Purpose:11C-Choline–positron emission tomography (PET) has been exploited to detect the aberrant choline metabolism in tumors. Radiolabeled choline uptake within the imaging time is primarily a function of transport, phosphorylation, and oxidation. Rapid choline oxidation, however, complicates interpretation of PET data. In this study, we investigated the biologic basis of the oxidation of deuterated choline analogs and assessed their specificity in human tumor xenografts.

Experimental Design:11C-Choline, 11C-methyl-[1,2-2H4]-choline (11C-D4-choline), and 18F-D4-choline were synthesized to permit comparison. Biodistribution, metabolism, small-animal PET studies, and kinetic analysis of tracer uptake were carried out in human colon HCT116 xenograft–bearing mice.

Results: Oxidation of choline analogs to betaine was highest with 11C-choline, with reduced oxidation observed with 11C-D4-choline and substantially reduced with 18F-D4-choline, suggesting that both fluorination and deuteration were important for tracer metabolism. Although all tracers were converted intracellularly to labeled phosphocholine (specific signal), the higher rate constants for intracellular retention (Ki and k3) of 11C-choline and 11C-D4-choline, compared with 18F-D4-choline, were explained by the rapid conversion of the nonfluorinated tracers to betaine within HCT116 tumors. Imaging studies showed that the uptake of 18F-D4-choline in three tumors with similar radiotracer delivery (K1) and choline kinase α expression—HCT116, A375, and PC3-M—were the same, suggesting that 18F-D4-choline has utility for cancer detection irrespective of histologic type.

Conclusion: We have shown here that both deuteration and fluorination combine to provide protection against choline oxidation in vivo. 18F-D4-choline showed the highest selectivity for phosphorylation and warrants clinical evaluation. Clin Cancer Res; 18(4); 1063–72. ©2012 AACR.

Translational Relevance

11C-Choline–positron emission tomography (PET) is a marker of choline kinase expression and activity, which is upregulated during carcinogenesis. To date, 11C-choline–PET has been used for the detection of a range of human cancers and has emerged as a viable alternative to 18F-2-fluoro-2-deoxyglucose for the imaging of prostate adenocarcinoma. 11C-choline, however, is rapidly oxidized to betaine in an unwanted side reaction, complicating data interpretation. Here, we designed novel choline analogs and tested their metabolic profiles and sensitivity for cancer detection. The doubly fluorinated and deuterated analog 18F-D4-choline showed lowest betaine oxidation. This radiotracer could be used for cancer detection, irrespective of histologic type. Therefore, the development of new choline radiotracers with an improved metabolic profile should provide a means to simplify interpretation of clinical PET data, while increasing selectivity for phosphorylation.

Choline is required for the biosynthesis of phosphatidylcholine, a key component of the plasma membrane. Following transport into the cell, choline is phosphorylated by choline kinase to phosphocholine and then is further metabolized to phosphatidylcholine via CDP-choline, known as the Kennedy pathway. Once phosphorylated, phosphocholine is trapped within the cell. Diacylglycerol, a product of phosphatidylcholine degradation, is mitogenic, playing a role in the regulation of cell-cycle progression from G1 to S via increased cyclin D1 and cyclin D3 expression (1). Furthermore, aberrant activation and expression of several oncogenes results in elevated choline kinase activity and intracellular levels of phosphocholine (2–4). Choline kinase overexpression is a common feature of several human cancers (5) and in early stage non–small cell lung cancer, choline kinase has been shown to have prognostic significance (6). The expression of choline transporters, including CTL1 and OCT3, is also increased following malignant transformation and may contribute to radiotracer uptake (7, 8), with choline transport closely associated with cell growth (9). 11C-choline has become a viable alternative to 18F-2-fluoro-2-deoxyglucose for positron emission tomography (PET) imaging of the prostate (10–12), in which the increased choline kinase activity in tumors provides the basis for tumor-specific contrast in comparison with surrounding nonneoplastic tissues. A fluorinated analog, 18F-fluoromethylcholine, has also been developed for PET imaging of choline metabolism (13), with the longer half-life of fluorine-18 (109.8 vs. 20.4 minutes for carbon-11), potentially enabling more widespread adoption of choline imaging in the clinic and the ability to image at later time points posttracer injection.

Within the imaging time window (60 minutes), tumor radiolabeled choline uptake is a function of perfusion, transport of the radiotracer from the extracellular space into cells, where it is either converted into phosphocholine by the action of choline kinase or oxidized by choline oxidase to betaine. Further incorporation of phosphocholine to membrane phosphatidylcholine is negligible within this time window (7, 8, 14, 15). Chromatographic analysis also indicates that further betaine metabolism or conversion to acetylcholine is negligible (16, 17). Hence, radiotracer uptake broadly represents transport and phosphorylation on the one hand, and transport and oxidation on the other. One key limitation of choline–PET is the rapid oxidation to radiolabeled betaine, making it difficult to assess choline kinase–specific trapping of activity (as phosphocholine) within tumors without plasma metabolite evaluation using complex kinetic analysis. We have recently developed a novel tracer, 18F-fluoromethyl-[1,2-2H4]-choline (18F-D4-choline), with reduced in vivo oxidation to betaine and improved sensitivity, for the detection of choline metabolism in comparison with the nondeuterated 18F-fluoromethylcholine (16). This improved metabolic profile was shown (16, 17) to be based on the deuterium isotope effect (18–21). Here, we sought to further evaluate the structural determinants of deuteration on substrate metabolism, as well as the effect of deuteration on tumor-specific uptake. To this end, we developed a novel choline tracer, 11C-[1,2-2H4]-choline (11C-D4-choline) and compared its in vivo tumor uptake, kinetics, and metabolic profile to 11C-choline and 18F-D4-choline PET tracers.

