Hypoxia occurs to a variable extent in most rodent and human solid tumours (Moulder
and Rockwell, 1984; Höckel and Vaupel, 2001). It results from an inadequate and disorganised
tumour vasculature and blood flow, and hence impaired oxygen delivery (Vaupel et al,
1989). Detection of hypoxia is important because hypoxia is a powerful trigger for
gene expression and thus clonal selection of a more aggressive phenotype, for example,
diminished apoptotic potential (Gräber et al, 1996; Koong et al, 2000). Of immediate
clinical importance, hypoxia reduces local tumour control by external beam radiotherapy
(Horsman and Overgaard, 1992) and predicts general treatment outcome, including metastatic
potential and survival following radio/chemotherapy and surgery in a number of human
cancers (Höckel et al, 1993; Brizel et al, 1996). Thus, detection of hypoxia could
improve patient selection for therapy with bioreductive agents (Stratford and Workman,
1998), modified radiotherapy regimes such as accelerated radiotherapy with carbogen
and nicotinamide ‘ARCON’ (Hoskin and Saunders, 1994), conformal radiotherapy, and
hypoxia-targeted gene therapy (Dachs et al, 1997). Beyond oncology, potential applications
exist for diagnosis of stroke, ischemic heart disease, peripheral vascular disease,
arthritis, and anaerobic infection.
Studies with fine needle oxygen electrodes provided proof that low pO2 leads to poor
outcome after treatment (Höckel et al, 1996; Brizel et al, 1997; Nordsmark and Overgaard,
2000; Fyles et al, 2002). Immunohistochemical parameters obtained by staining biopsy
sections with antibodies to EF5, pimonidazole, hypoxia-inducible factor 1α (HIF-1α
3), and carbonic anhydrase IX have also shown promise (Evans et al, 1996; Wykoff et
al, 2000; Aebersold et al, 2001; Kaanders et al, 2002). Utility in a routine clinical
setting would, however, favour a simple, convenient, noninvasive method for detecting
tumour hypoxia that yields results, which correlate with clinical outcome. In this
regard, a number of probes have been developed for imaging hypoxia by positron emission
tomography (PET) and single-photon emission tomography in nuclear medicine. These
include 18F-labelled 2-nitroimidazoles such as [18F]FMISO (Koh et al, 1995; Rasey
et al, 1996), [18F]EF5 (Ziemer et al, 2003), [18F]FETNIM (Yang et al, 1995), [18F]FAZA
(Sorger et al, 2003), and N-(2-([18F]fluoroethyl)-2-(2-nitroimidazol-1-yl)-acetamide
([18F]FETA) (Rasey et al, 1999); copper bis-thiosemi-carbazones such as [60Cu]ATSM
(Fujibayashi et al, 1997; Dehdashti et al, 2003); technetium-based probes such as
[99mTc]HL-91 (Honess et al, 1998); and iodine-based probes such as [123I]IAZA (Mannan
et al, 1991). Due to various limitations, none of these radiotracers have found their
way into routine clinical use (Workman and Brown, 1981; Nunn et al, 1995; Rasey et
al, 1999; Bentzen et al, 2002).
Of the numerous possibilities in nuclear medicine, appropriately labelled 2-nitroimidazole
probes are particularly attractive for imaging tumour hypoxia (pO2 <10 mmHg (Höckel
et al, 1996; Nordsmark et al, 1996); radiobiological hypoxia, pO2 <1 mmHg (Rasey et
al, 1990)). A combination of pharmacological and physical properties that need to
be considered as part of an ideal design goal include: (i) a nitro group with appropriate
redox potential (E1/7 of ∼−380 to −390 mV) for selective reduction and binding in
hypoxic tumour cells; (ii) lipophilicity that is high enough to enable diffusion across
cellular membranes to the site of metabolism (octanol–water partition coefficient
of ∼⩾0.1 (Brown and Workman, 1980; Workman, 1982), but low enough (∼⩽2) to assure
rapid systemic elimination and, hence, convenient imaging times (within 2 h); (iii)
stability to hypoxia-independent degradation; and (iv) high photon flux (and low energy)
to assure high detection sensitivity and spatial image resolution. The last property
would favour 18F-based PET radiotracers. In this manuscript we report on preclinical
studies that underpin the development of [18F]FETA ([18F]fluoroetanidazole), an extremely
promising hypoxia probe for imaging by PET. We demonstrate for the first time in mouse
models of cancer that the retention of [18F]FETA-derived radioactivity correlates
with pO2 and radiation sensitivity.
MATERIALS AND METHODS
Radiosynthesis of [18F]FETA
Unless otherwise noted, all reagents, including anhydrous solvents, were obtained
from Sigma-Aldrich (Poole, UK). The synthesis of [18F]FETA was based on the method
of Tewson (1997), with some modifications. N-[2-(toluene-4-sulphonyloxy)-ethyl]-phthalimide
(I in Figure 1
Figure 1
Radiochemical synthesis of [18F]FETA. I, II, III, and IV represent N-[2-(toluene-4-sulphonyloxy)-ethyl]-phthalimide,
2,3,5,6-Tetrafluorophenyl 2-(2-nitroimidazol-1-yl) acetate, [18F]Fluoroetanidazole,
and [18F]Fluoroethylamine.
