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Cancer Research 68, 7183, September 1, 2008. doi: 10.1158/0008-5472.CAN-08-0095
© 2008 American Association for Cancer Research
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Systems Biology and Emerging Technologies

Manipulation of [11C]-5-Hydroxytryptophan and 6-[18F]Fluoro-3,4-Dihydroxy-L-Phenylalanine Accumulation in Neuroendocrine Tumor Cells

Oliver C. Neels1, Klaas P. Koopmans1, Pieter L. Jager1, Laya Vercauteren4, Aren van Waarde1, Janine Doorduin1, Hetty Timmer-Bosscha2, Adrienne H. Brouwers1, Elisabeth G.E. de Vries2, Rudi A.J.O. Dierckx1, Ido P. Kema3 and Philip H. Elsinga1

Departments of 1 Nuclear Medicine and Molecular Imaging, 2 Medical Oncology, and 3 Pathology and Laboratory Medicine, University Medical Center Groningen and University of Groningen, Groningen, the Netherlands; and 4 Department of Pharmacy, University of Ghent, Ghent, Belgium

Requests for reprints: Philip H. Elsinga, Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen and University of Groningen, P.O. Box 30.001, 9700 RB Groningen, the Netherlands. Phone: 31-50-3613247; Fax: 31-50-3611687; E-mail: p.h.elsinga{at}ngmb.umcg.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 References
 
[11C]-5-Hydroxytryptophan ([11C]HTP) and 6-[18F]fluoro-3,4-dihydroxy-L-phenylalanine ([18F]FDOPA) are used to image neuroendocrine tumors with positron emission tomography. The precise mechanism by which these tracers accumulate in tumor cells is unknown. We aimed to study tracer uptake via large amino acid transporters, peripheral decarboxylation (inhibited by carbidopa), and intracellular breakdown by monoamine oxidase (MAO). [11C]HTP and [18F]FDOPA tracer accumulation was assessed in a human neuroendocrine tumor cell line, BON. The carbidopa experiments were done in a tumor-bearing mouse model. Intracellular [11C]HTP accumulation was 2-fold higher than that of [18F]FDOPA. Cellular transport of both tracers was inhibited by amino-2-norbornanecarboxylic acid. The MAO inhibitors clorgyline and pargyline increased tracer accumulation in vitro. Carbidopa did not influence tracer accumulation in vitro but improved tumor imaging in vivo. Despite lower accumulation in vitro, visualization of [18F]FDOPA is superior to [11C]HTP in the neuroendocrine pancreatic tumor xenograft model. This could be a consequence of the serotonin saturation of BON cells in the in vivo model. [Cancer Res 2008;68(17):7183–90]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 References
 
Because of their high sensitivity, positron emission tomography (PET) studies for imaging of neuroendocrine tumors have recently raised interest (1–7). To visualize these tumors, the principle of amine precursor uptake and decarboxylation (8) and imaging of the somatostatin receptor plays a major role. Neuroendocrine tumors possess the unique property of synthesis, storage, and secretion of biogenic amines. Clinically applied tracers to visualize this are 6-[18F]fluoro-3,4-dihydroxy-L-phenylalanine ([18F]FDOPA) and [11C]-5-hydroxytryptophan ([11C]HTP).

Despite the clinical utility of these tracers, little is known about the precise mechanisms that govern their accumulation in tumor cells. Several factors are potentially involved in this accumulation. The first factor is the fact, as both tracers are amino acids, that they may well be a substrate for transmembrane amino acid transporters. The Na+-independent transporters large amino acid transporter (LAT)-1, LAT2, LAT3, and LAT4 are defined as system L transporters and can be blocked by the model substrate amino-2-norbornanecarboxylic acid (BCH; ref. 9). LAT transporters are responsible for the transport of large neutral amino acids across the cellular membrane.

Second, the precise effects of the peripheral decarboxylation inhibitor carbidopa, generally given to patients before [11C]HTP or [18F]FDOPA tracer injection to improve uptake, are poorly understood.

A third factor involved can be monoamine oxidase (MAO). It plays a major role in the metabolism of tryptophan and L-DOPA and is the enzyme responsible for the degradation of serotonin (5-HT) to 5-hydroxyindole acetic acid (5-HIAA) and of dopamine to homovanillic acid. Based on inhibitor sensitivity and substrate selectivity, MAOs are subtyped as MAO A and B (10, 11). Whereas MAO A is mainly responsible for the degradation of 5-HT, MAO B breaks down both 5-HT and dopamine (12). With the use of selective and nonselective MAO inhibitors like clorgyline (MAO A) and pargyline (MAO A and B), the effect of the MAOs on tracer trapping can be examined (13, 14). An increased accumulation due to reduced intracellular metabolism of [11C]HTP and [18F]FDOPA can be expected by the use of MAO inhibitors.