Cell lines

HCT116 colorectal carcinoma (LGC Standards) and PC3-M prostate adenocarcinoma cells (kind donation from Dr Matthew Caley, Prostate Cancer Metastasis Team, Imperial College London, United Kingdom) were grown in RPMI-1640 media, supplemented with 10% fetal calf serum (FCS), 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). A375 malignant melanoma cells were a kind donation from Professor Eyal Gottlieb, Beatson Institute for Cancer Research, Glasgow, United Kingdom, and were grown in high glucose (4.5 g/L) Dulbecco's modified Eagle's medium media, supplemented with 10% FCS, 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

Western blots

Western blotting was done using standard techniques (22, 23). For detailed methodology, see Supplementary Materials.

In vivo tumor models

All animal experiments were conducted by licensed investigators in accordance with the United Kingdom Home Office Guidance on the Operation of the Animal (Scientific Procedures) Act 1986 and within the newly published guidelines for the welfare and use of animals in cancer research (24). Male BALB/c nude mice (aged 6–8 weeks; Charles River) were used. Tumor cells (2 × 106) were injected subcutaneously on the back of mice and animals were used when the xenografts reached approximately 100 mm3. Tumor dimensions were measured continuously using a caliper and tumor volumes were calculated by the equation: volume = (π/6) × a × b × c, in which a, b, and c represent 3 orthogonal axes of the tumor.

In vivo tracer metabolism

Radiolabeled metabolites from plasma and tissues were quantified using a method adapted from Smith and colleagues (17). Briefly, tumor-bearing mice under general anesthesia (2.5% isofluorane; nonrecovery anesthesia) were administered a bolus intravenous injection of one of the following radiotracers: 11C-choline, 11C-D4-choline (∼18.5 MBq), or 18F-D4-choline (∼3.7 MBq), and sacrificed by exsanguination via cardiac puncture at 2, 15, 30, or 60 minutes postradiotracer injection. For automated radiosynthesis methodology see Supplementary Materials. Tumor, kidney, and liver samples were immediately snap-frozen in liquid nitrogen. Aliquots of heparinized blood were rapidly centrifuged (14,000 × g, 5 minutes, 4°C) to obtain plasma. Plasma samples were subsequently snap-frozen in liquid nitrogen and kept on dry ice prior to analysis.

For analysis, samples were thawed and kept at 4°C immediately before use. To ice-cold plasma (200 μL) was added ice-cold methanol (1.5 mL) and the resulting suspension centrifuged (14,000 × g; 4°C; 3 minutes). The supernatant was then decanted and evaporated to dryness on a rotary evaporator (bath temperature, 40°C), then resuspended in high-performance liquid chromatography (HPLC) mobile phase [solvent A: acetonitrile/water/ethanol/acetic acid/1.0 mol/L ammonium acetate/0.1 mol/L sodium phosphate (800/127/68/2/3/10); 1.1 mL]. Samples were filtered through a hydrophilic syringe filter (0.2-μm filter, Millex PTFE filter, Millipore) and the sample (∼1 mL) then injected via a 1-mL sample loop onto the HPLC for analysis. Tissues were homogenized in ice-cold methanol (1.5 mL) using an Ultra-Turrax T-25 homogenizer (IKA Werke GmbH and Co. KG) and subsequently treated as per plasma samples.

Samples were analyzed on an Agilent 1100 series HPLC system (Agilent Technologies), configured as described above, using the method of Leyton and colleagues (16). A μBondapak C18 HPLC column (7.8 × 3,000 mm; Waters), stationary phase and a mobile phase comprising of solvent A (vide supra) and solvent B [acetonitrile/water/ethanol/acetic acid/1.0 mol/L ammonium acetate/0.1 mol/L sodium phosphate (400/400/68/44/88/10)], delivered at a flow rate of 3 mL/min were used for analyte separation. The gradient was set as follows: 0% B for 5 minutes; 0% to 100% B in 10 minutes; 100% B for 0.5 minutes; 100% to 0% B in 2 minutes; 0% B for 2.5 minutes.

PET imaging studies

Dynamic 11C-choline, 11C-D4-choline, and 18F-D4-choline imaging scans were carried out on a dedicated small animal PET scanner (Siemens Inveon PET module, Siemens Medical Solutions USA, Inc.) following a bolus intravenous injection in tumor-bearing mice of either approximately 3.7 MBq for 18F studies or approximately 18.5 MBq for 11C, accommodating for substantially shorter half-life of 11C (20.38 minutes for 11C vs. 109.77 minutes for 18F). Dynamic scans were acquired in list mode format over 60 minutes. The acquired data were then sorted into 0.5-mm sinogram bins and 19 time frames for image reconstruction (4 × 15, 4 × 60, and 11 × 300 seconds), which was done by filtered back projection. For input function analysis, data were sorted into 25 time frames for image reconstruction (8 × 5, 1 × 20, 4 × 40, 1 × 80, and 11 × 300 seconds). The Siemens Inveon Research Workplace software was used for visualization of radiotracer uptake in the tumor; 30- to 60-minute cumulative images of the dynamic data were employed to define 3-dimensional (3D) regions of interest (ROI). Arterial input function was estimated as follows: a single voxel 3D ROI was manually drawn in the center of the heart cavity using 2 to 5 minutes of cumulative images. Care was taken to minimize ROI overlap with the myocardium. The count densities were averaged for all ROIs at each time point to obtain a time versus radioactivity curve (TAC). Tumor TACs were normalized to injected dose, measured by a VDC-304 dose calibrator (Veenstra Instruments) and expressed as percentage injected dose per mL tissue. The area under the TAC, calculated as the integral of %ID/mL from 0 to 60 minutes, and the normalized uptake of radiotracer at 60 minutes (%ID/mL60) were also used for comparisons.

