research article
18F-FET and 18F-FCH uptake in human glioblastoma T98G cell lines
Marco Giovanni Persico1, Federica Eleonora Buroni1, Francesca Pasi2, Lorenzo Lodola1, Carlo Aprile1, Rosanna Nano3, Marina Hodolic4
1	Department of Oncohaematology, Nuclear Medicine Unit, IRCCS San Matteo Hospital Foundation, Pavia, Italy
2	Department of Oncohaematology, Radiotherapy Unit, IRCCS San Matteo Hospital Foundation, Pavia, Italy
3	Department of Biology and Biotecnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy
4	Nuclear medicine research department, Iason, Graz, Austria
Radiol Oncol 2016; 50(2): 153-158.
Received 26 November 2015 Accepted 18 March 2016
Correspondence to: Lorenzo Lodola, Fondazione IRCCS Policlinico San Matteo, V. le Golgi 19, 27100 Pavia, Italy. Phone: +39 038 250 1666; Fax: +39 0382501669; E-mail: l.lodola@smatteo.pv.it
Disclosure: No potential conflicts of interest were disclosed.
M.G.P., F.E.B. and F.P. have contributed equally.
Background. Despite complex treatment of surgery, radiotherapy and chemotherapy, high grade gliomas often recur. Differentiation between post-treatment changes and recurrence is difficult. 18F-methyl-choline (18F-FCH) is frequently used in staging and detection of recurrent prostate cancer disease as well as some brain tumours; however accumulation in inflammatory tissue limits its specificity. The 18F-ethyl-tyrosine (18F-FET) shows a specific uptake in malignant cells, resulting from increased expression of amino acid transporters or diffusing through the disrupted blood-brain barrier. 18F-FET exhibits lower uptake in machrophages and other inflammatory cells. Aim of this study was to evaluate 18F-FCH and 18F-FET uptake by human glioblastoma T98G cells.
Material and methods. Human glioblastoma T98G or human dermal fibroblasts cells, seeded at a density to obtain 2 x 105 cells per flask when radioactive tracers were administered, grew adherent to the plastic surface at 37°C in 5% CO2 in complete medium. Equimolar amounts of radiopharmaceuticals were added to cells for different incubation times (20 to 120 minutes) for 18F-FCH and 18F-FET respectively. The cellular radiotracer uptake was determined with a gamma counter. All experiments were carried out in duplicate and repeated three times. The uptake measurements are expressed as the percentage of the administered dose of tracer per 2 x 105 cells. Data (expressed as mean values of % uptake of radiopharmaceuticals) were compared using parametric or non-parametric tests as appropriate. Differences were regarded as statistically significant when p<0.05.
Results. A significant uptake of 18F-FCH was seen in T98G cells at 60, 90 and 120 minutes. The percentage uptake of
18F-FET in comparison to 18F-FCH was lower by a factor of more than 3, with different kinetic curves. 18F-FET showed a more rapid initial uptake up to 40 minutes and 18F-FCH showed a progressive rise reaching a maximum after 90 minutes. Conclusions. 18F-FCH and 18F-FET are candidates for neuro-oncological PET imaging. 18F-FET could be the most useful oncological PET marker in the presence of reparative changes after therapy, where the higher affinity of 18F-FCH to inflammatory cells makes it more difficult to discriminate between tumour persistence and non-neoplastic changes. Additional studies on the influence of inflammatory tissue and radionecrotic cellular components on radiopharma-ceutical uptake are necessary.
Key words:
Introduction
The human brain is made up of approximately 100 billion nerve cells. Already in 19th century
there was a statement that nervous system is held together by specific cells called glia (in Greek language: glia=glue). More than insulating one neuron from another and prevent neuronal injury, glia
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doi:10.1515/raon-2016-0022
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Persico MG et al. / 18F-FET and 18F-FCH uptake in human glioblastoma
supply oxygen and nutrients to neurons, destroy pathogens and remove dead neurons. In the brain, glial cells are more numerous than nerve cells (ratio of app. 3:1).J
Approximately 30% of all brain tumours and app. 80% of malignant ones arise from glial cell (gliomas). Different oncogenes and genetic disorders are most commonly mentioned as causes of gliomas. Despite complex treatment of surgery, radiotherapy and chemotherapy, high grade gliomas almost always recur.23 Before additional systemic or local therapies are performed, precise localization of recurrent tumour is essential. Differentiation between postsurgical, postradiotherapy changes and recurrent tumour is still a difficult diagnostic task.