The aim of the present study was the analysis of the uptake of [11C]HTP and [18F]FDOPA by LAT, peripheral decarboxylation by amino acid decarboxylase (AADC), and intracellular breakdown by MAO in vitro. Finally, experiments with carbidopa were extended to a tumor-bearing animal model to get a better understanding of the in vivo metabolism using microPET.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosure of Potential...
 References
 
Tracers. Synthesis of [18F]FDOPA was carried out as described earlier (15) with an average specific activity of 9.8 GBq/mmol after end of synthesis. The tracer was diluted with 0.9% saline to the required concentration of ~100 nmol/mL. [18F]FDOPA at 0.19 ± 0.01 mL, corresponding to a dose of 6.17 ± 0.63 MBq, was injected per animal. [11C]HTP was synthesized via an enzymatic method with an average specific activity of 30,000 GBq/mmol after end of synthesis as recently described (16). It was used at a concentration of ~0.67 nmol/mL. [11C]HTP at 0.24 ± 0.02 mL, corresponding to a dose of 10.79 ± 1.18 MBq, was injected per animal.

Chemicals. 5-HTP, 5-HT, 5-HIAA, carbidopa, clorgyline, pargyline hydrochloride, and BCH were purchased from Sigma. GMC (5.6 mmol/L D-glucose, 0.49 mmol/L MgCl2, 0.68 mmol/L CaCl2) was added to PBS solution (140 mmol/L NaCl, 2.7 mmol/L KCl, 6.4 mmol/L Na2HPO4, 0.2 mmol/L KH2PO4). The pH of the resulting PBS-GMC solutions was adjusted to 7.4 with sodium hydroxide. PBS-GMC was used to deplete internal amino acid pools (17, 18). Matrigel was purchased from BD Biosciences.

Cell culture. Experiments were done with the human neuroendocrine pancreatic tumor cell line BON (19). Cells were maintained in 25-cm2 culture flasks in 5-mL DMEM/F-12 (1:1) medium supplemented with 10% FCS containing the amino acids L-phenylalanine (215 µmol) and L-tryptophan (44 µmol) among others. Cells were grown in a humidified atmosphere containing 5% CO2 and were routinely subcultured every 3 to 4 d. Cultures grown to a cell density of 1.0 x 106 to 1.4 x 106 cells/mL were used for the experiments.

Cells were harvested by trypsin treatment, resuspended, and diluted 3:7 in culture medium on 12-well plates 1 d before the experiments (1 mL/well). The viability and number of cells were determined by the trypan blue exclusion technique. Cell viability 1 to 2 h after experiments was >90%. At the day of experiment, cells were washed with warm PBS (3 x 2 mL) and 1 mL of culture medium or PBS-GMC buffer was added per well. The cells were then placed in a water bath for 1 h at 37°C before start of the experiments to allow depletion of internal amino acids. Accumulation experiments were started by addition of 150-µL [18F]FDOPA (~15 nmol) or 60-µL [11C]HTP (~0.04 nmol) of the solution in each well.

Determination of intracellular tracer accumulation. After completion of the experiment, buffer was removed and cells were washed with ice-cold PBS (3 x 2 mL) and harvested by addition of 200 µL trypsin per well. One milliliter of growth medium per well was added; then cells were resuspended, transferred to 10-mL tubes, and counted in a gamma counter (Compugamma, LKB Wallac). Measurements of tracer accumulation were expressed as percentage of the radioactive dose per 1 x 105 cells. All results were corrected for nonspecific accumulation. For the determination of nonspecific tracer accumulation, all washing procedures were done with ice-cold PBS. Experiments were done in ice-cold culture medium or PBS-GMC buffer and plates were placed on ice. Tracer accumulation at 0°C was considered as nonspecific binding. Results represent the mean of three to four experiments (±SE). Individual experiments were done in duplicate.

Inhibition experiments. Various concentrations of the blocking agent BCH (0–20 mmol/L) were applied to determine an adequate blocking concentration. To maintain cell viability, carbidopa was used at a maximum concentration of 0.08 mmol/L because higher concentrations of carbidopa induced apoptosis. Clorgyline and pargyline were added at a concentration of 0.1 mmol/L (20, 21). Inhibition experiments were carried out by adding 1 mL of culture medium (control and carbidopa only) or PBS-GMC containing the blocking agent in the relevant concentration to each well. Afterward, cells were incubated for 1 h to achieve the required amino acid depletion. Subsequently, tracer incubation was done and intracellular accumulation determined as described above. A tracer incubation time of 15 min was used for the blocking agent BCH. Tracer incubation times ranging from 5 to 60 min with AADC and MAO inhibitors carbidopa, clorgyline, and pargyline were used. Due to the short half-life of 11C, incubation periods longer than 60 min were not applied.