Kinetic analysis in HCT116 tumors

A 2-tissue irreversible compartmental model was employed to fit the TACs, as has been previously established for 11C-choline (25, 26), described extensively in Supplementary Data. Here, both a Single Input 3k model (irreversible binding of the parent) and Double Input [3+2]k model (irreversible binding of the parent, reversible binding of the metabolite) were used to describe radiotracer kinetics. K1 (radiotracer delivery; mL/mL/min) and k2 (1/min) are the rate constants of transfer from plasma to tissue and from tissue to plasma, respectively. k3 (1/min) represents the rate at which the parent tracer is phosphorylated. In this model the irreversible uptake rate constant Ki (mL/mL/min) can be expressed as a function of the microparameters as K1k3/(k2 + k3). K1′ (mL/mL/min) and k2′ (1/min) are the rate constants of transfer from plasma to tissue and from tissue to plasma of labeled betaine. A schematic describing the kinetic models used here is described in Supplementary Fig. S1.

Statistics

Data were expressed as mean ± SEM, unless otherwise shown. The significance of comparison between 2 data sets was determined using Student t test. ANOVA was used for multiparametric analysis (Prism v5.0 software for windows, GraphPad Software). Differences between groups were considered significant if P ≤ 0.05.

Deuteration leads to enhanced renal radiotracer uptake

Time course biodistribution was done in nontumor–bearing male nude mice with 11C-choline, 11C-D4-choline, and 18F-D4-choline tracers. Supplementary Fig. S2 shows tissue distribution at 2, 15, 30, and 60 minutes. There were minimal differences in tissue uptake between the 3 tracers over 60 minutes, with uptake values in broad agreement with data previously published for 18F-choline and 18F-D4-choline (13, 17). In all tracers there was rapid extraction from blood, with the majority of radioactivity retained within the kidneys, evident as early as 2 minutes postinjection. Deuteration of 11C-choline led to a significant 1.8-fold increase in kidney retention over 60 minutes (P < 0.05; Supplementary Fig. S2A and B), with a 3.3-fold increase in kidney retention observed for 18F-D4-choline when compared with 11C-choline at this time point (P < 0.01; Supplementary Fig. S2A and C, respectively). There was a trend toward increased urinary excretion for 11C-D4-choline and 18F-D4-choline, in comparison with the nature identical tracer, 11C-choline, although this increase did not reach statistical significance.

Deuteration of 11C-choline results in modest resistance to oxidation in vivo

Tracer metabolism in tissues and plasma was done by radio-HPLC (Fig. 1). Peaks were assigned as choline, betaine, betaine aldehyde, and phosphocholine, using enzymatic (alkaline phosphatase and choline oxidase) methods to determine their identity (Supplementary Figs. S3 and S4, respectively; ref. 16).

Figure 1.

Metabolic profile of 11C-choline (A and D), 11C-D4-choline (B and E), and 18F-D4-choline (C and F) in the liver (A–C) and kidney (D–F) of BALB/c nude mice. Radiolabeled metabolite profile was assessed at 2, 15, 30, and 60 minutes after intravenous injection of parent radiotracers using radio-HPLC. Mean values (n = 3) and SEM are shown. a, P < 0.05 when 11C-D4-choline is compared with 11C-choline; b, P < 0.05 when 18F-D4-choline is compared with 11C-choline; c, P < 0.05 when 18F-D4-choline is compared with 11C-D4-choline. Bet-ald, betaine aldehyde; p-Choline, phosphocholine.

Figure 1.

Metabolic profile of 11C-choline (A and D), 11C-D4-choline (B and E), and 18F-D4-choline (C and F) in the liver (A–C) and kidney (D–F) of BALB/c nude mice. Radiolabeled metabolite profile was assessed at 2, 15, 30, and 60 minutes after intravenous injection of parent radiotracers using radio-HPLC. Mean values (n = 3) and SEM are shown. a, P < 0.05 when 11C-D4-choline is compared with 11C-choline; b, P < 0.05 when 18F-D4-choline is compared with 11C-choline; c, P < 0.05 when 18F-D4-choline is compared with 11C-D4-choline. Bet-ald, betaine aldehyde; p-Choline, phosphocholine.

Close modal

In the liver, both 11C-choline and 11C-D4-choline were rapidly oxidized to betaine (Fig. 1A and B), with 49.2 ± 7.7% of 11C-choline radioactivity already oxidized to betaine by 2 minutes. Deuteration of 11C-choline provided significant protection against oxidation in the liver at 2 minutes postinjection, with 24.5 ± 2.1% radioactivity as betaine (51.2% decrease in betaine levels; P = 0.037), although this protection was lost by 15 minutes. Notably, a high proportion of liver radioactivity (∼80%) was present as 18F-D4-phosphocholine by 15 minutes with 18F-D4-choline (Fig. 1C). This corresponded to a much reduced liver-specific oxidation when compared with the 2 carbon-11 tracers (15.0 ± 3.6% of radioactivity as betaine at 60 minutes; P = 0.002).

In contrast to the liver, deuteration of 11C-choline resulted in protection against oxidation in the kidney over the entirety of the 60-minute time course (Fig. 1D and E). With 11C-D4-choline there was a 20% to 40% decrease in betaine levels over 60 minutes when compared with 11C-choline (P < 0.05), corresponding to a proportional increase in labeled phosphocholine (P < 0.05). As shown in Fig. 1F, 18F-D4-choline was more resistant to oxidation in the kidney than both carbon-11–labeled choline tracers. There was a relationship between levels of radiolabeled phosphocholine and kidney retention when data from all 3 tracers were compared (R2 = 0.504; Supplementary Fig. S5). In the plasma, the temporal levels of betaine for both 11C-choline and 11C-D4-choline were almost identical; it should be noted that total radioactivity levels were low for all radiotracers. At 2 minutes, 12.1 ± 2.6% and 8.8 ± 3.8% of radioactivity was in the form of betaine for 11C-choline and 11C-D4-choline, respectively, rising to 78.6 ± 4.4% and 79.5 ± 2.9% at 15 minutes. Betaine levels were significantly reduced with 18F-D4-choline, with 43.7 ± 12.4% of activity present as betaine at 15 minutes. A further increase in plasma betaine was not observed with 18F-D4-choline over the remainder of the time course.

