Introduction
The radiolabeled amino acid L-[
methyl-
11C]methionine ([
11C]MET) is widely used for tumor imaging. However, [
11C]MET is taken up not only by tumors but also by various other lesions [
1‐
3]. For this reason, there is much interest in the development of positron emission tomography (PET) tracers with greater tumor specificity than [
11C]MET. A successful alternative is the amino acid analog
O-2-[
18F]fluoroethyl-L-tyrosine ([
18F]FET) [
4]. Another amino acid analog which has been proposed for the same purpose is
S-[
11C]methyl-L-cysteine ([
11C]MCYS) [
5,
6]. MCYS is a naturally occurring derivative of the amino acid L-cysteine. Large amounts of this substance are present in many vegetables [
7,
8]. Biodistribution studies in nude mice indicated a somewhat higher uptake of [
11C]MCYS in Hepa1–6 tumors compared to [
11C]MET, but a lower uptake in turpentine-induced sterile inflammations [
5]. Thus, [
11C]MCYS was claimed to offer superior differentiation of tumor from inflammation and to have considerable potential as an oncologic PET tracer.
In order to examine this claim, we performed longitudinal PET studies with [
11C]MCYS and [
11C]MET in various animal models: immune-competent rats with orthotopically implanted gliomas (see [
9]) which were either untreated or received radiotherapy, rats which were injected with bacterial lipopolysaccharide (LPS) in the right striatum (see [
10]), and sham-injected rats which received physiological saline instead of LPS. Our data indicate that [
11C]MCYS accumulates less in some non-tumor tissues (salivary glands, Harderian glands, nasal epithelium, healing wounds) than [
11C]MET, but shows a higher uptake in the healthy brain, resulting in lower tumor-to-brain contrast of [
11C]MCYS scans. Moreover, irradiation of gliomas results in an acute, 5- to 6-fold increase of the volume with significant [
11C]MCYS uptake which does not reflect the presence of viable tumor cells. Based on these results, [
11C]MCYS and [
11C]MET appear to reflect different aspects of
in vivo biology.
Materials and Methods
Reagents
L-[
methyl-
11C]methionine was prepared by methylation of L-homocysteine thiolactone with [
11C]methyl iodide. The radiochemical yield was 60 %, and specific activities were greater than 2 TBq/mmol [
1]. S-[
11C]methyl-L-cysteine was synthesized by [
11C]methylation of L-cysteine [
5], using [
11C]methyl triflate rather than [
11C]methyl iodide. 2-Deoxy-2-[
18F]fluoro-D-glucose ([
18F]FDG) was produced by the Hamacher method [
11]. Bacterial lipopolysaccharide (LPS) was purchased from Sigma Aldrich (cat.no. L6529).
Animals
All actions with experimental animals were performed by licensed investigators. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Groningen (protocol 6561A). Male Wistar rats were acquired from Harlan. They were housed at 21 ± 2 °C under a fixed 12-h light–dark regime. Standard laboratory chow and water were available
ad libitum. The animals received a special high-energy diet on the day of the surgery and the following day, as well as 2 days before and after brain irradiation (see below). After a 7-day acclimation period, the animals were randomly divided in five different groups (see Table
1):
1 (
Pilot): C6 glioma cells were stereotactically injected in the right striatum. [
11C]MCYS and [
11C]MET scans were made after 8 days and a [
18F]FDG scan after 15 days;
2 (
Tumor-bearing,
untreated): C6 glioma cells were injected as in group 1, but [
11C]MCYS and [
11C]MET scans were made after 6, 9, and 12 days;
3 (
Tumor-bearing,
radiotherapy): As group 2, but the lesion-containing hemisphere was irradiated after 8 days;
4 (
Saline-injected): 2 μl of physiological saline was stereotactically injected into the right striatum and [
11C]MCYS and [
11C]MET scans were made after 3 days;
5 (
LPS-injected): As group 4, but 2 μl of a solution of LPS in saline was stereotactically injected into the right striatum in order to induce neuroinflammation.
