]FHPG PET.
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Materials and Methods |
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Preparation of [18F]FHPG.
[18F]FHPG was prepared as described by Alauddin
et al. (14)
. However, the deprotection was
performed at 90°C for 5 min (15)
, and the
radiopharmaceutical was purified by HPLC over a semipreparative Alltima
C18 reverse-phase column (5 µ, 10 x 250 mm; Alltech) using 2% ethanol and 10
mM
NaH2PO4 in 0.9% NaCl as
the eluent (flow rate, 6 ml/min, Rt,
21 min). The product was obtained with a specific activity of >20
TBq/mmol and a radiochemical purity of >99% (HPLC).
Cell Lines.
C6 rat glioma cells obtained from the American Type Culture Collection
were cultured in monolayers in DMEM supplemented with 10% FCS in a
humidified atmosphere with 5% CO2 at 37°C.
HSV-tk-positive C6 cells (C6tk+) were obtained by transfection of the
C6 cells with supernatant of PA317-tk packaging cells containing
replication incompetent retroviruses carrying the HSV-tk gene and the
NeoR gene. Subsequent G418 selection resulted in the C6tk+ cell
line. Stable resistance in the C6tk+ cells was assured by culturing
this cell line in the presence of G418 (0.5 mg/ml).
Ganciclovir Sensitivity Assay.
Ganciclovir sensitivity was tested in vitro with a
cytotoxicity assay. In a 6-well plate, 500 cells per well were
incubated with ganciclovir. The concentration range of ganciclovir was
10-3 to 103 µg/ml. On
day 7, the number of cells was counted. Three independent experiments
were performed, each in triplicate. From the survival curves, the
ganciclovir concentrations that inhibited cell survival by 50%
(IC50) were determined.
Animal Model.
Female nude rats (HSD Ham RNU rnu; Harlan, the Netherlands; body
weight, 140–200 g; age, 6 weeks) were injected with tumor cells.
Before injection, C6 and C6tk+ cells (5 x 106cells/0.1 ml of DMEM/10% FCS) were mixed with
0.1 ml of Matrigel. Subsequently, the C6 and C6tk+ cell suspensions
were injected s.c. into the right and left flank close to the forelegs
of the nude rat, respectively. Ten to 14 days after injection of the
tumor cells, a solid tumor nodule of 0.7–3 cm in diameter had grown in
each flank. In this experimental setting, the animal carried both the
HSV-tk-containing tumor and the control tumor, which minimizes the
effects of biodiversity because each animal serves as its own control.
All studies were carried out in compliance with the local ethical
guidelines for animal experiments. The protocols were approved by the
local Animal Ethics Committee.
Tumor Histology.
To allow collection of histological data from the C6 and C6tk+ tumors,
these tumors were excised and frozen in liquid nitrogen. Four-µm
slices were cut from the frozen tissue. The cell type and viability
were determined on H&E-stained slides.
Immunohistochemistry for HSV-tk Expression.
The HSV-tk expression in cell lines and tumor tissue was tested
immunohistochemically with the HSV-tk-directed antibody. Frozen tissue
sections were fixed in acetone for 10 min and air-dried. After three
washes with PBS, sections were incubated in 0.075% hydrogen peroxide
in PBS [140 mM NaCl, 9 mM
Na3PO4, 1.3 mM
NaH2PO4 (pH 7.4)] for 30
min and washed again. The sections were reacted for 1 h at room
temperature with rabbit antiserum against the HSV-tk protein in 1% BSA
in PBS at a 1:100 dilution and the cell lines at a dilution of 1:500.
The sections were reacted with swine anti-rabbit biotinylated IgG
(1:300 dilution; DAKO, Glostrup, Denmark) and streptavidin
(1:300; DAKA, Denmark) each for 30 min.
3-Amino-9-ethyl-carborole was used as the chromogen, which provided a
red stain color in areas of HSV-tk expression. Sections were
counterstained with hematoxylin.
Accumulation of [18F]FHPG in Tumor Cells.