Fluorination protects against choline oxidation in tumor

11C-choline, 11C-D4-choline, and 18F-D4-choline metabolism were measured in HCT116 tumors (Fig. 2). With all tracers, choline oxidation was greatly reduced in the tumor in comparison with levels in the kidney and liver. At 15 minutes, both 11C-D4-choline and 18F-D4-choline had significantly more radioactivity corresponding to labeled phosphocholine than 11C-choline; 43.8 ± 1.5% and 45.1 ± 3.2% for 11C-D4-choline and 18F-D4-choline, respectively, in comparison with 30.5 ± 4.0% for 11C-choline (P = 0.035 and P = 0.046, respectively). By 60 minutes, the majority of radioactivity was phosphocholine for all 3 tracers, with labeled phosphocholine levels increasing in the order of 11C-choline < 11C-D4-choline < 18F-D4-choline. There was no difference in the tumor metabolic profile for 11C-choline and 11C-D4-choline at 60 minutes, although reduced choline oxidation was observed for 18F-D4-choline; 14.0 ± 3.0% betaine radioactivity with 18F-D4-choline compared with 28.1 ± 2.9% for 11C-choline (P = 0.026).

Figure 2.

Metabolic profile of 11C-choline, 11C-D4-choline, and 18F-D4-choline in HCT116 tumors. Radiolabeled metabolite profile in HCT116 tumor xenografts was assessed at 15 (A) and 60 minutes (B) after intravenous injection of parent radiotracers using radio-HPLC. Mean values (n = 3) and SEM are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001. p-Choline, phosphocholine.

Figure 2.

Metabolic profile of 11C-choline, 11C-D4-choline, and 18F-D4-choline in HCT116 tumors. Radiolabeled metabolite profile in HCT116 tumor xenografts was assessed at 15 (A) and 60 minutes (B) after intravenous injection of parent radiotracers using radio-HPLC. Mean values (n = 3) and SEM are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001. p-Choline, phosphocholine.

Close modal

Choline tracers have similar sensitivity for imaging tumors by PET

Despite the low systemic oxidation of 18F-D4-choline, tumor radiotracer uptake in mice by PET was no higher than with 11C-choline or 11C-D4-choline (Fig. 3). Figure 3A–C shows typical (0.5 mm) transverse PET image slices showing accumulation of all 3 tracers in HCT116 tumors. For all 3 tracers, there was heterogeneous tumor uptake, but tumor signal-to-background levels were identical, confirmed by normalized uptake values at 60 minutes and values for the tumor area under the time versus radioactivity curve (data not shown). It should be noted that the PET data represent total radioactivity. In the case of 11C-choline or 11C-D4-choline, a significant proportion of this radioactivity is betaine (Fig. 2).

Figure 3.

11C-choline (○), 11C-D4-choline (▴), and 18F-D4-choline (▪) PET image analysis. HCT116 tumor uptake profiles were examined following 60-minute dynamic PET imaging. A–C, representative axial PET-CT images of HCT116 tumor–bearing mice (30–60 minutes of summed activity) for 11C-choline (A), 11C-D4-choline (B), and 18F-D4-choline (C). Tumor margins, indicated from CT image, are outlined in red. D, the tumor TAC. Mean ± SEM (n = 4 mice per group).

Figure 3.

11C-choline (○), 11C-D4-choline (▴), and 18F-D4-choline (▪) PET image analysis. HCT116 tumor uptake profiles were examined following 60-minute dynamic PET imaging. A–C, representative axial PET-CT images of HCT116 tumor–bearing mice (30–60 minutes of summed activity) for 11C-choline (A), 11C-D4-choline (B), and 18F-D4-choline (C). Tumor margins, indicated from CT image, are outlined in red. D, the tumor TAC. Mean ± SEM (n = 4 mice per group).

Close modal

Tumor tracer kinetics

Despite there being no difference in overall tracer retention in the tumor, the kinetic profiles of tracer uptake varied between the 3 choline tracers, detected by PET (Fig. 3D). The kinetics for the 3 tracers were characterized by rapid tumor influx over the initial 5 minutes, followed by stabilization of tumor retention. Initial delivery of 18F-D4-choline over the first 14 minutes of imaging was higher than for both 11C-choline and 11C-D4-choline (expanded TAC for initial 14 minutes shown in Supplementary Fig. S6). Slow wash-out of activity was observed with both 18F-D4-choline and 11C-D4-choline between 30 and 60 minutes, in contrast to the gradual accumulation detected with 11C-choline. Parameters for the irreversible trapping of radioactivity in the tumor, Ki and k3, were calculated from a 2-tissue irreversible model, using metabolite-corrected TAC from the heart cavity as input function (Fig. 4A and B). A double input model, accounting for the contribution of metabolites to the tissue TAC, was used for kinetic analysis, described in Supplementary Data. There was no significant difference in flux constant measurements between deuterated and undeuterated 11C-choline. Addition of methylfluoride, however, resulted in 49.2% (n = 3; P = 0.022) and 75.2% (n = 3; P = 0.005) decreases in Ki and k3, respectively; that is, when 18F-D4-choline was compared with 11C-D4-choline. K1′ values were similar between all 3 tracers: 0.106 ± 0.026, 0.114 ± 0.019, and 0.142 ± 0.027 for 11C-choline, 11C-D4-choline, and 18F-D4-choline, respectively. It is possible that intracellular betaine formation (not just presence of betaine in the extracellular space) led to a higher than expected irreversible uptake; there was a significant 388% and 230% increase in the ratio of betaine:phophocholine at 15 and 60 minutes, respectively (P = 0.045 and 0.036) with 11C-choline in comparison with 18F-D4-choline (Fig. 4C).