Nonlabeled tracer accumulation. Because of the short half-life of PET isotopes, the detection and quantification of radioactive 5-HTP metabolites like 5-HT or 5-HIAA is limited. A sensitive automatic detection method of 5-HTP and its metabolites used in carcinoid patients was used for the detection of nonlabeled tracer in vitro (22). For these experiments, culture medium was removed from the 25-cm2 culture flasks. Cells were then washed with PBS (3 x 2 mL). Five milliliters of PBS-GMC buffer containing the carbidopa (0.08 mmol/L), the MAO A inhibitor clorgyline (0.1 mmol/L), or the nonselective MAO inhibitor pargyline (0.1 mmol/L) were added. After the 1-h depletion period, non–radioactive-labeled 5-HTP (55 nmol/L) was added in 5-mL PBS-GMC buffer.

After 15 and 60 min of tracer incubation, PBS-GMC buffer was removed. The buffer supernatant was analyzed for the amounts of 5-HIAA. Preconcentration was done by liquid-liquid extraction in the following way. Per sample, 2 drops of glacial acetic acid, 1 g of NaCl, and 5 mL of diethyl ether were added and slightly shaken. After centrifugation at 2,000 x g for 5 min, the organic phase was transferred into a test tube and diethyl ether evaporated in a slight stream of nitrogen. Samples were dissolved in eluent and analyzed as described earlier (22).

For the analysis of 5-HTP metabolites, supernatant was removed and the cells were harvested with 1-mL trypsin and resuspended in 1-mL PBS containing 10% FCS. Cells were washed thrice with ice-cold PBS (1 mL) and centrifuged for 10 min at 10,000 x g. Cells were lysed using liquid nitrogen. The concentrations of 5-HTP, 5-HT, and 5-HIAA present in the lysed BON cells dissolved in 1-mL saline were determined by the method described above. Cellular accumulation of nonlabeled tracer was defined as the amount of substance detected in lysed cells divided by the total amount of 5-HTP in incubation medium at the start of the experiment. All results were represented as the mean of three experiments (±SE).

Animals. Nude male mice (BALB/c, age 6–8 wk, body weight 18–24 g) were obtained from Harlan Netherlands BV. Experimental groups consisted of four to five animals to perform microPET scanning after injection of [11C]HTP and [18F]FDOPA. The total number of studied animals was 36. Animals were housed in temperature- and humidity-controlled rooms with 12-h day and 12-h night cycles and were provided with forage and water ad libitum. Animals were housed in HEPA-filtered cages in the animal research facility of the University Medical Center Groningen under controlled water, lab chow, humidity, and temperature conditions. At least 2 h before starting the experiments, the animals were acclimated to laboratory conditions. All animal experiments were done by licensed investigators in compliance with the Law on Animal Experiments of the Netherlands. The study protocol was approved by the Committee on Animal Ethics of the University of Groningen.

MicroPET scanning. Sixty-minute dynamic scanning followed by 10-min transmission scanning was done using a Concorde microPET Focus 220 system equipped with microPET manager for data acquisition in list mode and ASIPro for preparing sinograms and image reconstruction. Using ASIPro's clipping tool, areas with very high activity that were not relevant to the current study, such as the liver region, were removed as to yield a more cleaned up version of the scan that is, therefore, easier to evaluate. Ordered subset expectation maximization (OSEM2D) statistics was applied for the quantitative analysis. The PET acquisition data were fully corrected for dead time, random coincidences, attenuation, and scatter. PET image size was 128 x 128 x 95 voxels.

In all experiments, cells were harvested with trypsin and resuspended in 1-mL growth medium/Matrigel (1:1) and injected s.c. (1 x 106 cells per injection) into the right shoulder of the animals to establish tumors. Growth of tumors was checked thrice a week. After ~3 wk of growth, a 10-mm tumor size was reached and considered suitable for the experiments. I.v. injection and scanning procedure were done under isoflurane anesthesia. Four to five animals from each group received carbidopa (1 mg/kg) i.p. in the abdominal region 1 h before tracer injection. Thereafter, radioactive tracers ([11C]HTP, 0.50 ± 0.24 MBq/g body weight; [18F]FDOPA, 0.29 ± 0.13 MBq/g body weight) were injected i.p. in the abdominal region or i.v. via the penile vein. After scanning for 70 min (60-min dynamic scanning, 10-min transmission scan), animals were sacrificed to determine tracer accumulation in tumor tissue and in different body parts (e.g., liver, kidney, pancreas, intestines, and brain). Radioactivity was measured in a gamma counter. Measurements of tracer accumulation were expressed as percentage of the injected dose per gram of body weight.