Magnetic resonance imaging (MRI) is well established imaging modality for diagnosis of recurrent disease in patients with gliomas.4-6 18F-fluorodeoxyglucose (18F-FDG) Positron Emission Tomography (PET) in brain tumours was the first application of this modality in oncology78, however because of the high physiologic glucose uptake of normal brain tissue, 18F-FDG did not gain widespread use in brain tumours imaging.910
PET imaging with [11C]- and [18F]-labelled cho-line derivates is frequently used in the staging and detection of recurrent prostate cancer disease due to the increased choline kinase expression in this malignancy. Moreover, choline kinase dysregula-tion can be frequently found, not only in prostate cancer cells but in a large panel of human tumours such as lung, colorectal, and brain tumours.11-13 Following intravenous injection of choline derivatives in rats and mice, the brain uptake is less than 0.2% of the injected dose.14 However, choline accumulation in inflammatory tissue limits the specificity of choline PET for tumour detection.15
In the last decades, radiolabelled amino acids are attracting increasing interest in nuclear medicine because amino acid tracers appear to be more specific for brain tumour imaging than tracers like [11C]- and [18F]-labelled choline derivates or 3,4-Dihydroxy-6-[18F]fluoro-l-phenylaIanine (18F-DOPA). Results on cellular uptake of O-(2-[18F] fluoroethyl)-l-tyrosine (18F-FET) has been studied in vitro and in vivo already in the 1960's.16 The uptake mechanism of 18F-FET in malignantly transformed cells can either be active or probably result from increased expression of amino acid transporters or passive, whereby the accumulation is slightly higher in tumour tissue with a disrupted blood-brain barrier. In contrast to 18F and 11C-choline, 18F-FET exhibits lower uptake in machrophages and other inflammatory cells.1718 Also 11C-methionine, la-
belled amino acid for PET imaging of central nervous system tumours, showed very good results. But because of short half-life of 11C (20.4 min), this tracer can be used just in the centres with on-site cyclotron. In the last years many articles supported statement that 18F-FET PET/CT is valuable modality for individual treatment decision in patients with low grade gliomas.19-24 The T98G cells are the most radio resistant cell line available derived from a human glioblastoma multiform tumour.25 T98G are arrested in G1 phase under stationary phase conditions, so they also exhibit the transformed characteristics of anchorage independence and immortality.26
In our previous study27, we compared the uptake of 18F-FCH and 18F-FDG by T98G cells and fibroblasts; also for evaluation its influence on cellular radiopharmaceutical uptake competition experiments with cold choline were performed.
Aim of this study was to evaluate 18F-FCH and 18F-FET uptake on T98G cell lines derived from a human glioblastoma multiforme tumour.
Material and methods Cell lines
Human glioblastoma T98G cells were purchased from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and cultured in Eagle's Minimum Essential Medium (EMEM, Euroclone SpA, MI, Italy) supplemented with 10% fetal bovine serum, 100 units/mL penicillin/streptomycin, 2 mM L-glutamine and 0.01% sodium pyruvate at 37°C in a humidified atmosphere of 5% CO2 in air. Human dermal fibroblasts were used as non-pathological control cell types. Primary cultures of human dermal fibroblasts were derived from biopsies of healthy donors after obtaining informed consent. Primary cultures of fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM, Euroclone SpA, MI, Italy) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 g/ mL streptomycin, 2 mM L-glutamine at 37°C in a humidified atmosphere of 5% CO2 in air. Stock cultures of both cell lines were maintained in exponential growth as monolayers in 25 cm2 Corning plastic tissue-culture flasks (Sigma-Aldrich, St Louis, MO, USA).
Radioactive tracer incubation
18F-FCH and 18F-FET were obtained from IASON GmbH (Graz-Seiersberg, Austria). Synthesis of 18F-FCH was performed as follows: The precur-
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sor was reacted with 18F and the intermediate was evaporated via a solid phase cartridge. After the gas phase reaction, the product was trapped and purified by solid phase cartridges and passed through a sterilized filter, synthesis of 18F-FET was performed as follows: The precursor (in acetonitrile) was reacted with 18F. After 18F incorporation, acetonitrile was removed under pressure, and hydrolysis was carried out with 1 M HCl. The final solution was neutralized and purified by solid phase cartridges and passed through a sterilized filter.
Cells, seeded at a density to obtain 2 x 105 cells per flask when radioactive tracers were administered, grew adherent to the plastic surface at 37°C in 5% CO2 in complete medium. Radioactive tracer experiments were performed 20-22 hours postseeding in order to use the cells in the exponential phase of growth. The medium was renewed before performing studies. Cells were incubated at 37°C with 100 kBq (100 |jL) equimolar amounts of 18F-FCH or 18F-FET, added in 2 mL of medium in each flask for varying incubation times (20, 40, 60, 90, 120 min for 18F-FCH; 20, 40, 60, 80, 100, 120 min for 18F-FET) under 5% CO2 gaseous conditions. For experiments with 18F-FCH and 18F-FET, radiotracer incubation was done in complete medium. Control samples underwent the same procedure as other samples, but they were incubated with 100 |jL of saline instead of a radiotracer.