Figure 4.

Pharmacokinetics of 11C-choline, 11C-D4-choline, and 18F-D4-choline in HCT116 tumors. A, modified compartmental modeling analysis, taking into account plasma metabolites and their flux into the exchangeable space in tumor, was used to derive Ki, a measure of irreversible retention within the tumor. B, the kinetic parameter, k3, an indirect measure of choline kinase activity, was calculated using a 2-site compartmental model as previously described (36, 37). C, ratio of betaine to phosphocholine in tumors. Metabolites were quantified by radio-HPLC at 15 and 60 minutes of postinjection of tracer. Mean values (n = 4) and SEM are shown. *, P < 0.05; ***, P < 0.001. p-choline, phosphocholine.

Figure 4.

Pharmacokinetics of 11C-choline, 11C-D4-choline, and 18F-D4-choline in HCT116 tumors. A, modified compartmental modeling analysis, taking into account plasma metabolites and their flux into the exchangeable space in tumor, was used to derive Ki, a measure of irreversible retention within the tumor. B, the kinetic parameter, k3, an indirect measure of choline kinase activity, was calculated using a 2-site compartmental model as previously described (36, 37). C, ratio of betaine to phosphocholine in tumors. Metabolites were quantified by radio-HPLC at 15 and 60 minutes of postinjection of tracer. Mean values (n = 4) and SEM are shown. *, P < 0.05; ***, P < 0.001. p-choline, phosphocholine.

Close modal

18F-D4-choline shows good sensitivity for the PET imaging of prostate adenocarcinoma and malignant melanoma

Having confirmed that 18F-D4-choline has the most desirable metabolic profile for in vivo studies, with good sensitivity for the imaging of colon adenocarcinoma, we wanted to evaluate its suitability for cancer detection in other models of human cancer, including malignant melanoma A375 and prostate adenocarcinoma PC3-M. In vitro uptake of 18F-D4-choline was similar in the 3 cell lines over 30 minutes (Supplementary Fig. S7), relating to near-identical levels of choline kinase expression (Supplementary Fig. S7 insert). Retention of radioactivity was shown to be dependent on both choline transport and choline kinase activity, as treatment of cells with the dual choline transport and choline kinase inhibitor, hemicholinium-3, resulted in more than 90% decrease in intracellular tracer radioactivity in all 3 cell lines. Similar intracellular trapping of 18F-D4-choline in these cancer models were translated to their uptake in vivo (Fig. 5A), showing similar values for flux constant measurements, including rates of delivery (K1; Supplementary Table S1) and other and PET imaging variables. There was a trend toward increased tumor retention of 18F-D4-choline in the order of A375 < HCT116 < PC3-M, reflected by the expression of choline kinase in these lines (Fig. 5C). There was no discernible difference in tumor metabolite profiles between the 3 cell cancer models at either 15 or 60 minutes of postinjection (Fig. 5B).

Figure 5.

Dynamic uptake and metabolite analysis with 18F-D4-choline in tumors of different histologic origin. A, the tumor TAC obtained from 60-minute dynamic PET imaging. Mean ± SEM (n = 3–5 mice per group). B, metabolic profile of 18F-D4-choline in tumors. Radiolabeled metabolite profile in HCT116 tumor xenografts was assessed post-PET imaging using radio-HPLC. Mean values (n = 3) and SEM are shown. C, choline kinase expression in malignant melanoma, prostate adenocarcinoma, and colon carcinoma tumors. Representative Western blot from tumor lysates (n = 3 xenografts per tumor cell line). Actin was used as a loading control. CKα, choline kinase alpha; %ID, percentage injected dose.

Figure 5.

Dynamic uptake and metabolite analysis with 18F-D4-choline in tumors of different histologic origin. A, the tumor TAC obtained from 60-minute dynamic PET imaging. Mean ± SEM (n = 3–5 mice per group). B, metabolic profile of 18F-D4-choline in tumors. Radiolabeled metabolite profile in HCT116 tumor xenografts was assessed post-PET imaging using radio-HPLC. Mean values (n = 3) and SEM are shown. C, choline kinase expression in malignant melanoma, prostate adenocarcinoma, and colon carcinoma tumors. Representative Western blot from tumor lysates (n = 3 xenografts per tumor cell line). Actin was used as a loading control. CKα, choline kinase alpha; %ID, percentage injected dose.

Close modal

Aberrant phospholipid metabolism is a hallmark of many cancers (5), resulting in upregulated mitotic signaling and an increase in plasma membrane biosynthesis. One such mediator of phospholipid metabolism, choline kinase, has been shown to be a biomarker of malignant transformation (2). Proton and phosphorous magnetic resonance spectroscopic (MRS) techniques have provided a means to measure the product of choline kinase activity, phosphocholine, from tumor tissue biopsies ex vivo and from noninvasive spectroscopic imaging measurements in vivo (27). MRS, however, is hampered by inherently poor in vivo sensitivity, making it difficult to resolve individual choline metabolite resonances, complicating data interpretation, whereas ex vivo measurements requires invasive sampling from a small, possibly unrepresentative, region of interest. Given the current drawbacks of choline metabolite analysis by MRS, a more viable alternative has been the use of radiolabeled choline for noninvasive tumor imaging. PET-labeled choline tracers provide vastly improved sensitivity, when compared with MRS, and enable dynamic measurements of choline metabolism, but without the chemical resolution of MRS. To date, 11C-choline has successfully been used for the clinical imaging of prostate, brain, breast, and esophageal carcinomas (10, 25, 28–30).