) was prepared according to the literature method (Tewson, 1997). The precursor, 2,3,5,6-Tetrafluorophenyl
2-(2-nitroimidazol-1-yl) acetate, (II) was prepared by a novel method, published separately
(Wilson, 2003). [18F]fluoride was prepared by the 18O(p,n)18F reaction using H2
18O enriched water (Ruth and Wolf, 1979). A measure of 250 μl 0.1 M K2CO3 and 3 mg
[2,2,2]Kryptofix in 500 μl acetonitrile were added to the [18F]fluoride. The fluoride
was dried in a glassy carbon vessel at 125°C, under N2, for 35 min (with two further
additions of 500 μl acetonitrile), before cooling. In total, 15 mg of compound (I)
was dissolved in 500 μl acetonitrile. This was added to the reaction vessel, which
was sealed at 100°C for 10 min before evaporation of the acetonitrile. A measure of
75 μl hydrazine hydrate was added to the reaction vessel. [18F]fluoroethylamine (IV)
was distilled from the reaction vessel (75°C, N2 flow ∼3 ml min−1) into a vial containing
4 mg precursor (II) in 400 μl of ice cold acetonitrile. The vial was sealed and stirred
for 30 min at room temperature. The resulting product was purified by high-performance
liquid chromatography (HPLC) (Waters μ-Bondapak C18 column) and eluted with water
(Baxter Healthcare Ltd, Compton, UK; 3 ml min−1). The radioactive product having the
same retention time as fluoroetanidazole was collected (reference material synthesised
according to Tewson, 1997). The product was evaporated to dryness and taken up in
0.9% saline (Phoenix Pharma Ltd, Gloucester, UK) for injection. The (manual) preparation
time for [18F]FETA (synthesis, purification, formulation) from the end of bombardment
was approximately 3 h.
Lipophilicity measurements
The lipophilicity of [18F]FETA was determined by measuring the octanol–water partition
coefficient. For this, 80–100 μCi (2.96–3.70 MBq) of [18F]FETA was dissolved in distilled
H2O to a final volume of 0.5 ml and 0.5 ml of octan-1-ol (BDH Chemicals Ltd, Poole,
UK) was added. The resulting mixture was processed in an orbital shaker (Sanyo Gallenkamp
PLC, Loughborough, UK) for 10 min at 300 rpm and subsequently centrifuged for 30 min
(12 000 rpm; Rotina 35R, Hettich Zentrifugen, Tuttlingen, Germany). Aliquots (200 μl)
of the resulting top layer (representing [18F]FETA dissolved in octanol) and bottom
layer ([18F]FETA dissolved in H2O) were taken and the radioactivity in these samples
was measured using a Cobra II Auto-Gamma counter (Packard Instruments, Meriden, CT,
USA). Six octanol–H2O mixtures were analysed and the complete experiment was repeated
once. The octanol–water partition coefficient was calculated by dividing the octanol-containing
radioactivity by the water-containing radioactivity.
Cellular uptake of [18F]FETA
The inhibitory effect of oxygen on [18F]FETA uptake by cells was studied in vitro
in RIF-1 tumour cells. Uptake was defined as the transport of radiotracer into cells
and its specific binding to cellular macromolecules (and glutathione). The cells were
cultured (5% CO2 incubator at 37°C) in T75 flasks in RPMI growth medium (Life Technologies,
Strathclyde, UK) supplemented with 10% foetal bovine serum (Sigma) and antibiotics.
Exponentially growing cells were harvested, dissolved in phosphate-buffered saline
(PBS) and placed in a custom-made hypoxic chamber (Aboagye et al, 1997a), which was
positioned in a 37°C shaking water bath and incubated with pure nitrogen (O2 <20 ppm;
BOC Gases, Guildford, UK) or with air as control, each flowing at 2 l min−1. After
pregassing for 8 min, the cells were incubated with 80–100 μCi (2.96–3.7 MBq) of [18F]FETA.
No correction for the total amount of stable etanidazole in the reaction mixture was
made between experiments. Immediately after addition of the radioactivity, and at
15, 30, 45, 60, 90, and 120 min, the cells were harvested and centrifuged (5000 rpm
for 5 min) to obtain a pellet. The pellet was washed twice with PBS (5 ml) and centrifuged
as above. The cell number in the resulting pellet was determined by counting cells
that excluded trypan blue. The total radioactivity added to each hypoxic chamber and
the cell-bound radioactivity were measured on a Cobra II Auto-Gamma counter (Packard
Instruments, Meriden, Connecticut, SA, USA) to determine the degree of radioactivity
binding. Percentage of bound radioactivity was calculated as (bound radioactivity
in 106 cells × 100)/total radioactivity. These experiments were carried out in triplicate
and repeated independently three times.
Animals and tumours
The mice used in this work were obtained from Harlan UK Ltd (Bicester, UK). To obtain
tumours, 2 × 105–2 × 106 tumour cells harvested from exponentially growing in vitro
cultures were injected into the dorsum subcutis of normal C3H/Hej and Balb/c mice
(for RIF-1 mouse fibrosarcoma and EMT6 mouse mammary carcinoma tumours, respectively)
or Balb/c nude mice (for HT1080 human fibrosarcoma and MCF-7 human mammary carcinoma
tumours). In the case of the HT1080 tumours, two subclones (1-3C, 26.6) with different
capacities for producing vascular endothelial growth factor (VEFG) were studied. As
published recently by our group, the 26.6-subclone produces two- to four-fold more
VEGF than the 1-3C-subclone (Collingridge et al, 2002). To obtain MCF7 tumours, a
60-day slow release 17-β-oestradiol pellet (Innovative Research of America, Sarasota,
FL, USA; 0.72 mg pellet−1) was inserted subcutaneously on the contralateral flank
of the mouse at the time of inoculation of the tumour cells to maintain blood levels
of 300–400 pg ml−1. All the animal work was performed by licensed investigators in
accordance with the United Kingdom's ‘Guidance on the Operation of Animals (Scientific
Procedures) Act 1986’ (HMSO, London, UK, 1990) and in full compliance with government
regulations and the UKCCCR guidelines on animal welfare in experimental neoplasia
(Workman et al, 1998). Tumours were selected for experiments when they had reached
5–8 mm in diameter (100–300 mg). In order to obtain comparable results, all biodistribution
studies, PET imaging studies, and pO2 measurements were performed in anaesthetised
mice. For that, a general neuropleptanalgesia comprising of a mixture of fentanyl
citrate/fluanisone (Janssen-Cilag Ltd, Saunderton, UK; 0.79 and 25 mg kg−1 respectively)
and midazolam hydrochloride (Phoenix Pharma, Ltd, Gloucester, UK; 12.5 mg kg−1) was
injected i.p. 5 min before the start of the experiments. Irradiation of mice, in studies
to determine radiobiological hypoxic fraction, were performed in unanaesthetised mice.