Monolayers of C6 and C6tk+ cells in 3 ml of DMEM supplemented with 10%
FCS were incubated with 3–4 MBq of [18F]FHPG
for 2 h at 37°C. Thereafter, the culture medium was removed, and
the monolayers were washed three times with 1.5 ml of ice-cold PBS. The
cells were harvested from the culture plates by treatment with 0.3 ml
of 0.5% trypsin for 5 min and resuspended in 1.5 ml of culture medium
to neutralize the trypsin. The cellular accumulation of radioactivity
was measured in a gamma counter (LKB Wallac, Turku, Finland) and
normalized to the number of viable cells in the monolayer.
Biodistribution.
Ten to 14 days after inoculation of the HSV-tk-transduced C6tk+ tumor
cells and the wild-type C6 cells, the tumor-bearing nude rats were
anesthetized with sodium pentobarbital (60 mg/kg).
[18F]FHPG (7–9 MBq in 0.4–0.9 ml of HPLC
eluent) was injected in the tail vein of the rats. After 2 h, the
rats were sacrificed by extirpation of the heart. Tumors and several
organs were dissected, and the radioactivity accumulated in these
tissues was measured with a gamma counter.
Analysis of [18F]FHPG Metabolites in Tumors
and Plasma.
The tumors were homogenized in 1 ml of PBS with 20 µl of 70%
HClO4. The homogenate was centrifuged (5 min,
1000 x g), and the pellet was washed twice
with 1 ml of PBS with 20 µl of HClO4. The
radioactivity in the acid-soluble and acid-insoluble fractions was
measured with a gamma counter. The acid-soluble fraction of the tumor
and plasma samples was analyzed by gradient ion-exchange HPLC
[Nucleosil 100–5 SB column (5 µ, 4 x 250 mm); flow
rate, 1.5 ml/min; mobile phase ammonium phosphate (pH 6.5); gradient,
0.1 M (0–5 min), 0.1–0.5
M (linear gradient; 5–25 min), and 0.5
M (25–35 min)]. The eluate was collected
in fractions of 1.5 ml during a 35-min period and counted in a gamma
counter. More than 95% of the radioactivity injected on the HPLC
column was recovered in the eluate.
PET Studies.
PET studies were performed using a Siemens Exact HR+ positron camera
(Siemens/CTI, Knoxville, TN) with a resolution of about 5 mm
(FWMH). Transmission and emission scans were obtained in
two-dimensional mode. The tumor-bearing rats were anesthetized
with sodium pentobarbital (60 mg/kg) and positioned in the PET camera
with their long axis parallel to the transaxial plane of the tomograph.
A 20-min transmission scan was obtained to correct for attenuation of
511 keV photons by tissue. A
H215O PET scan was performed to
identify differences in perfusion between the tumors that could
influence tracer accumulation. Because both tumors were present in the
same animal, correction for the
H215O input was not necessary.
Thus, approximately 20 MBq of
H215O in 0.3 ml of 0.9% NaCl
was injected into the tail vein, and data acquisition was started. At
least 15 min after the H215O PET
data acquisition was completed, about 8 MBq of
[18F]FHPG was injected into the tail vein. For
2 h, data were acquired. Emission scans were corrected for
radioactive decay, attenuation, random counts, and dead time. Data
analysis was performed using Siemens ECAT 7.1 software. In frame 10
(3.5 min p.i.) of the [18F]FHPG PET
images and frame 8 (2.5 min p.i.) of the
H215O PET images, ROIs were
drawn around both tumors in all coronal slices where the tumor was
visible. The ROI activities were summed, and time-activity curves were
calculated. Tracer accumulation is expressed as standardized uptake
values (SUV), which can be defined as:
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The radioactivity concentration in tissue was calculated by dividing the tissue activity in the ROI by the volume of the ROI.
Statistical Analysis.
Differences in tracer accumulation between C6 and C6tk+ tumor cells
were analyzed using an unpaired Student’s t test for
in vitro studies and a paired Student’s t test
for in vivo experiments. P < 0.05
was considered significant.
