Introduction
The use of positron emission tomography (PET) for imaging of cancer has a successful history spanning more than three decades. Suitable radiopharmaceuticals have been designed to track molecular events in the body, to monitor the time course of disease and to assess treatment outcome. Although
18F-FDG and other metabolic PET tracers (
18F-FLT,
11C-methionine,
11C-choline, etc.) are widely available for the diagnosis of cancer and monitoring of treatment response, these tracers are not tumour specific and/or have only moderate cellular uptake [
1,
2]. For this reason, there is much interest in the development and validation of novel radiopharmaceuticals with greater tumour selectivity. Attractive candidates are sigma ligands since sigma receptors are strongly overexpressed in rapidly proliferating cells [
3‐
6].
Sigma receptors are proteins with a highly conserved sequence (87–92% identity and 90–93% homology for sigma-1 receptor) [
7]. They are found in kidney, liver, immune, endocrine and reproductive organs [
8]. Furthermore, sigma receptors are widely distributed in the brain and are implied in memory function, cognition, drug addiction, depression and schizophrenia [
9]. Importantly, sigma receptors were demonstrated to play a role in tumour cell proliferation and cancer cell death. These binding sites are therefore important targets in the development of novel anti-cancer drugs [
10]. Although the endogenous ligands for sigma receptors have not yet been identified, steroid hormones (in particular progesterone) are potential candidates [
11,
12].
Recent work has demonstrated regulation of sigma-1 receptor availability by endogenous steroids. Such steroids modulate the efficacy of a sigma-1 receptor agonist in stress and depression [
12]. Binding of neurosteroids to sigma receptors affects the release of substance P from nociceptor endings in mice [
13] and the release of glutamate and norepinephrine in rat prelimbic cortex and hippocampus [
14,
15]. Furthermore, such binding reduces subjective craving in cocaine addiction [
16]. Based on their effects on substance P and norepinephrine release, progesterone was proposed to be a sigma antagonist, whereas dehydroepiandrosterone-3-sulphate (DHEA-S) and pregnenolone sulphate were considered as sigma agonists [
13,
15]. Multiple sequence analyses revealed the presence of two steroid-like binding domains (SBDLI and SBDLII) in the guinea pig sigma-1 receptor (amino acids 91–109 and 176–194, respectively) [
17,
18]. Recently, it was shown that administration of steroids reduces in vivo binding of the sigma-1 receptor ligand
18F-FPS in rodent brain [
19].
Although an interaction between cerebral sigma receptors and steroids has been established, our knowledge about competition between sigma ligands and endogenous steroids in cancer cells is still rudimentary. Moreover, it remains necessary to examine how steroid hormones affect the binding of radiopharmaceuticals other than
18F-FPS, since sigma ligands may bind to different receptor subtypes [
20] and to different binding sites within the sigma-1 receptor molecule (e.g. the agonist binding site and the phenytoin-binding site [
21]).
Here, we examine the impact of steroid competition on binding of the sigma-1 agonist
11C-SA4503.
11C-SA4503 has been used extensively for PET studies of animal tumours [
2,
22‐
24] and the human brain [
25‐
28]. For in vitro tests of steroid competition, we used C6 rat glioma cells, a tumour line which expresses both sigma-1 and sigma-2 receptors [
3]. As an in vivo model we used C6 glioma-bearing Wistar rats. Tumour cells were either repeatedly washed and incubated in steroid-free medium or exposed to five different exogenous steroids. Rats were either repeatedly injected with pentobarbital, a treatment known to reduce the levels of endogenous steroids [
29], or injected with progesterone. If the binding of
11C-SA4503 is sensitive to competition by endogenous steroids, we expected to find increased tracer binding after prolonged anaesthesia and decreased binding after steroid addition. Such competition, if present, could result in intrasubject variability of the binding potential of
11C-SA4503 in women during the menstrual cycle and also in intersubject variability in aged subjects which may show widely different endogenous steroid concentrations.
Materials and methods
Dulbecco’s minimum essential medium (DMEM), fetal calf serum (FCS) and trypsin were products of Invitrogen. Allopregnanolone acetate (5α-pregnan-3β-ol-20-one 3β-acetate), dehydroepiandrosterone 3-sulphate sodium salt (DHEA-S), haloperidol, progesterone (cell culture tested, P8783) and trypan blue (0.4% solution in phosphate-buffered saline) were purchased from Sigma. Androstanolone (5α-androstan-17β-ol-3-one) and testosterone were obtained from Fluka. Matrigel
R came from Becton Dickinson. Arachid oil was a product of Levo BV, Franeker, Holland. Stock solutions of steroids and of haloperidol were prepared in ethanol unless otherwise indicated. The radioligand
11C-SA4503 was prepared by reaction of
11C-methyl iodide with the appropriate 4-
O-methyl precursor [
30]. The decay-corrected radiochemical yield was 9–11%, the specific radioactivity >11 TBq/mmol at the moment of injection and radiochemical purity >95%.