Biodistribution of [18F]FETA
Anaesthetised tumour-bearing mice were injected intravenously via the lateral tail
vein with 100 μl of [18F]FETA (10–100 μCi; 0.37–3. 7 MBq). Mice were killed by exsanguination
via cardiac puncture 60 min after injection of the radiotracer. Aliquots of heparinised
blood were rapidly centrifuged (2000 g for 5 min) to obtain plasma. The radioactivity
contained in tumour, liver, kidney, spleen, lungs, heart, small intestines, brain,
muscle, bone, blood, plasma, faeces, and urine was determined in a Cobra II Auto-Gamma
counter and expressed as % ID g−1 (percentage of injected dose per gram of tissue).
A minimum of six mice was used for each tumour model.
Biodistribution studies were also carried out in mice following modulation of tumour
hypoxia. In RIF-1 tumour-bearing mice, the degree of tumour hypoxia was modulated
by administration of carbogen gas (95% O2, 5% CO2), which is known to decrease tumour
hypoxia (Powell et al, 1999). There are a number of reports in the literature demonstrating
an increase in oxygenation or perfusion in RIF-1 tumours under carbogen breathing.
These studies include direct pO2 measurements, as well as magnetic resonance and 86Rb
extraction methods in RIF-1 tumours implanted in the leg (Hasegawa et al, 1987; Song
et al, 1987; Ilangovan et al, 2002) or flank (Helmer et al, 1998; Dardzinski and Sotak
1994; Honess and Bleehen, 1995). Carbogen gas was delivered via a nose cone at 2.0 l min−1,
starting 2 min before radiotracer injection and for 60 min after radiotracer injection
(n=5). Untreated RIF-1 tumour-bearing mice served as controls (n=8). Mice were injected
with [18F]FETA and killed at 60 min postinjection (p.i.), as described above. Successful
modulation of tumour pO2 was verified by means of OxyLite probes as described below.
Small animal [18F]FETA-PET imaging
In parallel with the 60-min biodistribution studies above, the time course of [18F]FETA
distribution was studied in vivo in tumour-bearing mice by means of a second-generation
dedicated small animal PET scanner (Quad-HIDAC; Oxford Positron Systems, Weston-on-the-Green,
UK (Jeavons et al, 1999)). Mice were anaesthetised as described above and placed prone
on a thermostatically controlled bed within the scanner. All anaesthetised animals
(in the PET imaging, biodistribution and OxyLite pO2 studies) were placed on thermal
platforms to maintain their body temperature at ∼37°C. Dynamic PET scans were acquired
over 60 min after intravenous (i.v.) injection of 40–100 μCi (1.48–3.7 MBq) of [18F]FETA
via a tail vein cannula. The imaging and image-data processing protocol has been recently
described in detail elsewhere (Barthel et al, 2003). In brief, data were acquired
in list mode format and image reconstruction was performed by filtered back-projection
using a 2D Hamming filter (cutoff 0.6). The image data sets obtained were transferred
to a SUN workstation (Ultra 10; SUN Microsystems, Santa Clara, CA, USA) and visualised
using the ANALYZE software (Version 5.0; Biomedical Imaging Resource, Mayo Clinic,
Rochester, NY, USA). Region(s) of interest (ROIs) were defined for tumour and heart
cavity (to provide information on the delivered radioactivity) on three to six transverse
planes for tumour and three planes for heart cavity. Time vs radioactivity curve(s)
(TACs) from the ROIs were averaged for each tumour and normalised to the integral
of the heart cavity TAC. Altogether, four HT1080/1-3C and three HT1080/26.6 tumour-bearing
mice were scanned.
In vivo metabolism of [18F]FETA
Plasma, urine, liver, gall bladder, muscle, and tumour samples obtained from RIF-1
tumour-bearing C3H/Hej mice were assessed for putative [18F]FETA metabolites by HPLC.
For this, plasma samples (100 μl) were deproteinated by adding 2.0 ml ice-cold methanol
and centrifuged (3000 g, 10 min, 4°C). Liver, muscle, and tumour samples were cut
into small pieces and homogenised in 2.0 ml of ice-cold methanol using an Ultra-Thurrax
homogeniser (IKA, Staufen, Germany) and the resultant homogenate was centrifuged (3000 g,
10 min, 4°C). The supernatants from plasma and tissues were evaporated to dryness
in a rotary evaporator (50°C) under vacuum, reconstituted in 1.5 ml of mobile phase
(methanol/H2O (20/80% v v−1)), centrifuged and filtered (0.2 μm). Unlike plasma and
tissue samples, urine and gall bladder samples were diluted with 1.5 ml of mobile
phase and clarified by filtration (0.2 μm). Aliquots of each filtrate (1.0 ml) and
of the injected dose solution diluted in 1.5 ml of mobile phase were analysed by HPLC.
The samples were separated on a C18 μBondapak column (7.8 × 300 mm, size 10 μm; Waters,
Milford, MA, USA) that was eluted with the aforementioned mobile phase at a flow rate
of 2.0 ml min−1. The radioactivity of the eluents was monitored. Peak areas were integrated
and corrected for physical decay and background radioactivity.
Direct measurement of pO2
Oxygenation in the established tumour models was measured using the OxyLite pO2 system
(Oxford Optronix Ltd, Oxford, UK) (Young et al, 1996; Collingridge et al, 1997; Seddon
et al, 2001). The OxyLite probes measure pO2 by using a fluorescence quenching technique
(Young et al, 1996). These experiments were performed under general neuroleptanalgesia.