Despite its relative success, 11C-choline-PET imaging has not been widely adopted in the clinic; the short half-life of carbon-11 requires an on-site cyclotron and rapid metabolism of the choline tracer presents complications for data interpretation and limits the imaging time frame to early time frames (25). We have recently developed a fluorinated choline analog, 18F-D4-choline, labeled with a longer lived isotope and with improved metabolic profile, required for late tumor imaging (16). The substitution of deuterium for hydrogen on the ethyl alcohol portion of choline resulted in a large observed isotope effect in the oxidation of choline to betaine by choline oxidase. Further studies showed that the magnitude of the 1H/2D isotope effect was more profound when all protons were substituted for deuterium, in comparison with partial deuteration of the fluorocholine (17). Urinary radioactivity, however, is higher with fluorinated choline analogs relative to 11C-choline (16, 31), potentially limiting their use for the detection of pelvic cancers, such as prostate adenocarcinoma. Here, we developed a novel choline tracer, 11C-D4-choline, which, based on previous work with fluorinated and deuterated choline tracers, was predicted to have reduced oxidation to betaine and a favorable urinary excretion profile.

The kidney has high choline kinase activity along the nephron (32), shown to exhibit the greatest tissue retention for choline-PET and, therefore, is the radiation-dose-critical organ (13, 17). Kidney retention increased in the order of 11C-choline < 11C-D4-choline < 18F-D4-choline over the 60-minute time course (Supplementary Fig. S2), with total kidney radioactivity shown to be proportional to the % radioactivity retained as labeled phosphocholine (Supplementary Fig. S5; R2 = 0.504). The increased conversion of choline to phosphocholine with 11C-D4-choline relative to 11C-choline corresponded with a significant decrease in choline oxidation to betaine and could be ascribed to increased substrate availability for phosphorylation. Further attenuation of choline oxidation was observed with 18F-D4-choline, indicating that the magnitude of the 1H/2D isotope effect is influenced by fluorination. Protection against choline oxidation by deuteration of 11C-choline was shown to be tissue specific, with a decrease in betaine radioactivity measured in the liver at just 2 minutes postinjection when compared with 11C-choline (Fig. 1). This effect is presumably due to the lower capacity of choline oxidase in rodent kidney compared with liver. 18F-D4-choline provided substantially reduced betaine oxidation in the liver over 60 minutes postinjection, again suggesting that fluorination, in part, drives this reduced capacity to oxidize choline pseudosubstrates to betaine.

Despite systemic protection against choline oxidation with 18F-D4-choline, the reduction in the rate of choline oxidation was much more subtle in implanted HCT116 tumors (Fig. 2). At 15 minutes postinjection, there were 43.6% and 47.9% higher levels of radiolabeled phosphocholine when 11C-D4-choline and 18F-D4-choline, respectively, were injected relative to 11C-choline. By 60 minutes there was no difference in labeled phosphocholine levels between the 3 tracers, although there was a significant decrease in betaine-specific radioactivity with 18F-D4-choline. This equilibration of phosphocholine-specific activity can be explained by a saturation effect, with parent tracer levels reduced to a minimum by 60 minutes, severely limiting substrate levels available for choline kinase activity. Lower betaine levels were observed in the tumor with all 3 tracers over the entire time course when compared with liver and kidney, likely resulting from a lower capacity for choline oxidation or increased washout of betaine. It should be noted that the capacity of rodents to metabolize choline is substantially higher than that of humans (14, 33). The slower metabolic rate in humans may, therefore, provide a better differential between these choline tracers. Despite this, deuteration of 11C-choline per se provided less than expected protection against choline oxidation in the liver, tumor, and kidney, especially in the context of improved metabolic profile shown with deuterated fluorocholine versus nondeuterated fluorocholine (16, 17)

Comparison of the 3 choline radiotracers by PET showed no significant differences in overall tumor radiotracer uptake and hence sensitivity (Fig. 3), despite large changes observed in other organs. Initial tumor kinetics (at time points when metabolism was lower), however, varied between tracers, with 18F-D4-choline characterized by rapid delivery over approximately 5 minutes, followed by slow wash-out of activity from the tumor. This compared with the slower uptake, but continuous tumor retention of 11C-choline. At 60 minutes, a 2.7-fold and 4.0-fold higher unmetabolized parent tracer was seen with 18F-D4-choline in tumor compared with 11C-choline and 11C-D4-choline, respectively, (Fig. 2). Deuteration did not, however, alter total tumor radioactivity levels and the modeling approach used did not distinguish between different intracellular species. Although all tracers were converted intracellularly to phosphocholine, the higher rate constants for intracellular retention (Ki and k3; Fig. 3A and B) of 11C-choline and 11C-D4-choline, compared with 18F-D4-choline, were explained by the rapid conversion of the nonfluorinated tracers to betaine within HCT116 tumors, indicating greater specificity with 18F-D4-choline. Compared with 18F-D4-choline, the tumor betaine-to-phosphocholine metabolite ratio increased by 388% (P = 0.045) and 259% (P = 0.061, nonsignificant) for 11C-choline and 11C-D4-choline, respectively (Fig. 4C). It is also important to note that the compartmental modeling is subject to some minor experimental limitations. In humans and larger animals, a more accurate input function can be obtained by continuous blood sampling following radiotracer injection. Individual plasma metabolite data can also be easily obtained and fitted instead of the averaged data taken from a separate cohort of animals used here.