Statistics. Differences between various groups were tested for statistical significance using Student's t test for independent samples. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosure of Potential...
 References
 
Time course of tracer accumulation. Cellular accumulation of [18F]FDOPA and [11C]HTP of cells incubated in culture medium over a period of 60 minutes was rapid but low ([18F]FDOPA, 0.07 ± 0.01%/105 cells; [11C]HTP, 0.15 ± 0.01%/105 cells). However, in amino acid–free PBS-GMC buffer both tracers showed very rapid accumulation, and under these conditions considerably higher levels of accumulation were reached. [18F]FDOPA was accumulated to 1.2 ± 0.2%/105 cells after 15 minutes and remained constant up to the end of the 60-minute incubation period. [11C]HTP accumulation was much higher than that of [18F]FDOPA (ratio, 5:1) with a maximum tracer accumulation of 5.3 ± 0.8%/105 cells at 60 minutes (Fig. 1 ).


Figure 1
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  		Figure 1. Accumulation of [11C]HTP (top) and [18F]FDOPA (bottom) in BON cells. Tracer accumulation was measured in amino acid–free medium and was corrected for nonspecific binding. Points, mean of three to four experiments; bars, SE. *, P ≤ 0.05, compared with control.

 
Inhibition experiments. Incubation of cells with carbidopa did not affect accumulation of both [11C]HTP and [18F]FDOPA after 60 minutes, neither in full culture medium nor in PBS-GMC buffer. Clorgyline preincubation led to significantly higher accumulation compared with control for [11C]HTP (14.2 ± 3.8%/105 cells after 60 minutes) and [18F]FDOPA (9.2 ± 2.9%/105 cells after 60 minutes) in PBS-GMC buffer. With pargyline, slightly higher accumulation compared with control was obtained for [18F]FDOPA (3.7 ± 0.7%/105 cells after 60 minutes; Fig. 1).

Accumulation experiments with BCH were done at the time point of 15 minutes because [18F]FDOPA reached a plateau phase of accumulation at 15 minutes in untreated BON cells. At 1.0 mmol/L BCH, the accumulation of [18F]FDOPA and [11C]HTP was inhibited to levels of 0.43 ± 0.02%/105 cells ([18F]FDOPA) and 0.22 ± 0.08%/105 cells ([11C]HTP) and suppressed at 20 mmol/L BCH. The BCH IC50 value for [11C]HTP is 0.12 mmol/L (Fig. 2 ). The lowest used concentration of 0.03 mmol/L BCH resulted in a reduction of tracer accumulation to 45% compared with control for [18F]FDOPA and a BCH IC50 value of 0.01 mmol/L.


Figure 2
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  		Figure 2. Accumulation of [11C]HTP and [18F]FDOPA in BON cells after inhibition of LAT. Tracer accumulation was measured in amino acid–free medium and was corrected for nonspecific binding. A concentration of 10–10 mmol/L BCH is set as control. Incubation time, 15 min. Points, mean of three experiments; bars, SE. BCH IC50, 0.12 mmol/L ([11C]HTP) and 0.01 mmol/L ([18F]FDOPA).

 
Nonlabeled tracer accumulation. In BON cells (control), apart from 5-HTP, both 5-HT and 5-HIAA were detected 15 and 60 minutes after starting incubation. Only low cellular 5-HT levels (0.6 ± 0.1% after 60 minutes) were found compared with 5-HTP (12.0 ± 0.0% after 60 minutes) and 5-HIAA (11.9 ± 0.8% after 60 minutes) levels. Treatment with carbidopa neither increased 5-HTP (3.0 ± 0.9% after 60 minutes) nor decreased the cellular levels of 5-HT (3.0 ± 0.3% after 60 minutes) or 5-HIAA (11.8 ± 1.4% after 60 minutes). Clorgyline, however, increased 5-HT (18.4 ± 2.9% after 60 minutes) and decreased 5-HIAA levels (0.9 ± 0.0% after 60 minutes). Similar results were obtained after pargyline treatment (5-HT, 29.3 ± 5.0%; 5-HIAA, 2.4 ± 0.2% after 60 minutes; Fig. 3 ).


Figure 3
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  		Figure 3. 5-HTP and metabolites in BON cells after incubation with 5-HTP. Top, 15-min incubation time. Bottom, 60-min incubation time. Columns, mean of three experiments; bars, SE. *, P ≤ 0.05, compared with control.