The animals were placed on a thermal blanket (37°C) and a maximum of four (depending
on the tumour size) oxygen probes were simultaneously implanted into the mouse tumours.
The probes were moved forward manually ∼0.2 cm every 10 min (total measurement time
30–50 min), which resulted in 45–125 pO2 measurements per tumour. All oxygen measurements
were postcalibrated to account for tumour temperature, as previously reported (Young
et al, 1996; Collingridge et al, 1997).
Measurement of vessel density
In parallel studies in HT1080 (grown from 1-3C and 26.6 subclones) tumour-bearing
mice, tumours were excised for histological examination of vessel density. For this,
each of seven 1-3C and 26.6 tumours were excised, fixed in formalin, embedded in paraffin,
and cut into 5.0 μm sections. Adjacent sections were stained with haematoxylin–eosin
(H&E). Sections from three different regions of each tumour, separated by at least
1.0 mm, were used for the analyses. Vessels were counted in five randomly selected
fields of view (0.23 mm2) per section using a BX51 Olympus microscope (Olympus Optical,
Tokyo, Japan) at × 200 magnification.
Determination of radiobiological hypoxic fraction
In addition to the pO2 measurements, radiobiological hypoxia was evaluated for the
26.6. and 1-3C subclones of the HT1080 tumours by performing clonogenic assays. Clonogenic
cell survival, which is still regarded as gold standard for measuring tumour hypoxia
(Rockwell and Moulder, 1990), was determined using an established protocol (Collingridge
and Rockwell, 2000). In brief, the tumour-bearing mice were irradiated with 5, 15,
or 25 Gy (mean dose rate=4.3 Gy min−1) using an IBL 637 irradiator (CIS bio international,
Gif/Yvette Cedex, France). One group of mice breathed air during the irradiation;
the other group comprised of mice killed 10 min before the irradiation (n=12 per group).
Immediately following irradiation, the tumours were excised, minced, and digested
for 45 min in a 37°C shaking water bath with a 10 ml solution of foetal bovine serum-free
RPMI growth medium containing 2 mg ml−1 collagenase type IV (Sigma, Poole, UK) and
0.2 mg ml−1 DNAse I (Sigma). After centrifugation, cell density was determined by
trypan blue exclusion and defined numbers of cells were plated in Petri dishes containing
complete growth medium. The dishes were incubated at 37°C in a humidified incubator
in an atmosphere of 95% air/5% CO2 for 10–14 days. The resulting colonies were fixed
and stained with crystal violet made up in 70% ethanol and subsequently counted. The
survival fraction was calculated by dividing the plating efficiency of the irradiated
mice by that of unirradiated control mice (n=4). Using these survival curves, radiobiological
hypoxia fraction was calculated from the vertical displacement of the aerobic curve
from the anoxic curve according to the equation loghypoxia fraction=logaerobic survival
fraction−loganoxic survival fraction (Moulder and Rockwell, 1984). The determination
of hypoxic fraction in this way is only possible if the survival curves are parallel.
Pooled tumours from four animals were used for each irradiation level and tumour type.
Nitroreductase activity
Tumour retention of an ideal radiolabelled 2-nitroimidazole should depend solely on
intratumoural oxygenation levels, and not on the levels of nitroreductase activities.
In this regard, we investigated the influence of tumour nitroreductase activities
on [18F]FETA tumour retention. The activities of cytochrome P450 reductase, cytochrome
b
5
reductase, and DT-diaphorase were determined in excised, snap-frozen tumours using
a standard cytochrome c assay, as previously described by Sharp et al (2000). Briefly,
enzyme activities were determined on a UV-spectrophotometer (BioMate 5; Thermo Spectronic,
Cambridge, UK) by measuring the rate of reduction of cytochrome c at 550 nm (Walton
et al, 1991). Tumour samples were thawed and homogenised in an equivalent volume of
50 mM Tris – 150 mM KCl–HCl buffer (pH 7.4). The homogenates were centrifuged (10 000 rpm
for 30 min, 4°C) and the resulting supernatant, which represented the S9 fraction,
was taken for the further analysis. The protein content of the S9 fractions was determined
using a commercial kit (BCA protein assay kit; Pierce, Rockford, IL, USA). Samples
of the S9 fraction (5–30 μl) were added to 1 ml of a reaction mixture containing 50 mM
(final concentration) Tris HCl buffer (pH 7.4), 20 μ
M menadione (Sigma) as initial electron acceptor, 70 μ
M cytochrome c (Sigma) as terminal electron acceptor, 200 μ
M NADH (Sigma) as electron donor, and 1.4 mg ml−1 bovine albumin fraction V (Sigma).
The solutions were prewarmed at 37°C and the experiment was performed with or without
10 μ
M dicumarol (Sigma), an established inhibitor of DT-diaphorase (Hollander and Ernster,
1975). DT-diaphorase activity was taken as dicumarol-inhibitable activity and expressed
as nmol of cytochrome c reduced per minute per mg of protein. The activities of cytochrome
P450 reductase and cytochrome b
5
reductase were determined as above, but without menadione/dicumarol, and with 200 μ
M NADPH (Sigma) instead of NADH in the case of cytochrome P450 reductase assay (Fitzsimmons
et al, 1996; Winski et al, 1998).