Cell culture
C6 rat glioma cells obtained from the American Type Culture Collection were grown as monolayers in DMEM (high glucose) supplemented with 7.5% FCS in a humidified atmosphere of 5% CO2/95% air at 37°C. Before each experiment, the cells were seeded in 12-well plates (Costar). An equal number of cells were dispensed in each well in 1.1 ml of serum-containing medium: DMEM (high glucose) supplemented with 7.5% FCS.
Binding studies
Binding studies were performed 48 h after seeding cells in 12-well plates when confluency had reached 80–90%. In some experiments, cells were steroid-depleted by removal of the normal medium, repeated (3 ×) washing with phosphate-buffered saline (PBS, 1 ml) and addition of DMEM (high glucose) without serum and phenol red, 1 h before addition of the radiotracer. Various concentrations of an unlabelled competitor (haloperidol or a steroid) were dispensed to the culture medium in the wells. Steroids investigated were allopregnanolone, androstanolone, DHEA-S, progesterone and testosterone. After 2 min, 4 MBq of 11C-SA4503 in <30 µl of saline (containing 30% ethanol) were added to each well. After 45–60 min of incubation, the medium was quickly removed and the monolayer was washed 3 times with PBS. Cells were then treated with 0.2 ml of trypsin. When the monolayer had detached from the bottom of the well, 1 ml of DMEM (high glucose) supplemented with 7.5% FCS was added to stop the proteolytic action. Cell aggregates were resolved by repeated (at least tenfold) pipetting of the trypsin/DMEM mixture. Radioactivity in the cell suspension (1.2 ml) was assessed using a gamma counter (Compugamma 1282 CS, LKB-Wallac, Turku, Finland). A sample of the suspension was mixed with trypan blue solution (1:1 v/v) and was used for cell counting. Cell numbers were determined manually, using a phase contrast microscope (Olympus, Tokyo, Japan), a Bürker bright-line chamber (depth 0.1 mm; 0.0025 mm2 squares) and a hand tally counter. All experiments were performed as a quadruplicate study with at least two repeats.
Animal model
The animal experiments were performed by licensed investigators in compliance with the Law on Animal Experiments of The Netherlands. The protocol was approved by the Committee on Animal Ethics of the University of Groningen.
C6 glioma cells [2.5 × 106, in a 1:1 mixture of Matrigel and DMEM (high glucose with 7.5% FCS)] were subcutaneously injected into the right shoulder of male Wistar rats. The animals were maintained at a 12 h light/12 h dark regime and were fed standard laboratory chow ad libitum. They were scanned after 9–10 days, when tumours had grown to above 0.5 g.
MicroPET scanning
Two rats were scanned simultaneously in each scan session, using a Siemens/Concorde microPET camera (Focus 220). A list mode protocol was used (60 min, brain, tumour and upper half of both lungs in the field of view). These organs were clearly visualized, as reported previously [
24]. The scanning was started during injection into the first rat; the second rat was injected 30 s later. Body temperature of anaesthetized animals in the scanner was kept at 37.5 ± 0.5°C, using rectal probes, individual temperature controllers and electronic heating (M2M Imaging).
Two microPET scans of five rats were made in order to explore the effect of anaesthesia duration on the in vivo binding of 11C-SA4503 (23–27 MBq administered as a 0.3 ml bolus, tail vein catheter, mass <2 nmol). For the first scan, the tracer was injected shortly (<20 min) after the induction of pentobarbital anaesthesia (first scan = control condition). When this scan had been finished, the animal was kept under anaesthesia and remained at a fixed position in the microPET scanner. 11C-SA4503 was once more injected after prolonged anaesthesia [four intraperitoneal injections of a sodium pentobarbital solution in water (60 mg/ml): first injection 1 ml/kg body weight, and then three subsequent 0.3 ml/kg body weight at 30–40 min intervals, total anaesthesia duration >3.5 h] in order to acquire the second scan.
For examination of the effect of an exogenous steroid on 11C-SA4503 binding, microPET scans were made of four pairs of rats. One rat of each pair was treated with progesterone (three intraperitoneal injections of 10 mg each at 1.5-h intervals, in arachid oil, progesterone-treated group), whereas the other animal was treated with carrier (arachid oil) only (control group). In the arachid oil- and progesterone-treated rats, the tracer 11C-SA4503 was injected shortly (< 20 min) after the induction of pentobarbital anaesthesia.