 
Animal experiments. Thirty-six mice were divided into eight groups of four to five animals (control and carbidopa) and given [18F]FDOPA or [11C]HTP, i.p. or i.v. In 33 of 36 mouse microPET scans, tumors were visualized. Tumor weights on day of experiments ranged from 19 to 311 mg (average, 118 mg) after decapitation. In three mouse scans ([18F]FDOPA i.p. control, [18F]FDOPA i.v. carbidopa, and [11C]HTP i.v. carbidopa), tumors were not visualized. In the tumor region following i.v. and i.p. administration, [18F]FDOPA in combination with carbidopa pretreatment gave the highest specific uptake values (SUV; Fig. 4 ). Within 5 minutes after i.v. tracer injection, the plateau phase of the maximum tumor SUV for all experiments was reached, except for [18F]FDOPA combined with carbidopa given i.p. in the abdominal region. I.p. tracer injection showed increasing tumor SUV over time, reaching a plateau phase later than 1 hour. Treatment with carbidopa resulted in higher SUVs for tumors compared with controls for both tracers within 60 minutes. For i.v. injections in both carbidopa treated and control, [18F]FDOPA generated significantly higher tumor SUVs than those obtained after [11C]HTP.


Figure 4
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  		Figure 4. Tracer accumulation in tumor after i.p. (top) and i.v. (bottom) injection expressed as SUV. Points, mean of four to five animals; bars, SE. *, P ≤ 0.05, [18F]FDOPA: carbidopa versus control; **, P ≤ 0.05, control: [11C]HTP versus [18F]FDOPA; ***, P ≤ 0.05, control: [18F]FDOPA versus [11C]HTP; ****, P ≤ 0.05, carbidopa: [18F]FDOPA versus [11C]HTP.

 
Biodistribution data after scanning showed high SUVs for liver, kidney, and pancreas for both tracers (i.v.). SUVs were not significantly altered after carbidopa treatment compared with control (Fig. 5 ). Although tumor SUVs in microPET images were higher for [18F]FDOPA, [11C]HTP resulted in higher uptake in most organs; significant differences in uptake were noted for spleen, RBC, and blood plasma irrespective of carbidopa treatment.


Figure 5
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  		Figure 5. Accumulation of [11C]HTP and [18F]FDOPA in different organs 70 min after i.p. (top) and i.v. (bottom) injection, expressed as SUV. In separate experiments, animals were pretreated with carbidopa. Columns, mean from four to five animals; bars, SE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosure of Potential...
 References
 
Cellular accumulation of [11C]HTP and [18F]FDOPA in neuroendocrine tumor cells is rapid and reaches a plateau after 15 minutes in vitro and within <5 minutes in the tumor in animal model after i.v. injection. We identified LAT, AADC, and MAO as factors affecting intracellular tracer accumulation. Inhibition of amino acid transport resulted in a nearly complete shutdown of accumulation, illustrating that this mechanism is a key factor in intracellular accumulation. Carbidopa did not influence cellular accumulation of both tracers in tumor cells in vitro but did increase tumor accumulation of radioactivity in animals. In vitro, selective inhibition of MAO A by clorgyline induced increased accumulation of both tracers, confirming that MAO is a third important factor affecting their biodistribution. Nonselective inhibition of MAO by pargyline only increased [18F]FDOPA accumulation.

We performed in vitro cellular accumulation experiments in culture medium because the levels of large amino acids as present in DMEM/F-12 culture medium are similar to the ones in blood plasma (23). The very low rates of [18F]FDOPA and [11C]HTP accumulation suggest that other amino acids in culture medium are competing for accumulation with these radiolabeled tracers. Thereafter the amino acid–free PBS, in which cells remain viable during the 2-hour test period, was used. PBS was supplemented with D-glucose, magnesium chloride, and calcium chloride as used by Jager and colleagues (18). [11C]HTP was accumulated twice as much as [18F]FDOPA over a period of 60 minutes. This could be a consequence of the fact that BON cells produce more 5-HT than dopamine (24, 25). The retention of neurotransmitters is considered to be the result of uptake (LAT), decarboxylation (AADC), and granular storage by vesicular monoamine transporters (VMAT). The latter process prevents enzymatic breakdown in the cytoplasm (MAO) and subsequent secretion. One of the most important factors of VMAT activity is the amount of 5-HT or catecholamines in secretory vesicles (26, 27). In our cell studies, the medium was free of amino acids and metabolites, tentatively resulting in reduced filling of secretory granules. This theoretically led to higher VMAT-substrate activity. Separate granular storage of 5-HT and catecholamines may explain the differences in retention for [11C]HTP and [18F]FDOPA (28–30).

The cellular accumulation of [11C]HTP and [18F]FDOPA is blocked by low concentrations of BCH, a conventional inhibitor of the amino acid transporter system L (18). An interesting aspect about the growth of BON tumor cells is its dependence on large amino acids. If the supply of those amino acids could be disrupted (e.g., by BCH), proliferation would possibly be slowed down or stopped. Recently, a dose-dependent inhibition of growth of C6 glioma cells was observed following BCH exposure in vitro and in vivo (31).