Cell kinetic studies and uptake evaluation
The cellular radiotracer uptake was determined with a 3 x 3'' NaI(Tl) pinhole 16 x 40 mm gamma counter (Raytest, Straubenhardt, Germany). All measurements were carried out under the same counting position along with a standardized source to verify the counter's performance and the data were corrected for background and decay. Total radioactivity was counted when the radiotracer was added to the medium in each flask (time 0). After 20, 40, 60, 90, 120 min for 18F-FCH and 20, 40, 60, 80, 100, 120 min for 18F-FET from time 0, the medium was harvested, the cells were rapidly washed three times with 1 mL of phosphate-buffered saline (PBS) and radiopharmaceutical uptake for each sample was assessed. All experiments were carried out in duplicate and repeated three times. The uptake measurements are expressed as the percentage of the administered dose of tracer per 2 x 105 cells after correction for negative control uptake (flasks containing no cells with complete medium and incubated with radiopharmaceutical).
Uptake of FCH by T98G cells or fibroblasts
time (min)
FIGURE 1. Uptake of 18F-methyl-choline (18F-FCH) by T98G cells and human dermal fibroblasts.
Cell viability assay
At the end of quantitative gamma spectrometry, adherent cells were harvested with 1% trypsin-ED-TA solution and supernatants with adherent cells were counted with Burker's chamber. Trypan Blue dye assay was performed to assess cell viability as standard protocol.
Statistical analysis
In vitro binding experiments were conducted in duplicate and repeated three times. Data (expressed as mean values of % uptake of radiopharmaceuticals) were compared using parametric or non-parametric tests as appropriate. Differences were regarded as statistically significant when p<0.05. All values are expressed as mean values with confidence interval CI 95% and report the uptake of radiotracers as a function of the incubation period. All values are shown as a percentage of the administered dose per 200,000 cells (mean ± CI 95%). Therefore, if error bars on the Y axis do not overlap, the two points are considered significantly different.
Results
Radiopharmaceuticals binding assay
A significant uptake of 18F-FCH was seen in T98G cells after 60 minutes, with a percentage of uptake of 1.8 ± 0.3%, 3.6 ± 0.4% and 3.6 ± 0.6% at 60, 90 and 120 min respectively. Human dermal fibro-blasts did not seem to accumulate 18F-FCH specifically; at each incubation time the percentage of the administered dose in the cells was lower than 1%. Human dermal fibroblast uptake was significantly lower than in the T98G cell uptake in all incubation times (Figure 1).
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Uptake of FET by T98G
0,0
0	20	40	60	80	100 120 140
time (min)
FIGURE 2. Uptake of 18F-ethyl-tyrosine (18F-FET) by T98G cells.
Uptake of FCH and FET by T98G
60 80 time (min)
FIGURE 3. Uptake of 18F-methyl-choline (18F-FCH) and 18F-ethyl-tyrosine (18F-FET) by T98G cells.
Uptake of FDG, FCH, FET by T98G
if S. S
0	20	40	60	80	100 120 140
time (mln)
FIGURE 4. Uptake of 18F-fluorodeoxyglucose (18F-FDG), 18F-methyl-choline (18F-FCH) and 18F-ethyl-tyrosine (18F-FET) by T98G cells.
Figure 2 shows the kinetic uptake of 18F-FET by T98G cells. Despite the trend represented by the curve, the uptake is quite low in terms of radiotracer uptake (% / 200000 cells).
Figure 3 shows that the uptake by T98G cells is increased for 18F-FCH in comparison to 18F-FET. The trend of the two kinetic curves are quite different: the uptake by T98G cells is increased for 18F-FCH over 18F-FET and the accumulation kinetic is not superimposable (see discussion).
Figure 4 illustrates the comparison of 18F-FDG (data derived from our previous study27, 18F-FCH and 18F-FET uptake in T98G cells. At 40 min and at the following time points there is not overlapping of the confidence bars for 18F-FDG and 18F-FET, and the 18F-FET uptake is always lower than 18F-FDG. 18F-FCH uptake at time points after 60 min, is higher in comparison to the other radiopharmaceuti-cals.
As a negative control, flasks containing medium without cells were incubated under the same conditions and did not show a significant uptake of radiotracers.
Cell viability
Exposure to the gaseous mixture was maintained throughout the experiment and the cells' viability was calculated to be approximately 90% under all experimental conditions (data not shown).