Statistics
Statistical analyses were performed using the software SPSS for Windows, version 10.0.7
(SPSS Inc., Chicago, IL, USA). Differences in pO2 data, ex vivo [18F]FETA biodistribution,
and nitroreductase activities were tested for significance using analysis of variance
(ANOVA). Two-sided Student's t-test for independent samples was employed to test for
differences in [18F]FETA uptake in vitro under normoxic and hypoxic conditions, pO2
values, and ex vivo [18F]FETA retention in RIF-1 tumours treated with carbogen vs
untreated controls, as well as differences between vessel density, clonogenic survival,
and fractional radiotracer retention obtained from dynamic PET scanning in 26.6 and
1-3C subclones of HT1080 tumours. Associations between [18F]FETA tumour retention,
pO2 values, and nitroreductase activities in the established tumour models were tested
for correlations by means of linear regression analysis. pO2 values obtained from
the OxyLite measurements were expressed as median values for all investigated mouse
tumours. Survival fractions from clonogenic assays were calculated relative to the
corresponding plating efficiency and are given as mean±1 s.e.m. Survival curves were
fitted by regression analysis and compared at the midpoints of the curves. Unless
stated, data are expressed as mean±1 s.e.m. The P-values of ⩽0.05 were considered
significant.
RESULTS
Radiochemistry and in vitro characteristics of [18F]FETA
Beginning with 30–50 mCi (1.11–1.85 GBq) [18F]fluoride, [18F]FETA yields were typically
0.6–3.0 mCi (22–111 MBq), which represents a final (decay corrected to end of bombardment)
radiochemical yield of approximately 10–20%. High-performance liquid chromatography
analyses of the [18F]FETA dose solutions were performed 60 min after preparation of
the radiotracer. [18F]FETA eluted at ∼9.5 min. The radiochemical purity was 94.6±1.5%
(n=4). The lipophilicity of [18F]FETA expressed as octanol–water partition coefficient
was determined to be 0.16±0.01 (n=12).
We studied the uptake of [18F]FETA in air vs nitrogen in RIF-1 cells. The uptake of
the radiotracer by normoxic cells was very low. In contrast, there was a rapid time-dependent
linear increase of [18F]FETA uptake by RIF-1 cells under nitrogen, which was 3.0-
and 4.3-fold as compared to the normoxia values at 60 and 120 min p.i., respectively.
In this model system the hypoxia-dependent increase in [18F]FETA uptake reached significance
after 60 min (Figure 2
Figure 2
In vitro binding of [18F]FETA to RIF-1 cells under hypoxic (-•-) and normoxic (-○-)
conditions. The cells were incubated with [18F]FETA under nitrogen gas or air for
0–120 min and washed to remove unbound radioactivity. The percentage of bound radioactivity
was calculated as (bound radioactivity in 106 cells × 100)/total radioactivity. Data
are mean±s.e.m. (n=3), *
P⩽0.05.
).
In vivo tissue distribution and metabolism of [18F]FETA
The normal tissue retention of the radiotracer at 60 min postinjection was low (<5%
ID g−1) in lung, heart, brain, and bone; intermediate (5–8% ID g−1) in plasma, liver,
spleen, small intestines, and muscle; and high (>8% ID g−1) in kidney, bile, and urine
(Figure 3
Figure 3
Tumour and normal tissue distribution of [18F]FETA at 1 h p.i. in HT1080/26.6 tumour-bearing
male Balb/c nu/nu mice (n=10). Inset: radiotracer uptake in the different tumour types
(n=5–10). Data are mean % ID g−1±s.e.m.
). The 60-min tumour retention of [18F]FETA ranged from 6 to 10% ID g−1 for the different
xenografts (P=0.023; one-factorial ANOVA; Figure 3 inset). Other than differences
in tumour retention, no significant differences in radiotracer accumulation were found
for normal tissues of the different mouse strains (data not shown).
Radiochromatograms of plasma, liver, tumour, muscle, gall bladder, and urine that
were obtained at 10 (plasma, liver, and tumour) and 60 min (all tissues and body fluids)
after the administration of [18F]FETA are shown in Figure 4
Figure 4
Reversed-phase high-performance liquid chromatograms of [18F]FETA and its putative
metabolites at an early time-point (10 min p.i.) in plasma (A), liver (D), and tumour
(G), and a late time-point (60 min p.i.) in plasma (B), muscle (C), liver (E), gall
bladder (F), tumour (H), and urine (I). Filled arrows are parent [18F]FETA compound,
and double-lined arrows are metabolites.
. As with the analysis of the dose solution, the parent compound ([18F]FETA) eluted
at a retention time of ∼9.5 min. Parent [18F]FETA comprised 88% of the total radioactivity
in plasma at 10 min p.i. In tumour, a minor radioactive peak at a retention time of
∼6 min that comprised 18% of the total radioactivity was detected at 10 min p.i.,
whereas most of the radioactivity (92%) in the liver was associated with the metabolite(s)
eluting at ∼6 min. At 60 min p.i., metabolite peaks with retention times of ∼6 to
7 min were detected in the plasma (7% of total radioactivity), muscle (50%), liver
(99%), bile (82%), tumour (52%), and urine (26%) (Figure 4). These HPLC data represent
profiles of soluble parent and metabolites. The acid-insoluble fraction obtained from
PCA extraction (representing bound radioactivity) was 69.2% for tumour and 63.7% for
liver tissues at 60 min p.i.
[18F]FETA tumour retention correlates with pO2 but not nitroreductase activity
Various threshold pO2 values (<1, 2.5, 5, and 10 mmHg) have been used to describe
the degree of hypoxia in tissues. In the tumour xenografts used here, the relative
frequency of pO2 values <1.0 mmHg as determined by the OxyLite method ranged from
28±13 to 93±9% (P=0.008; one-factorial ANOVA). The relative frequency of pO2 values
<2.5 mmHg ranged from 51±13 to 100±0% (P=0.057). In HT1080 tumours, the relative frequency
of hypoxic pO2 values was significantly higher for the 1-3C subclone compared to the
26.6 subclone at a cutoff pO2 of 1.0 mmHg (93±9 vs 55±15%; P=0.023) and 2.5 mmHg (100±0
vs 66±16%; P=0.045).
For all the tumour models, the relative [18F]FETA retention, expressed as tumour-to-muscle
ratio, was positively correlated with the relative frequency of pO2 values <5 mmHg.