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Results |
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HSV-tk Expression and Histology.
HSV-tk expression in the C6tk+ cell line and the C6 control cell line
was tested with the HSV-tk-directed antibody. A heterogenous HSV-tk
expression was detected in the C6tk+ cell line. Counting 1000 cells on
cytospins revealed staining in all these cells, with 28% of the cells
being more clearly stained. The control C6 cells were negative. H&E
staining showed growing tumor cells in both the s.c. C6 and C6tk+ tumor
nodules. The HSV-tk directed antibody clearly stained the cells in the
C6tk+ tumors, whereas the control C6 tumors were negative.
Cellular Accumulation of [18F]FHPG.
To assess the feasibility of [18F]FHPG as a
tracer for HSV-tk activity, in vitro accumulation studies
were performed. After 2 h of incubation, in vitro
cellular accumulation of [18F]FHPG in C6 and
C6tk+ rat glioma cells was 0.016 ± 0.002 and
0.56 ± 0.07% injected
dose/106 cells, respectively. Thus, uptake
in HSV-tk-containing C6tk+ cells was 35 ± 5 times
higher than that in C6 control cells (P < 0.0005).
Biodistribution.
Two h after injection of the tracer, the
[18F]FHPG content in several tissues was
determined by ex vivo counting. As shown in Fig. 1
, the level of radioactivity in both proliferative and nonproliferative
tissues was similar to blood plasma levels. High levels of
radioactivity were found in the kidneys and the urine as a result of
the rapid renal excretion of the tracer. In contrast to the C6 control
tumor, high levels of [18F]FHPG had accumulated
in the HSV-tk-containing C6tk+ tumor (tissue:plasma ratio,
12.3 ± 0.8). After only 2 h of biodistribution,
the uptake of radioactivity in the C6tk+ tumor already exceeded the
uptake in the C6 control tumor by a factor of 15 ± 5
(n = 4; P < 0.0005).
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. However, in the acid-soluble fraction of C6tk+ tumors, only
17 ± 6% (n = 3) of tissue
radioactivity represented the parent compound. In addition to
[18F]FHPG, three negatively charged metabolites
were found. The elution pattern of these metabolites and published data
(8
, 11)
suggest that these metabolites could be
[18F]FHPG-monophosphate,
[18F]FHPG-diphosphate, and
[18F]FHPG-triphosphate. Like ganciclovir
(10)
, [18F]FHPG is expected to be
converted by HSV-tk to FHPG-monophosphate, which will then be further
phosphorylated by host cellular kinases. These phosphates are
negatively charged and cannot cross the cell membrane. Thus,
accumulation of radioactivity in the tumor represents HSV-tk enzyme
activity.
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. The consecutive PET images show that the tracer is rapidly cleared
from nontarget tissues into the urine. After 120 min, the control tumor
and other nontarget tissues were no longer visible, whereas the C6tk+
tumor, the kidneys, and the bladder were clearly delineated. Even a
C6tk+ tumor weighing only 0.2 g could clearly be visualized with
[18F]FHPG PET. The
[18F]FHPG time-activity curves showed that the
accumulation of radioactivity in both tumors reaches a maximum at about
4 min postinjection (Fig. 4)
. Thereafter, the level of radioactivity in the HSV-tk-containing tumor
remained virtually constant. The radioactivity in the control tumor, on
the other hand, decreased exponentially with a half-life of
28.1 ± 1.4 min. The ratio between the activity in the
C6tk+ tumor and the activity in the control tumor, as determined by
PET, was 12 ± 4 after 2 h of tracer distribution.
