18F-FET and 18F-FCH uptake in human glioblastoma T98G cell lines
Article Category: Research Article
Publié en ligne: 19 avr. 2016
Pages: 153 - 158
Reçu: 26 nov. 2015
Accepté: 18 mars 2016
DOI: https://doi.org/10.1515/raon-2016-0022
Mots clés
© 2016 Radiol Oncol
This article is distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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.4-618F-fluorodeoxyglucose (18F-FDG) Positron Emission Tomography (PET) in brain tumours was the first application of this modality in oncology7,8, 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
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.
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).
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.
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).
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.
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.
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).
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).
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).
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.
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.
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).
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 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 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
Both Hebermaier
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.