This correlation was found to be linear (y=0.04x–1.5, r=0.805) and significant (P=0.027;
Figure 5A
Figure 5
Association between tumour oxygenation as measured by OxyLite probes and [18F]FETA
tumour uptake determined by ex vivo biodistribution studies. (A) Relative [18F]FETA
uptake expressed as tumour-to-muscle ratio vs relative frequency of pO2 values <5 mmHg.
(B) [18F]FETA uptake vs relative frequency of pO2 values <2.5 mmHg.
). A trend towards a significant positive correlation was also observed for the association
between tumour [18F]FETA retention and relative frequency of pO2 values <2.5 mmHg
(r=0.691, P=0.064; Figure 5B). Of interest, [18F]FETA radiotracer retention at 60 min
p.i. was higher in the less VEGF-producing HT1080/1-3C subclone compared to the 26.6
subclone (9.29±1.36 vs 5.98±0.50% ID g−1; P=0.015). In addition, tumour/muscle ratios
obtained from the [18F]FETA biodistribution studies were significantly higher for
the 1-3C subclone as compared to the 26.6 subclone (1.94±0.36 vs 1.05±0.03, P<0.001).
The activities of cytochrome P450 reductase, cytochrome b5 reductase, and DT-diaphorase
for the different tumours are given in Table 1
Table 1
Nitroreductase activities in established tumours
Tumour type
Cytochrome P450 reductase (nmol min−1 mg−1)
Cytochrome b5 reductase (nmol min−1 mg−1)
DT-diaphorase (nmol min−1 mg−1)
RIF-1
2.21±0.18
50.0±1.8
1.38±0.23
HT1080/26.6
3.39±0.36
36.8±1.4
0.93±0.01
HT1080/1-3C
4.15±0.34
35.3±2.5
0.21±0.05
EMT6
2.79±0.37
52.2±1.9
0.62±0.15
MCF-7
2.01±0.17
20.8±1.0
0.00±0.00a
P (ANOVA)
0.001
<0.001
<0.001
Enzyme activities were determined spectroscopically by measuring reduction of cytochrome
c at 550 nm, as described in Materials and Methods. Units are nmol of cytochrome c
reduced per minute per mg of sample protein and are given as mean±s.e.m. (n=5–7).
a
No activity detected within effective range of assay (lower limit of detection=0.05 nmol min−1 mg−1).
. There were significant intertumour differences (one-factorial ANOVA) in enzyme activity.
The range of these values, however, were ⩽2.5 fold for all the reductive enzymes tested
(Table 1). There were no correlations between tumour enzyme profiles (cytochrome P450
reductase, cytochrome b5 reductase, and DT-diaphorase) and ex vivo [18F]FETA tumour
retention or tumour pO2 obtained from OyxLite measurements.
Acute changes in pO2 are detectable with [18F]FETA
The ability to detect acute changes in tumour oxygenation was investigated in RIF-1
tumours after application of a physiological modulator, for which the resultant effect
is well documented. Carbogen breathing decreased [18F]FETA tumour retention to 4.70±0.26%
ID g−1, compared to untreated controls (7.41±0.98% ID g−1; P=0.028). In tissues other
than tumours, [18F]FETA retention was not altered by carbogen breathing. Administration
of carbogen decreased the relative frequency of pO2 values <2.5 mmHg in comparison
to untreated controls (40±11 vs 51±13%). However, these differences did not reach
significance level (P=0.551).
Small animal PET imaging with [18F]FETA
Whole body PET scanning was carried out in HT1080/26.6 (n=3) and HT1080/1-3C tumour-bearing
mice (n=4). For illustration purposes, the 30–60 min summed images are presented (Figure
6
Figure 6
[18F]FETA-PET images of HT1080 tumour-bearing mice acquired on the small animal quad-HIDAC
scanner (pixel size 0.5 × 0.5 × 0.5 mm3). (A) Three-dimensional (volume-rendered)
image of an HT1080/1-3C tumour-bearing mouse (30–60 min p.i. summed) showing a dorsal
view of the mouse. Here, pixel values are defined by the maximum voxel value in corresponding
lines in the z-axis. Arrows point to tumour (Tm), Kidneys (Ki), small intestine (In),
and urinary bladder (Bl). (B) Sagittal (0.5 mm) slice of 30–60 min p.i. summed [18F]FETA-PET
images from the same mouse as in (A) at the midplane level, showing low radiotracer
uptake in brain (Br) and spinal cord (Co), as well as high accumulation in urinary
bladder. (C) Transverse (0.5 mm) slice of 30–60 min p.i. summed [18F]FETA-PET images
from the same HT1080/1-3C tumour-bearing mouse as in (A) at the level of the maximal
tumour diameter. (D) Corresponding transverse slice from an HT1080/26.6 tumour-bearing
mouse, exhibiting lower tumour radiotracer uptake.
). In agreement with the biodistribution data determined by ex vivo γ-counting, the
PET images revealed high [18F]FETA accumulation in tumour, kidney, urinary bladder,
and proximal parts of the small intestine (Figure 6A). In contrast, radiotracer retention
was low in brain and spinal cord (Figure 6B). Also in agreement with the biodistribution
studies, the tumour retention of [18F]FETA was higher in all HT1080/1-3C tumours (Figure
6C) than in the HT1080/26.6 tumours (Figure 6D).
By drawing ROIs over the whole tumour, TACs were obtained from the dynamic PET data
sets (Figure 7
Figure 7
Tumour time–activity curves (TACs), normalised to the integral of the heart cavity
TAC. The curves were obtained from region of interest analysis of dynamic [18F]FETA-PET
images from HT1080/26.6 (-•-) and HT1080/1-3C (-○-) tumour-bearing mice. Data are
mean±s.e.m. (n=3–4 per group).