Table 1
Groups included in this study
1. Tumor-bearing (pilot study) | 4 | 8 and 15 days | – | 0 | 278 ± 8 | 27 ± 17 | 21 ± 13 |
2. Tumor-bearing (untreated) | 10 | 6, 9 and 12 days | – | 0 | 314 ± 23 | 23 ± 12 | 24 ± 13 |
3. Tumor-bearing (radiotherapy) | 7 | 6, 9 and 12 days | 8 days | 25 | 308 ± 13 | 21 ± 6 | 22 ± 5 |
4. Right striatum injected with saline | 9 | 3 days | – | 0 | 283 ± 19 | 33 ± 20 | 31 ± 17 |
5. Right striatum injected with LPS | 9 | 3 days | – | 0 | 283 ± 17 | 34 ± 19 | 28 ± 17 |
Stereotactic Injection of C6 Cells
Rats from groups 1, 2, and 3 were anesthetized by intraperitoneal injection of ketamine (25 mg/kg) and medetomidine (0.2 mg/kg) and placed in a stereotactic frame. Eye cream was applied and heating pads were used to maintain body temperatures close to the normal value. During surgery, the rats breathed oxygen from a nose mask, and pO
2 and heart rate were continuously monitored. A longitudinal incision was made along the medial line of the skull, the skin and fascia were pushed aside, and the skull was exposed. A hole was drilled at the coordinates of the striatum (from Bregma
A = − 0.30;
L = + 3.0), until the dura became visible and could be opened [
12]. Using a Hamilton injection needle, 5 × 10
5 C6 cells in 5 μl of sterile saline were slowly injected (in 10 min), at a depth of − 5.0 from Bregma. Before injection, the needle was wiped with 70 % alcohol and saline to avoid contamination of host tissue with tumor cells outside the desired area. The syringe was left in position for an additional 5 min and was then withdrawn. The skin was closed and saline was injected subcutaneously to prevent dehydration. Bupivacaine (5 mg/ml) was locally applied to suppress pain. Anti-sedan was used to wake up the animal and 0.03 mg/kg of s.c. Temgesic was given after 30 min.
Irradiation
Group 3 received special care to reduce the discomfort of radiotherapy as much as possible, starting 2 days before irradiation. This special treatment involved the feeding of a high-energy diet, the addition of sucrose to the drinking water, and the application of Bepanthen® cream to the skull after radiotherapy. On the day of treatment, rats were anesthetized by intraperitoneal injection of ketamine (25 mg/kg) and xylazine (20 mg/ml). Eye cream was applied, and the animals were placed in a special frame containing a lead collimator which shielded the non-lesioned hemisphere and all tissues outside the brain, including the eyes and the parotid glands. The right hemisphere was irradiated with a single X-ray fraction of 25 Gy. After irradiation (about 18 min), the rats were aroused with Anti-sedan, returned to their cages, and allowed to recover. The entire procedure was finished within 30 min.
Stereotactic Injection of Saline or LPS
Rats were injected with saline or LPS using the same procedure as for injection of C6 cells, but the injected volume was 2 μl and the sterile saline contained either nothing (group 4) or bacterial lipopolysaccharide (Escherichia coli 020:B6, 0.5 μg/μl, group 5).