Örlefors and colleagues (32) reported in patients an improved uptake of [11C]HTP in carcinoid tumors and decreased urinary 5-HIAA levels when [11C]HTP was administered after p.o. administration of carbidopa. This was suggested to be the result of decreased conversion of [11C]HTP and [18F]FDOPA to [11C]-5-HT/[18F]dopamine by activity of the AADC enzyme in peripheral tissues such as the liver and kidneys (33–35). They hypothesized that the degradation of [11C]HTP to [11C]-5-HT in peripheral organs is blocked by carbidopa and thus increases the availability of [11C]HTP, resulting in higher accumulation of radiolabeled tracer in tumor lesions. Our results validate this hypothesis. Carbidopa did not affect the accumulation of [11C]HTP or [18F]FDOPA in BON cells. This suggests that intracellular decarboxylation in BON cells was not inhibited. AADC activity was found to be up-regulated in neuroendocrine tumor cells (36). In nude mice bearing a BON tumor, carbidopa increased tracer tumor accumulation within the tumor, resulting in better imaging (Fig. 6 ). Our mouse PET images show high accumulation in the abdominal region. This is in line with our mice biodistribution data of high SUVs for organs such as liver and kidney located in this region. In the tumor model that we used, which was derived from a human pancreatic tumor cell line, [18F]FDOPA accumulation was higher than that of [11C]HTP. Due to the high 5-HT production by carcinoid tumors (22), the granules in tumor cells of our animals are expected to be saturated with 5-HT. This may reduce granular storage of [11C]-5-HT, which results in cytoplasmic breakdown (MAO) and subsequent excretion of metabolites. As noted above, different granular storage of 5-HT and catecholamines may explain the differences in tracer retention.


Figure 6
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  		Figure 6. A, [11C]HTP PET after i.v. injection, coronal view. Left, control 22.6 g, 9.9 MBq, tumor weight 23.5 mg. Right, carbidopa treated 21.6 g, 6.2 MBq, tumor weight 61.1 mg. B, [18F]FDOPA PET after i.v. injection, coronal view. Left, control 20.5 g, 8.4 MBq, tumor weight 109 mg. Right, carbidopa treated 23.6 g, 8.4 MBq, tumor weight 57.9 mg. Summed frames. Hotspots in abdominal region were cleaned up using ASIPro's clipping tool. Arrows, tumors located in the right shoulder.

 
Increased in vitro accumulation was noticed after exposure to the MAO A inhibitor clorgyline. This result differs from reported PET studies with the MAO inhibitors harmine and deprenyl. Harmine and deprenyl both decreased the accumulation of [18F]FDG and [11C]DOPA in BON cells (37). Degradation of [18F]fluorodopamine and [11C]-5-HT seems to be blocked by clorgyline. Pargyline gives a slightly higher [18F]FDOPA accumulation compared with control in accordance with the fact that pargyline is an inhibitor for MAO B. Higher concentrations of pargyline could probably also lead to an increased [11C]HTP accumulation.

In our institution, sensitive analytic methods to profile tryptophan-related plasma indoles in patients (22) are available. This allowed us, as the half-life of 11C is short, to determine the transformation of cold 5-HTP into 5-HT and 5-HIAA at two different time points (15 and 60 minutes) at equal concentrations as used for radioactive-labeled [11C]HTP. However, this method only reflects the concentrations of metabolites. To completely understand the metabolism, we suggest the use of stable isotopes in combination with mass spectroscopy as a follow-up study. The use of selective AADC and MAO inhibitors gave insight into intracellular processes. The presence of AADC in BON cells is confirmed by the intracellular synthesis of 5-HT. No increase in 5-HTP is noticed after carbidopa treatment, most likely because carbidopa is not a substrate for LAT transporters in BON cells and therefore not being transported into these cells. Neuroendocrine tumor cells express up-regulated AADC activity (36). Inhibition of AADC will therefore not be inhibited by carbidopa within the tumor cell, and intracellular conversion of 5-HTP into 5-HT will still be possible. Use of the MAO A inhibitor clorgyline and nonselective MAO inhibitor pargyline resulted in higher intracellular tumor 5-HT and lower 5-HIAA level. Once 5-HT is formed inside the cell, it does not seem to be transported outside the cell before being deaminated by MAO into 5-HIAA.