List mode data were reframed into a dynamic sequence of 4 × 60 s, 3 × 120 s, 4 × 300 s and 3 × 600 s frames. The data were reconstructed per time frame employing an interactive reconstruction algorithm (OSEM2D). The final data sets consisted of 95 slices with a slice thickness of 0.8 mm and an in-plane image matrix of 128 × 128 pixels of size 1 × 1 mm2. Data sets were fully corrected for random coincidences, scatter and attenuation. A separate transmission scan was acquired for attenuation correction. This scan was performed right after the last emission scan.
Three-dimensional regions of interest (3-D ROIs) were manually drawn around the entire tumour, brain and peripheral area of the right lung, avoiding hilar structures, as described previously [
24]. Time-activity curves (TACs) and volumes (cm
3) for the ROIs were calculated, using standard software (AsiPro VM 6.2.5.0, Siemens-Concorde, Knoxville, TN, USA). TACs were normalized for body weight and injected dose as indicated in the figure legends.
Biodistribution studies
After the scanning period, the anaesthetized animals were terminated. Blood was collected, and plasma and a cell fraction were obtained from the blood sample by short centrifugation (5 min at 1,000
g). Several tissues (see Table
1) were excised. The complete tumour was removed and separated from muscle and skin. All tissue samples were weighed. Radioactivity in tissue samples was measured using a gamma counter, applying a decay correction. The results were expressed as dimensionless standardized uptake values (SUVs). The parameter SUV is defined as: [tissue activity concentration (MBq/g) * body weight (g) / injected dose (MBq)].
Table 1
Effect of prolonged anaesthesia and progesterone administration on biodistribution of 11C-SA4503
Cerebellum | 1.66 ± 0.18 | 2.41 ± 0.33 | 1.69 ± 0.30 | < 0.01 | NS |
Cerebral cortex | 1.50 ± 0.40 | 2.38 ± 0.40 | 1.85 ± 0.33 | 0.02 | NS |
Rest of brain | 1.51 ± 0.38 | 2.12 ± 0.41 | 1.69 ± 0.37 | NS | NS |
Adipose tissue | 0.25 ± 0.12 | 0.38 ± 0.15 | 0.25 ± 0.08 | NS | NS |
Bladder | 0.67 ± 0.16 | 0.80 ± 0.31 | 0.55 ± 0.17 | NS | NS |
Bone | 0.27 ± 0.08 | 0.42 ± 0.09 | 0.34 ± 0.11 | < 0.05 | NS |
Bone marrow | 1.56 ± 0.36 | 2.15 ± 0.23 | 1.17 ± 0.20 | < 0.05 | NS |
Heart | 0.35 ± 0.09 | 0.48 ± 0.07 | 0.31 ± 0.04 | NS | NS |
Large intestine | 1.82 ± 0.36 | 2.62 ± 0.39 | 1.87 ± 0.39 | < 0.05 | NS |
Small intestine | 2.35 ± 0.28 | 3.48 ± 0.86 | 2.73 ± 0.53 | NS | NS |
Kidney | 4.07 ± 0.45 | 4.60 ± 0.19 | 4.07 ± 0.63 | NS | NS |
Liver | 7.53 ± 0.77 | 5.20 ± 0.28 | 7.16 ± 1.39 | 0.001 | NS |
Lung | 2.21 ± 0.59 | 2.51 ± 0.62 | 1.92 ± 0.50 | NS | NS |
Muscle | 0.18 ± 0.04 | 0.21 ± 0.01 | 0.20 ± 0.05 | NS | NS |
Pancreas | 3.78 ± 1.55 | 5.06 ± 1.22 | 5.16 ± 1.60 | NS | NS |
Plasma | 0.09 ± 0.03 | 0.10 ± 0.02 | 0.09 ± 0.01 | NS | NS |
Blood cells | 0.05 ± 0.01 | 0.07 ± 0.01 | 0.05 ± 0.01 | < 0.05 | NS |
Spleen | 2.26 ± 0.39 | 3.76 ± 0.95 | 2.07 ± 0.41 | < 0.05 | NS |
Submandibular gland | 2.91 ± 0.91 | 2.08 ± 0.17 | 2.59 ± 0.85 | NS | NS |
C6 tumour | 0.83 ± 0.11 | 1.52 ± 0.36 | 0.57 ± 0.14 | < 0.05 | < 0.05 |
Urine | 0.48 ± 0.20 | 0.75 ± 0.76 | 0.92 ± 0.29 | NS | < 0.05 |