This ratio is slightly lower than the ratio determined by ex
vivo counting (15 ± 5) because of spill-over and
partial volume effects. From the time-activity curves, it is clear that
the difference in uptake between control and C6tk+ tumors will increase
with time and is not yet maximal at 120 min.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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[124I]FIAU was the first radiopharmaceutical that allowed successful imaging of HSV-tk expression using PET (6) . Using this tracer, different levels of gene expression could be discriminated. However, widespread use of [124I]FIAU PET is not expected because of (a) the slow clearance of the tracer from nontarget tissue, which prevents 1-day protocols; (b) the ability of human thymidine kinases to phosphorylate FIAU (16) , which may give rise to significant nonspecific uptake in rapidly dividing cells; (c) the poor physical properties of 124I, which hamper quantification and high spatial resolution (17) ; and (d) the limited availability of 124I. A radiopharmaceutical labeled with 18F instead of 124I might overcome these restrictions.
Ganciclovir has been radiolabeled with 18F in two positions: (a) at C-8 of the purine ring (8-[18F]fluoroganciclovir; Ref. 18 ); and (b) in the side-chain ([18F]FHPG; Refs. 13 and 17 ). 8-[18F]Fluoroganciclovir has been applied to image adenovirus-directed hepatic HSV-tk expression in mice with PET (7) . Although the tracer was rapidly cleared from most of the nontarget tissues into the urine, significant troublesome background accumulation was found in the intestines.
In the present study, we have demonstrated in rats with both a HSV-tk-expressing tumor and a control tumor in their flanks that [18F]FHPG was rapidly and selectively trapped in the HSV-tk-expressing tumors due to phosphorylation of the tracer by the HSV-tk enzyme. This observation was supported by the presence of three negatively charged metabolites in HSV-tk-expressing tumors only. Hardly any accumulation of radioactivity in rapidly proliferating tissues, such as intestines, bone marrow, and control tumors, was observed, indicating that FHPG is not significantly phosphorylated by host kinases. The tracer was rapidly cleared from nontarget tissues into the urine. As a result, high target:background ratios were obtained after only 2 h of tracer distribution, which easily allows 1-day protocols. Although the sensitivity of this imaging technique remains to be determined, these excellent target:background ratios suggest that clinically relevant levels of HSV-tk expression can be monitored as well. Prolonged distribution times can improve the sensitivity of the technique.
In contrast to 8-[18F]fluoroganciclovir (18) , [18F]FHPG can be prepared with high specific activity (14 , 19) , which allows low-mass injections (<5 µg per 400 MBq). Because of the low dose administered, the rapid clearance, the absence of significant tracer accumulation in nontarget tissues, and the absence of recirculating metabolites, application of [18F]FHPG as a PET tracer is not expected to give rise to any cytotoxic effects. Pharmacokinetic studies in monkeys have shown that the radiation dose of [18F]FHPG is acceptable, with the bladder wall receiving the highest burden due to the rapid renal excretion (20) . The radiation dose and background accumulation in the bladder can significantly be reduced by urination or by applying a catheter.
In conclusion, we have demonstrated that we can noninvasively image HSV-tk expression in a double tumor-bearing rat model using [18F]FHPG PET. From the high target:background ratios observed, this imaging method is expected to have a clinically relevant sensitivity. The relatively short half-life of 18F allows sequential imaging of gene expression with [18F]FHPG PET. Imaging of the level and the distribution of gene expression over time will provide new and useful information for monitoring clinical gene therapy protocols in the future.
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FOOTNOTES |
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1 Supported by the De Cock Stichting. 
2 To whom requests for reprints should be
addressed, at Department of Medical Oncology, Groningen University
Hospital, P. O. Box 30.001, 9700 RB Groningen, the Netherlands. Phone:
31-50-361-61-61; Fax: 31-50-361-48-62; E-mail: g.a.p.hospers{at}int.azg.nl
3 The abbreviations used are: HSV-tk, herpes
simplex virus type 1 thymidine kinase; FHPG,
9-[(1-fluoro-3-hydroxy-2-propoxy)methyl]guanine; FIAU,
2'-fluoro-2'-deoxy-1-ß-D-arabinofuranosyl-5-iodo-uridine;
HPLC, high-performance liquid chromatography; PET, positron emission
tomography; ROI, region of interest; tk, thymidine kinase; p.i.,
post-injection.
Received 8/24/99. Accepted 2/ 3/00.
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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