   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosure of Potential...
 References
 
Inhibition of LAT in BON cells leads to decreased tracer accumulation in vitro. Carbidopa does not influence tracer accumulation in tumor cells in vitro but improves tumor imaging in vivo. [18F]FDOPA is superior to [11C]HTP in tumor SUVs in this neuroendocrine pancreatic tumor xenograft model. MAO inhibition improves tracer accumulation in vitro.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Dutch Cancer Society grant 2003-2936.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Ahlman (Department of Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden) for kindly providing BON cells, and E.E.A. Venekamp and J. Krijnen for assistance in the analysis of 5-HTP, 5-HT, and 5-HIAA samples.

Received 1/20/08. Revised 4/29/08. Accepted 5/24/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 References
 

  1. Rufini V, Calcagni ML, Baum RP. Imaging of neuroendocrine tumors. Semin Nucl Med 2006;36:228–47.[CrossRef][Medline]
  2. Eriksson B, Örlefors H, Öberg K, Sundin A, Bergström M, Långström B. Developments in PET for the detection of endocrine tumours. Best Pract Res Clin Endocrinol Metab 2005;19:311–24.[CrossRef][Medline]
  3. Öberg K, Eriksson B. Nuclear medicine in the detection, staging and treatment of gastrointestinal carcinoid tumours. Best Pract Res Clin Endocrinol Metab 2005;19:265–76.[CrossRef][Medline]
  4. Örlefors H, Sundin A, Garske U, et al. Whole-body 11C-5-hydroxytryptophan positron emission tomography as a universal imaging technique for neuroendocrine tumors—comparison with somatostatin receptor scintigraphy and computed tomography. J Clin Endocrinol Metab 2005;90:3392–4000.[Abstract/Free Full Text]
  5. Montravers F, Grahek D, Kerrou K, et al. Can fluorodihydroxyphenylalanine PET replace somatostatin receptor scintigraphy in patients with digestive endocrine tumors? J Nucl Med 2006;47:1455–62.[Abstract/Free Full Text]
  6. Koopmans KP, de Vries EGE, Kema IP, et al. Staging of carcinoid tumours with 18F-DOPA PET: a prospective, diagnostic study. Lancet Oncol 2006;7:728–34.[CrossRef][Medline]
  7. Sundin A, Garske U, Örlefors H. Nuclear imaging of neuroendocrine tumors. Best Pract Res Clin Endocrinol Metab 2007;21:69–85.[CrossRef][Medline]
  8. Pearse AG. The APUD concept and hormone production. Clin Endocrinol Metab 1980;9:211–2.[Medline]
  9. Fuchs BC, Bode BP. Amino acid transporters ASCT2 and LAT1 in cancer: partners in crime? Semin Cancer Biol 2005;15:254–66.[CrossRef][Medline]
  10. Johnston JP. Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem Pharmacol 1968;17:1285–97.[CrossRef][Medline]
  11. Knoll J, Magyar K. Some puzzling effects of monoamine oxidase inhibitors. Adv Biochem Psychopharmcol 1972;5:393–408.[Medline]
  12. Shih JC, Chen K, Ridd MJ. Monoamine oxidase: from genes to behavior. Annu Rev Neurosci 1999;22:197–217.[CrossRef][Medline]
  13. Saura J, Kettler R, Da Prada M, Richards JG. Quantitative enzyme radioautography with 3H-Ro 41-1049 and 3H-Ro 19-6327 in vitro: localization and abundance of MAO-A and MAO-B in rat CNS, peripheral organs, and human brain. J Neurosci 1992;12:1977–99.[Abstract]
  14. Saura J, Nadal E, van den Berg B, Vila M, Bombi JA, Mahy N. Localization of monoamine oxidase in human peripheral tissues. Life Sci 1996;59:1341–9.[CrossRef][Medline]
  15. De Vries EFJ, Luurtsema G, Brussermann M, Elsinga PH, Vaalburg W. Fully automated synthesis module for the high yield one-pot preparation of 6-[18F]fluoro-L-DOPA. Appl Radiat Isot 1999;51:389–94.[CrossRef]
  16. Neels OC, Jager PL, Koopmans KP, et al. Development of a reliable remote-controlled synthesis of β-[11C]-5-hydroxy-L-tryptophan on a Zymark robotic system. J Label Compd Radiopharm 2006;49:889–95.[CrossRef]
  17. Shotwell MA, Jayme DW, Kilberg MS, Oxender DL. Neutral amino acid transport systems in Chinese hamster ovary cells. J Biol Chem 1981;256:5422–7.[Abstract/Free Full Text]
  18. Jager PL, de Vries EGE, Piers DA, Timmer-Bosscha H. Uptake mechanisms of L-3-[125I]iodo-{alpha}-methyl-tyrosine in a human small-cell lung cancer cell line: comparison with L-1-[14C]tyrosine. Nucl Med Commun 2001;22:87–96.[CrossRef][Medline]
