¹⁸F-FET and ¹⁸F-FCH uptake in human glioblastoma T98G cell lines

Abstract

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 × 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 × 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 radiopharmaceutical uptake are necessary.

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 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).¹

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.^2,3 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 18}F-fluorodeoxyglucose (¹⁸F-FDG) Positron Emission Tomography (PET) in brain tumours was the first application of this modality in oncology^7,8, however because of the high physiologic glucose uptake of normal brain tissue, ¹⁸F-FDG did not gain widespread use in brain tumours imaging.^9,10

PET imaging with [¹¹C]- and [¹⁸F]-labelled choline 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 dysregulation 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-phenylalanine (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.^17,18 Also ¹¹C-methionine, labelled 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 precursor 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 × 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 post-seeding 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 μL) 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 μL of saline instead of a radiotracer.

Cell kinetic studies and uptake evaluation

The cellular radiotracer uptake was determined with a 3 × 3″ NaI(Tl) pinhole 16 × 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 × 10⁵ cells after correction for negative control uptake (flasks containing no cells with complete medium and incubated with radiopharmaceutical).

Cell viability assay

At the end of quantitative gamma spectrometry, adherent cells were harvested with 1% trypsin-EDTA 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 fibroblasts 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).

Figure 1.

Uptake of ¹⁸F-methyl-choline (¹⁸F-FCH) by T98G cells and human dermal fibroblasts.

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 2.

Uptake of ¹⁸F-ethyl-tyrosine (¹⁸F-FET) by T98G 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 3.

Uptake of ¹⁸F-methyl-choline (¹⁸F-FCH) and ¹⁸F-ethyltyrosine (¹⁸F-FET) by T98G cells.

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 radiopharmaceuticals.

Figure 4.

Uptake of 18F-fluorodeoxyglucose (18F-FDG), ¹⁸F-methyl-choline (¹⁸F-FCH) and ¹⁸F-ethyl-tyrosine (¹⁸F-FET) by T98G cells.

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 neoplastic 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 models^29,30 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 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 al³³ 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 al³³ 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.^37,38 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 perilesional reactive astrogliosis after radiotherapy could lead to an overestimation of tumor size.⁴⁰

Conclusion

The in vitro model used in these experiments allows direct comparison of different radiopharmaceuticals 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.

Disclosure: No potential conflicts of interest were disclosed.

M.G.P., F.E.B. and F.P. have contributed equally.