PET Imaging
On each scanning day, rats were anesthetized with isoflurane (2 % in medical air, flow rate 1 to 2 ml/min). [11C]MCYS or [11C]MET was injected via a tail or penile vein. A dynamic emission scan of 60 min was made with a Siemens/Concorde Focus 220 camera, using a list mode protocol. Two rats were scanned simultaneously, in transaxial position with their brains in the field-of-view. In some animals, a static rather than a dynamic scan was made for logistic reasons, lasting from 30 to 60 min after tracer injection. Finally, a transmission scan was made (515 s), using a Co-57 point source. Data from this scan were used for the correction of attenuation and scatter of 511-keV photons by tissue. During all scans, body temperature of the animals was maintained by heating mats, whereas heart rate and blood oxygenation were continuously monitored. A [11C]MCYS scan and a [11C]MET scan were made of each animal on each scanning day, with an interval of at least 2 h. The list mode data of the emission scans were reframed into a dynamic sequence: 6 × 10 s, 4 × 30 s, 2 × 60 s, 1 × 120 s, 1 × 180 s, 4 × 300 s, 3 × 600 s frames. Images were reconstructed employing ordered subset expectation maximization (OSEM 2D with Fourier rebinning, four iterations, and 16 subsets). The final datasets consisted of 95 slices with a thickness of 0.8 mm and an in-plane image matrix of 128 × 128 pixels. Voxel size was 0.5 × 0.5 × 0.8 mm. The linear resolution at the center of the field-of-view was 1.5 mm. Data sets were corrected for decay, random coincidences, scatter, and attenuation.
Data Analysis
Three-dimensional regions of interest (ROIs) were manually drawn in PET images, representing the tumor, LPS- or saline-injected striatum, contralateral healthy brain, and the wound on top of the skull. ROI volumes and levels of radioactivity (mean and maximum within the ROI) were calculated, using AsiPro® (Siemens). Tracer accumulation was expressed as a standardized uptake value (SUV): [tissue activity concentration (MBq/g) × body weight (g)/injected dose (MBq)], assuming a specific tissue gravity of 1 g/ml. Images were smoothed with a Gaussian filter (1.5 mm in both directions). The color scale of the images was set from SUV 0 to 2 (± 10 %), in order to clearly delineate the lesion. However, for visualization of LPS- or saline-injected striatum with [11C]MET, the maximum had to be set to SUV 1.4 (± 10 %). Care was taken to include in the ROIs only planes that were located within the brain, since wound tissue on top of the skull could strongly take up [11C]MET, particularly at short intervals (3–6 days) after surgery.
Statistics
Results reported in the Tables are expressed as mean ± SD. Error bars represent SEM. Group differences were examined using t test and two-way ANOVA, followed by a Bonferroni correction, where applicable. A P value < 0.05 was considered statistically significant.
Discussion
[
11C]MCYS has been evaluated in a heterotopic mouse model of hepatocellular carcinoma and in a human PET study involving a patient with recurrent glioma [
5]. Here, we compared the uptake of [
11C]MCYS and [
11C]MET in an orthotopic glioma model. The rat model which we employed mimics the invasion and growth, as well as the neuroinflammation and leukocyte infiltration which occur in a human glioblastoma [
9,
13]. Our pilot study (group 1) indicated rapid growth of implanted gliomas. Thus, each rat in the following groups was scanned at rather short intervals (6, 9, and 12 days) after inoculation.
The PET studies indicated a similar uptake of the two tracers in untreated gliomas at 6, 8, and 9 days and a slightly higher uptake of [
11C]MCYS at 12 days after inoculation (Table
2). However, [
11C]MCYS showed also higher uptake in healthy brain tissue than [
11C]MET (Table
2). For this reason, tumor-to-healthy brain ratios of [
11C]MCYS were 19 to 26 % lower than of [
11C]MET (Table
2). At short intervals between the inoculation of tumor cells and the PET scan (6 days), [
11C]MET was strongly accumulated in the wound on top of the skull and also in salivary glands and nasal epithelium, in contrast to [
11C]MCYS (Table
2, Fig.
1). The strong accumulation of [
11C]MET in healing wounds and at the injection spot of the brain was a complicating factor in the detection and delineation of small tumors. Methionine is not only a substrate for protein synthesis but is also involved in transmethylation [
14]. The label of [
11C]MET may therefore end up in various phospholipids, DNA, and RNA [
2], in contrast to the label of [
11C]MCYS which is not incorporated into macromolecules during a PET scan [
5]. This difference in the metabolism of the two tracers may explain why [
11C]MET (but not [
11C]MCYS) accumulates in healing wounds.