Discussion
Our research data on T98G human glioblastoma cell lines underscores the affinity of 18F-FET for ne-oplastic tissue, confirming its potential as a viable oncological PET marker. However, two aspects need to be discussed.
The percentage uptake of 18F-FET in comparison to 18F-FCH was lower by a factor of more than 3. Furthermore, both tracers showed a lower uptake of radioactivity under 60 minutes in comparison to values previously reported for 18F-FDG.2
A thorough literature search did not find any studies with direct comparisons between 18F-FCH and 18F-FET uptake in glioma cell cultures. However, papers related to in vivo uptake in experimental rat gliomas indicate a higher accumulation of 18F-FET in terms of Standard Uptake Value (SUV) as seen in both transplanted C628 or F98 glioma models29,30 in comparison to radio-labelled choline. Despite the different amounts of 18F-FCH and 18F-FET taken up by the same cell culture, the in vitro kinetic uptake is quite similar. 18F-FET did show a more rapid initial uptake up to 40 minutes and 18F-FCH showed a more progressive, continuous rise reaching a maximum activity plateau after
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90 minutes. Several factors render the comparison between our results and data found in the literature difficult, due to the differing characteristics of our T98G cells and other experimental cell lines. In particular, the accumulation kinetics of 18F-FET in T98G cells is quite different from that described in the 9L cancer cell line, where a wash-out is observable after 60 min of incubation.31 This phenomenon is less evident in F98 cell culture, with an initially fast uptake, peaking at 10 min, and followed by a nearly constant or slow wash-out rate during the incubation period of 60 min.32 On the other hand, Habermeier et al. described a progressive accumulation of non-radioactive FET in a NL229 human glioblastoma line up to 4 hours.33
Both Hebermaier et al.33 and Heiss et al.34 tested the release of FET. Heiss et al.34 demonstrated a quick efflux of 18F-FET from porcine SW707 colon cancer cells, only 7% of the original activity remained in the experimental cells after 6 min incubation time, when the culture medium was replaced with a new tracer-free medium. Different results were reported by Habermeier et al.33 demonstrating that, although 18F-FET is not incorporated into proteins, an intracellular metabolism could lead to another impermeable derivative trapped within the glioma cells. This would suggest an asymmetry of intra- and extracellular recognition by LAT1. The 18F-FCH kinetic pattern in our study was quite similar to that seen in 9L glioma cells35, both in the normoxic or hypoxic conditions, reaching maximum activity at 120 minutes. Bansal et al.35 reported a negligible washout of 18F-FCH of about 13% after 2 hours in the release experiments because this radiopharmaceutical remains trapped in the cells as phospho-FCH. This demonstrates the slow rate of dephosphorylation. Conversely, apparent discrepancies between our in vitro observations and the in vivo glioma rat model emerged, both in terms of relative uptake and tracer kinetics. These mismatches could be explained by different causes, including radiotracer accumulation detected by the external imaging device or direct measurement of the pathological specimen, which provides information not only of the true tumour uptake but also of the inflammatory cells. In this setting, 18F-FET accumulates predominantly in the tumour rather than in inflammatory cells, differing from 11C-MET and suggesting that different subtypes of the L system are involved.36 Contrarily, 18F-FCH accumulation has been demonstrated in brain radiation injuries and in murine atherosclerotic plaques - probably mediated by macrophages - as well as in a turpentine-induced sterile abscess.3738 In a rat
model of acute brain injury (cryolesion and proton-induced necrosis) 18F-FET uptake was mainly due to the disruption of the blood-brain-barrier while 18F-FCH was additionally taken up by inflammatory cells.39 Similarly, a comparison of 18F-FCH and 18F-FET in a rat glioma radionecrosis indicated 18F-FET as the superior discriminant between viable tumour and inflammatory changes30, although evidence of increased 18F-FET uptake in perilesion-al reactive astrogliosis after radiotherapy could lead to an overestimation of tumor size.40
Conclusions
The in vitro model used in these experiments allows direct comparison of different radiopharmaceuti-cals as potential candidates for neuro-oncological PET imaging. The results obtained indicate a superiority of 18F-FCH in terms of absolute uptake and in obtaining an optimal target to non-target ratio in the brain, whereas the major limitation of 18F-FDG is its physiological parenchymal uptake. However, a direct translation to clinical application is hampered by certain conflicting results reported in the literature. 18F-FET could be more useful in the presence of reparative changes after therapy, where the higher affinity of 18F-FCH to inflammatory cells makes it more difficult to discriminate between tumour persistence and non-neoplastic changes. Additional studies on the influence of inflammatory tissue and radionecrotic cellular components on radiopharmaceutical uptake will be necessary to elucidate these topics.
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