). HT1080/26.6 tumours were characterised by a rapid delivery of [18F]FETA up to 2 min
p.i., followed by a plateau with a minimal increase of the TACs up to 60 min p.i.
The delivery of the radiotracer into HT1080/1-3C tumours was comparatively slower,
but retention was higher (Figure 7). The fractional retention of [18F]FETA in tumours
(ratio of radioactivity at 60 min to that at 2 min) was significantly higher in the
1-3C tumours compared to 26.6 tumours (1.44±0.10 vs 1.10±0.01; P=0.05).
Biological basis and implication of [18F]FETA retention in HT1080 tumours
In order to explain the differences in [18F]FETA retention between the different HT1080
tumour types, we compared the retention of [18F]FETA in the tumours to vessel density,
oxygenation, and radiation sensitivity. The vessel density measured in histological
sections of the HT1080 tumours was significantly lower for the 1-3C subclone, which
contains less VEGF (Collingridge et al, 2002), compared to the 26.6 subclone (Figures
8A–C
Figure 8
Vessel density, pO2, and radiation sensitivity of HT1080/26.6 and HT1080/1-3 tumours.
(A–B) Typical 5 μm H&E-stained histological sections of HT1080/26.6 (A) and HT1080/1-3C
(B) tumours, with arrows pointing to vessels. (C) Summary data for vessel density
depicting average number of vessels from five randomly selected fields of view (0.23 mm2;
× 200 magnification) per section; three sections per tumour. (D) OxyLite pO2 measurements.
(E–F) Radiation sensitivity determined in clonogenic assays and expressed as radiobiological
hypoxic fraction of HT1080/26.6 (E) and HT1080/1.3C (F) tumours. Data are mean survival
(n=4). RHF, radiobiological hypoxic fraction. There was a statistically significant
difference between the RHF of 1-3C and 26.6 tumours (P=0.008).
). The lower vessel density in the 1-3C tumours was associated with a significantly
higher frequency of pO2 values <1 mmHg (Figure 8D). Radiation sensitivity was determined
for the two tumour types by measuring the fraction of clonogenic radiobiological hypoxic
cells. The plating efficiency (mean±s.e.m.) of HT1080/1-3C and/26.6 tumours were 0.50±0.03
and 1.70±0.41%, respectively. Overall, disaggregation of the tumours yielded (irradiated
vs control) 2 to 20 × 106 cells vs 4 to 20 × 106 cells for HT1080/1-3C and 1 to 11
× 106 and 2 to 9 × 106 cells for HT1080/26.6 cells, respectively. Figures 8E, F show
that hypoxic fraction was significantly higher in HT1080/1-3C than in HT1080/26.6
tumours (86.1±2.1 vs 68.1±0.2%, P=0.008; Figures 8E, F).
DISCUSSION
The partial pressure of oxygen has been directly determined in accessible tumours
such as cervix tumours, head and neck tumours, and sarcomas using Eppendorf oxygen
electrodes (Höckel et al, 1996; Brizel et al, 1997; Nordsmark and Overgaard, 2000;
Fyles et al, 2002). Such studies provided proof that low pO2 (hypoxia) leads to poor
outcome after cancer treatment. These findings, together with the well-known effect
of hypoxia on radiation sensitivity as well as the development of hypoxia-modifying
therapies, have strengthened the medical need for a noninvasive, clinically acceptable
method of measuring hypoxia in patient tumours. Of the numerous possibilities available,
the use of appropriately labelled 2-nitroimidazoles detectable by PET imaging is particularly
attractive. In this paper, we have demonstrated that [18F]FETA is a suitable radiotracer
for PET imaging of tumour hypoxia.
18F-labelled 1-(2-nitro-imidazolyl)-3-fluoro-2-propanol ([18F]fluoromisonidazole ([18F]FMISO)
(Jerabek et al, 1986; Koh et al, 1995; Rasey et al, 1996)) is the most widely studied
radiotracer for tumour hypoxia. This radiotracer has, however, failed to gain a wider
acceptance for routine clinical application in a PET setting because of a number of
limitations including: (i) slow accumulation in hypoxic tumours (scanning has been
performed up to 5 h p.i. (Nunn et al, 1995; Bentzen et al, 2002)); (ii) low target-to-background
contrast due to high nonspecific binding resulting from a relatively high lipophilicity
(octanol/water partition coefficient=0.41 (Workman and Brown, 1981)); and (iii) significant
non-oxygen-dependent metabolism (e.g. 50.0 and 36.2% of metabolites in plasma and
urine, respectively, at 2 h p.i. (Rasey et al, 1999)). The choice of an ideal hypoxia
marker would favour probes that have low lipophilicity and resistance to hypoxia-independent
degradation; in general 2-nitroimidazoles with an amide side chain typified by etanidazole
are more stable to nonhypoxic metabolism than those with a hydroxypropyl side chain
such as misonidazole (Aboagye et al, 1998). [18F]FETA possesses these favourable characteristics.
Its octanol–water partition coefficient, determined here to be 0.16, is higher than
nonfluorinated etanidazole (0.046 (Brown and Workman, 1980)), but lower than that
of [18F]FMISO. In vitro, [18F]FETA accumulated selectively in hypoxic, but not in
aerobic RIF-1 cells (4.3-fold differential at 120 min p.i.), consistent with the presence
of a 2-nitroimidazole moiety (with redox potentials (E1/7) around −380 to −390 mV
(Adams et al, 1979; Brown and Workman, 1980; Workman and Brown, 1981). The large variation
in uptake could be due to the differences in stable etanidazole levels in the reaction
mixture.