We also examined the impact of radiotherapy on tracer uptake in gliomas. Tumor-bearing rats were scanned 1 and 4 days after the application of a single X-ray fraction of 25 Gy on day 8 after inoculation. The uptake of both tracers showed a significant decline after radiotherapy (Table
3) which reflects a decrease of tumor metabolism. In untreated animals, [
11C]MCYS indicated 47 % larger tumor volumes than [
11C]MET (Fig.
3), but the close correlation between [
11C]MCYS and [
11C]MET volumes was lost after irradiation. In irradiated animals, [
11C]MCYS indicated 5- to 6-fold larger tumor volumes than [
11C]MET (Table
4). The tumor volume observed in [
11C]MCYS scans 1 day after irradiation was even much (1.8-fold) larger than the tumor volume observed with the same tracer in untreated rats (Table
4). The acute increase in apparent tumor volume after irradiation that we observed in [
11C]MCYS but not in [
11C]MET scans seemed therefore to be related to a treatment-induced artifact (increased blood flow, increased leakiness of the blood-brain barrier, brain-infiltrating leukocytes) rather than the presence of viable tumor cells.
Since apparent tumor volumes after radiotherapy may be affected by tracer uptake in activated microglia, we examined the uptake of [
11C]MET and [
11C]MCYS in a neuroinflammation model. The LPS model which we employed is associated with activated microglia and infiltration of leukocytes after 3 days [
10]. [
11C]MCYS showed a higher uptake than [
11C]MET both in LPS-injected and saline-injected striatum, but the uptake of [
11C]MCYS in healthy brain tissue was also higher. The lesioned-to-intact striatum ratios of the two tracers were therefore identical (Table
4). Tracer uptake in LPS-injected and saline-injected striatum was similar. This observation suggests that both tracers show negligible accumulation in activated microglia. However, an interesting difference was noted when the
volume with significant tracer accumulation was considered. [
11C]MET and [
11C]MCYS indicated identical lesion volumes in saline-injected animals, but [
11C]MCYS indicated a larger volume for LPS-injected striatum than [
11C]MET. The difference between LPS and saline was thus detectable in [
11C]MCYS but not in [
11C]MET scans (Table
4). Since the PET scans in the neuroinflammation model did not suggest a significant accumulation of [
11C]MCYS in leukocytes or activated microglia, radiotherapy and LPS injection appear to cause secondary damage to the area surrounding the tumor or primary lesion which facilitates uptake of [
11C]MCYS but not of [
11C]MET. The mechanism underlying this facilitation is unknown and can only be clarified by further study,
e.g., by pathologic analysis of the area with increased [
11C]MCYS uptake, 1 day after tumor irradiation.
Conclusion
[11C]MCYS has been proposed as an oncologic PET tracer with greater tumor selectivity than [11C]MET. In support of this claim, we observed a strong accumulation of [11C]MET in salivary glands, Harderian glands, nasal epithelium, and healing wounds whereas [11C]MCYS showed little accumulation in such tissues. However, (1) the tumor-to-brain contrast in [11C]MCYS scans was 19 to 26 % lower than the tumor-to-brain contrast of [11C]MET. This difference in image contrast was due to a higher uptake of [11C]MCYS in brain tissue; (2) an acute, 2-fold increase of the apparent tumor volume was observed in [11C]MCYS scans after irradiation which appeared to be related to a treatment-induced artifact; (3) the decrease of tumor metabolism induced by radiotherapy was detected earlier in [11C]MET than in [11C]MCYS scans; and (4) both [11C]MET and [11C]MCYS showed negligible accumulation in LPS-injected striatum, i.e., in activated microglia. For these reasons, [11C]MCYS and [11C]MET appear to reflect different aspects of in vivo biology. [11C]MCYS may offer the advantage of lower background accumulation in extracranial tissue whereas [11C]MET may exhibit a lower normophysiological signal in the brain. Since the current data were acquired in anesthetized animals, tracer distribution may be different in the absence of anesthesia. Future studies should be aimed at elucidating the processes affecting the uptake of both amino acids and identifying the advantage of one compound over another in various circumstances.