It has been reported elsewhere that fluorination increases urinary excretion in comparison with 11C-choline (13, 16). However, in this study, we did not observe these undesirable urinary excretion properties. This may be due to use of anesthesia for immobilizing mice during imaging. There was a trend toward increased urinary excretion in the two deuterated tracers (suggesting a trade-off between reduced oxidation and renal excretion) when compared with 11C-choline, although these did not reach significance. Low radioactivity levels in the urine prevented accurate metabolite analysis (data not shown). The low radioactivity levels in the urine should enable accurate prostate imaging with 18F-D4-choline, especially if patients void to reduce bladder radioactivity prior to late time point imaging. Given the favorable urinary excretion properties and greatly superior systemic metabolic profile of 18F-D4-choline, PET imaging was carried out in 2 further models of human cancer to assess generic utility in tumors of different origins: A375 malignant melanoma and PC3-M prostate adenocarcinoma. PC3-M cells were chosen as a clinically relevant model for choline imaging, whereas A375 have constitutively active mitogen-activated protein kinase (MAPK) due to a BRAFV600E mutation (34); the MAPK pathway is known to regulate choline kinase activity (35). 18F-D4-choline uptake in vitro (where betaine formation is negligible) was similar in all 3 cell lines, reflecting near-identical levels of choline kinase α expression. The delivery of 18F-D4-choline (K1) was similar between the different tumor types in vivo, suggesting that choline transporter expression was probably not deficient in any of the tumors. These in vitro findings translated well in vivo, with comparable tumor uptake, kinetics, choline kinase α expression, and metabolism for all the tumor types, suggesting that 18F-D4-choline may have utility for tumor detection, irrespective of histologic type.

In conclusion, we have shown here that deuteration of 11C-choline provides a smaller than expected protection against choline oxidation. Despite a significant increase in labeled phosphocholine at early time points, this did not increase the overall sensitivity for the detection of choline metabolism in vivo. More promising is the substantial decrease in betaine oxidation illustrated here with 18F-D4-choline, which may permit the clinical imaging of choline without invasive blood sampling. Fluorine-18 labeling may also lead to wider clinical adoption and permit imaging at late time points. We have further validated 18F-D4-choline using 3 models of human cancer, including a clinically relevant model of human prostate adenocarcinoma.

A patent on novel choline imaging agents has been filed.

The authors thank Dr. Magdy Khalil for his help with the PET imaging studies and for advice about kinetic analysis, and Dr. Matthew Caley and Professor Eyal Gottlieb for provision of cell lines.

This work was funded by Cancer Research UK–Engineering and Physical Sciences Research Council grant C2536/A10337. E.O. Aboagye's laboratory receives core funding from the UK Medical Research Council (MC US A652 0030).

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.