[18F]FETA accumulated in all tissues analysed. The retention of the radiotracer at
60-min postinjection was relatively low (<5% ID g−1) in lung, heart, brain, and bone;
intermediate (5–8% ID g−1) in plasma, liver, spleen, small intestines, and muscle;
and high (>8% ID g−1) in kidney, bile, and urine. The highest [18F]FETA accumulation
was found in the excretory organs. Good accumulation in tumours, 6–10% ID g−1, would
suggest the possibility of achieving high tumour-to-background contrast in several
tumour types, including breast, lung, head and neck, and brain tumours. Chromatographic
analysis of plasma, urine, and tissues provided some explanation for the differences
in tissue distribution. [18F]FETA was found to be stable in plasma (∼93% as parent
compound at 60 min p.i.) and also excreted mainly as the unchanged drug in urine (74%
at 60 min p.i.). The radiotracer was metabolised in liver to catabolite(s), which
appeared to be eliminated predominantly via the hepatobiliary route.
Having shown that [18F]FETA is taken up into all tissues, we investigated whether
differences in pO2 could be monitored with the radiotracer in vivo. Two validation
scenarios were explored. In the first instance, the 60-min retention of [18F]FETA
into all tumour types as determined by γ-counting was compared to measured pO2. Radiotracer
retention positively correlated with the percentage of pO2 values <5 mmHg (r=0.805,
P=0.027), indicating that [18F]FETA retention is related to pO2 levels. Using five
different tumour types enabled a wide range of oxygenation levels to be achieved (the
relative frequency of pO2 values <1.0 mmHg ranged from 28±13 to 93±9% (P=0.008; one-factorial
ANOVA), and the relative frequency of pO2 values <2.5 mmHg in a range from 51±13 to
100±0% (P=0.057)). In general, the binding of nitroimidazoles requires the presence
of nitroreductases, particularly cytochrome P450 reductase. We found no relationship
between [18F]FETA and levels of the nitroreductases studied. Large differences in
nitroreductase levels can confound measurement of hypoxia with 2-nitroimidazole-based
probes; thus, our findings of a lack of significant differences in nitroreductase
activities between the tumour types used is a relevant contribution to the interpretation
of [18F]FETA data. The range of nitroreductase levels observed here, ⩽2.5-fold, is
unlikely to influence radiotracer retention due to a square-root relationship between
the binding of 2-nitroimidazoles and cytochrome P450 reductase levels (Joseph et al,
1994). Rasey et al (1999) have shown that the half-maximal inhibition of [18F]FETA
binding by oxygen is fairly similar (up to two-fold) across cell lines in vitro. This
observation may be explained by the small differences in nitroreductase levels between
cell lines. In the second instance, we investigated the ability of [18F]FETA to detect
acute changes in tumour oxygenation. Carbogen breathing decreased the 60-min tumour
retention of [18F]FETA by 36%, in keeping with an 11% decrease of hypoxic pO2 values
measured by OxyLite electrodes. This finding is similar to those reported previously
for other 2-nitroimidazoles (Aboagye et al, 1997b, 1998; Rasey et al, 2000; Bentzen
et al, 2002; Seddon et al, 2002) and demonstrates that [18F]FETA can be used to detect
acute changes in pO2.
PET studies performed in tumour-bearing mice demonstrated the feasibility of imaging
hypoxia in vivo. Qualitatively, the PET images were in agreement with the quantitative
tissue radioactivity data determined by γ-counting. It was also possible to perform
semiquantitation of the dynamic PET data by ROI analysis. Interestingly, the tumour
model with higher VEGF-expression (HT1080/26.6) showed lower fractional retention
of the radiotracer compared to that with the lower VEGF-expression (HT1080/1-3C) despite
a higher delivery of [18F]FETA into the former. This finding is in contradiction to
the current dogma of ‘hypoxia inducing VEGF through the expression of HIF-1α’ (Harris,
2002; Semenza, 2003) and, thus the more hypoxic tumours being those exhibiting higher
levels of VEGF expression. This observation may be explained in part by the lower
(∼10-fold half-maximal inhibition) oxygen levels required to effect binding of 2-nitroimidazoles
to cellular macromolecules vs that required to induce VEGF production (Leith and Michelson,
1995); in this regard, both tumour models are sufficiently hypoxic to induce VEGF
production. The findings were, however, corroborated by a higher vessel density (explaining
the higher radiotracer delivery) and a higher pO2 (explaining the lower fractional
retention) in the HT1080/26.6 model. The impact of these findings on radiation sensitivity
was that the HT1080/26.6 tumours were more sensitive to radiation than the HT1080/1-3C
tumours as demonstrated by a lower clonogenic radiobiological hypoxic fraction of
the former. Since the pO2 (and radiobiological hypoxic fractions) of the human tumour
models used in this study have not been previously reported in the literature, an
assessment of this (as well as the radiobiological hypoxic fraction for the HT1080
tumours) was made; the high and low pO2 values obtained for EMT6 and RIF-1 tumours,
respectively (Moulder and Rockwell, 1984), corroborate our measurements.
Since the PET studies required the use of anaesthesia for immobilisation, all other
in vivo studies were performed under anaesthesia. It is conceivable that most anaesthesia
will decrease clearance of radiotracers from the body and give rise to a systematic
increase in hypoxia levels within tumours. This may explain the very high percentage
of pO2 values <2.5 mmHg seen in our experiments. These conditions have, however, been
successfully used in other hypoxia experiments in mice to demonstrate nitroimidazole
retention in tumours (Aboagye et al, 1997b). In translating this work into clinical
studies one needs to bear in mind the fact that patients would be un-anaesthetised.
The level of oxygenation may therefore be higher than that measured for the mouse
tumours.
In summary we have validated an advanced-generation 2-nitroimidazole marker of tumour
hypoxia in the mouse. Our findings indicate that [18F]FETA has suitable physicochemical
properties and is stable to nonhypoxic degradation in vivo. We have also demonstrated
that the tumour retention of the radiotracer is related to pO2 status and radiobiological
hypoxia. These properties, as well as the ability to image hypoxia (pO2 values at
1–10 mmHg) dynamically with PET at early time points are attractive features that
support the clinical development of [18F]FETA.