  19. Evers BM, Townsend CM, Upp JR, et al. Establishment and characterization of a human carcinoid in nude mice and effect of various agents on tumor growth. Gastroenterology 1991;101:303–11.[Medline]
  20. Pino R, Lyles GA. Effects of activity of semicarbazide-sensitive aminooxidases and cellular glutathione on the cytotoxic effect of allylamine, acrolein, and formaldehyde in human cultured endothelial cells. Vopr Med Khim 1997;43:537–47.[Medline]
  21. Basma AN, Morris EJ, Nicklas WJ, Geller HM. L-DOPA cytotoxicity to PC12 cells in is via its autooxidation. J Neurochem 1995;64:825–32.[Medline]
  22. Kema IP, Meijer WG, Meiborg G, Ooms B, Willemse PHB, de Vries EGE. Profiling of tryptophan-related plasma indoles in patients with carcinoid tumors by automated, on-line, solid-phase extraction and HPLC with fluorescence detection. Clin Chem 2001;47:1811–20.[Abstract/Free Full Text]
  23. Hagberg GE, Torstenson R, Marteinsdottir I, Fredrikson M, Långström B, Blomqvist G. Kinetic compartment modeling of [11C]-5-hydroxy-L-tryptophan for positron emission tomography assessment of serotonin synthesis in human brain. J Cereb Blood Flow Metab 2002;22:1352–66.[CrossRef][Medline]
  24. Parekh D, Ishizuka J, Townsend CM, et al. Characterization of a human pancreatic carcinoid in vitro: morphology, amine and peptide storage, and secretion. Pancreas 1994;9:83–90.[Medline]
  25. Lemmer K, Ahnert-Hilger G, Höpfner M, et al. Expression of dopamine receptors and transporter in neuroendocrine gastrointestinal tumor cells. Life Sci 2002;71:667–78.[CrossRef][Medline]
  26. Brunk B, Blex C, Rachakonda S, et al. The first luminal domain of vesicular monoamine transporters mediates G-protein-dependent regulation of transmitter uptake. J Biol Chem 2006;281:33373–85.[Abstract/Free Full Text]
  27. Höltje M, Winter S, Walther D, et al. The vesicular monoamine content regulates VMAT2 activity through G{alpha}q in mouse platelets. J Biol Chem 2003;278:15850–8.[Abstract/Free Full Text]
  28. Feldman JM. Monoamine and dimonoamine oxidase activity in the diagnosis of carcinoid tumors. Cancer 1985;56:2855–60.[Medline]
  29. Smith CC. Evidence for separate serotonin and catecholamine compartments in human platelets. Biochim Biophys Acta 1996;1291:1–4.[Medline]
  30. Eissele R, Anlauf M, Schärfer MKH, Eiden LE, Arnold R, Weihe E. Expression of vesicular monoamine transporters in endocrine hyperplasia and endocrine tumors of the oxyntic stomach. Digestion 1999;60:428–39.[CrossRef][Medline]
  31. Nawashiro H, Otani N, Shinomiya N, et al. L-type amino acid transporter 1 as a potential molecular target in human astrocytic tumors. Int J Cancer 2006;119:484–92.[CrossRef][Medline]
  32. Örlefors H, Sundin A, Lu L, et al. Carbidopa pretreatment improves image interpretation and visualisation of carcinoid tumours with 11C-5-hydroxytryptophan positron emission tomography. Eur J Nucl Med Mol Imaging 2006;33:60–5.[CrossRef][Medline]
  33. Hoffman JM, Melega WP, Hawk TC, et al. The effects of carbidopa administration on 6-[18F]fluoro-L-DOPA kinetics in positron emission tomography. J Nucl Med 1992;32:1472–7.
  34. Eriksson B, Bergström M, Örlefors H, Sundin A, Öberg K, Långström B. Use of PET in neuroendocrine tumors. In vivo applications and in vitro studies. Q J Nucl Med 2000;44:68–76.[Medline]
  35. Bergström M, Lu L, Eriksson B, et al. Modulation of organ uptake of 11C-labelled 5-hydroxytryptohan. Biogenic Amines 1996;12:477–85.
  36. Vachtenheim J, Novotná H. Expression of the aromatic L-amino acid decarboxylase mRNA in human tumour cell lines of neuroendocrine and neuroectodermal origin. Eur J Cancer 1997;33:2411–7.[CrossRef][Medline]
  37. Xing T, Wu F, Brodin O, Fasth KJ, Långström B, Bergström M. In vitro PET evaluations in lung cancer cell lines. Anticancer Res 2000;20:1375–80.[Medline]



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I. P. Kema, K.-P. Koopmans, P. H. Elsinga, A. H. Brouwers, P. L. Jager, and E. G.E. de Vries
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