1.
Ramirez de Molina
A
,
Gallego-Ortega
D
,
Sarmentero-Estrada
J
,
Lagares
D
,
Gomez Del Pulgar
T
,
Bandres
E
, et al
Choline kinase as a link connecting phospholipid metabolism and cell cycle regulation: implications in cancer therapy
.
Int J Biochem Cell Biol
2008
;
40
:
1753
63
.
2.
Aboagye
EO
,
Bhujwalla
ZM
. 
Malignant transformation alters membrane choline phospholipid metabolism of human mammary epithelial cells
.
Cancer Res
1999
;
59
:
80
4
.
3.
Hernandez-Alcoceba
R
,
Saniger
L
,
Campos
J
,
Nunez
MC
,
Khaless
F
,
Gallo
MA
, et al
Choline kinase inhibitors as a novel approach for antiproliferative drug design
.
Oncogene
1997
;
15
:
2289
301
.
4.
Liu
D
,
Hutchinson
OC
,
Osman
S
,
Price
P
,
Workman
P
,
Aboagye
EO
. 
Use of radiolabelled choline as a pharmacodynamic marker for the signal transduction inhibitor geldanamycin
.
Br J Cancer
2002
;
87
:
783
9
.
5.
Ramirez de Molina
A
,
Rodriguez-Gonzalez
A
,
Gutierrez
R
,
Martinez-Pineiro
L
,
Sanchez
J
,
Bonilla
F
, et al
Overexpression of choline kinase is a frequent feature in human tumor-derived cell lines and in lung, prostate, and colorectal human cancers
.
Biochem Biophys Res Commun
2002
;
296
:
580
3
.
6.
Ramirez de Molina
A
,
Sarmentero-Estrada
J
,
Belda-Iniesta
C
,
Taron
M
,
Ramirez de Molina
V
,
Cejas
P
, et al
Expression of choline kinase alpha to predict outcome in patients with early-stage non-small-cell lung cancer: a retrospective study
.
Lancet Oncol
2007
;
8
:
889
97
.
7.
Hara
T
,
Bansal
A
,
DeGrado
TR
. 
Choline transporter as a novel target for molecular imaging of cancer
.
Mol Imaging
2006
;
5
:
498
509
.
8.
Yoshimoto
M
,
Waki
A
,
Obata
A
,
Furukawa
T
,
Yonekura
Y
,
Fujibayashi
Y
. 
Radiolabeled choline as a proliferation marker: comparison with radiolabeled acetate
.
Nucl Med Biol
2004
;
31
:
859
65
.
9.
Wang
T
,
Li
J
,
Chen
F
,
Zhao
Y
,
He
X
,
Wan
D
, et al
Choline transporters in human lung adenocarcinoma: expression and functional implications
.
Acta Biochim Biophys Sin (Shanghai)
2007
;
39
:
668
74
.
10.
Hara
T
,
Kosaka
N
,
Kishi
H
. 
PET imaging of prostate cancer using carbon-11-choline
.
J Nucl Med
1998
;
39
:
990
5
.
11.
Kotzerke
J
,
Prang
J
,
Neumaier
B
,
Volkmer
B
,
Guhlmann
A
,
Kleinschmidt
K
, et al
Experience with carbon-11 choline positron emission tomography in prostate carcinoma
.
Eur J Nucl Med
2000
;
27
:
1415
9
.
12.
Richter
JA
,
Rodriguez
M
,
Rioja
J
,
Penuelas
I
,
Marti-Climent
J
,
Garrastachu
P
, et al
Dual tracer 11C-choline and FDG-PET in the diagnosis of biochemical prostate cancer relapse after radical treatment
.
Mol Imaging Biol
2010
;
12
:
210
7
.
13.
DeGrado
TR
,
Baldwin
SW
,
Wang
S
,
Orr
MD
,
Liao
RP
,
Friedman
HS
, et al
Synthesis and evaluation of (18)F-labeled choline analogs as oncologic PET tracers
.
J Nucl Med
2001
;
42
:
1805
14
.
14.
Bansal
A
,
Shuyan
W
,
Hara
T
,
Harris
RA
,
Degrado
TR
. 
Biodisposition and metabolism of [(18)F]fluorocholine in 9L glioma cells and 9L glioma-bearing fisher rats
.
Eur J Nucl Med Mol Imaging
2008
;
35
:
1192
203
.
15.
Kuang
Y
,
Salem
N
,
Corn
DJ
,
Erokwu
B
,
Tian
H
,
Wang
F
, et al
Transport and metabolism of radiolabeled choline in hepatocellular carcinoma
.
Mol Pharm
2010
;
7
:
2077
92
.
16.
Leyton
J
,
Smith
G
,
Zhao
Y
,
Perumal
M
,
Nguyen
QD
,
Robins
E
, et al
[18F]fluoromethyl-[1,2-2H4]-choline: a novel radiotracer for imaging choline metabolism in tumors by positron emission tomography
.
Cancer Res
2009
;
69
:
7721
8
.
17.
Smith
G
,
Zhao
Y
,
Leyton
J
,
Shan
B
,
Nguyen
QD
,
Perumal
M
, et al
Radiosynthesis and pre-clinical evaluation of [(18)F]fluoro-[1,2-(2)H(4)]choline
.
Nucl Med Biol
2011
;
38
:
39
51
.
18.
Fan
F
,
Gadda
G
. 
On the catalytic mechanism of choline oxidase
.
J Am Chem Soc
2005
;
127
:
2067
74
.
19.
Fan
F
,
Gadda
G
. 
An internal equilibrium preorganizes the enzyme-substrate complex for hydride tunneling in choline oxidase
.
Biochemistry
2007
;
46
:
6402
8
.
20.
Gadda
G
. 
pH and deuterium kinetic isotope effects studies on the oxidation of choline to betaine-aldehyde catalyzed by choline oxidase
.
Biochim Biophys Acta
2003
;
1650
:
4
9
.
21.
Nagel
ZD
,
Klinman
JP
. 
Tunneling and dynamics in enzymatic hydride transfer
.
Chem Rev
2006
;
106
:
3095
118
.
22.
Witney
TH
,
Kettunen
MI
,
Brindle
KM
. 
Kinetic modeling of hyperpolarized 13C label exchange between pyruvate and lactate in tumor cells
.
J Biol Chem
2011
;
286
:
24572
80
.
23.
Witney
TH
,
Kettunen
MI
,
Hu
DE
,
Gallagher
FA
,
Bohndiek
SE
,
Napolitano
R
, et al
Detecting treatment response in a model of human breast adenocarcinoma using hyperpolarised [1-13C]pyruvate and [1,4-13C2]fumarate
.
Br J Cancer
2010
;
103
:
1400
6
.
24.
Workman
P
,
Aboagye
EO
,
Balkwill
F
,
Balmain
A
,
Bruder
G
,
Chaplin
DJ
, et al
Guidelines for the welfare and use of animals in cancer research
.
Br J Cancer
2010
;
102
:
1555
77
.
25.
Kenny
LM
,
Contractor
KB
,
Hinz
R
,
Stebbing
J
,
Palmieri
C
,
Jiang
J
, et al
Reproducibility of [11C]choline-positron emission tomography and effect of trastuzumab
.
Clin Cancer Res
2010
;
16
:
4236
45
.
26.
Sutinen
E
,
Nurmi
M
,
Roivainen
A
,
Varpula
M
,
Tolvanen
T
,
Lehikoinen
P
, et al
Kinetics of [(11)C]choline uptake in prostate cancer: a PET study
.
Eur J Nucl Med Mol Imaging
2004
;
31
:
317
24
.
27.
Glunde
K
,
Bhujwalla
ZM
. 
Metabolic tumor imaging using magnetic resonance spectroscopy
.
Semin Oncol
2011
;
38
:
26
41
.
28.
Contractor
KB
,
Kenny
LM
,
Stebbing
J
,
Al-Nahhas
A
,
Palmieri
C
,
Sinnett
D
, et al
[11C]choline positron emission tomography in estrogen receptor-positive breast cancer
.
Clin Cancer Res
2009
;
15
:
5503
10
.
29.
Hara
T
,
Kosaka
N
,
Shinoura
N
,
Kondo
T
. 
PET imaging of brain tumor with [methyl-11C]choline
.
J Nucl Med
1997
;
38
:
842
7
.
30.
Kobori
O
,
Kirihara
Y
,
Kosaka
N
,
Hara
T
. 
Positron emission tomography of esophageal carcinoma using (11)C-choline and (18)F-fluorodeoxyglucose: a novel method of preoperative lymph node staging
.
Cancer
1999
;
86
:
1638
48
.
31.
DeGrado
TR
,
Coleman
RE
,
Wang
S
,
Baldwin
SW
,
Orr
MD
,
Robertson
CN
, et al
Synthesis and evaluation of 18F-labeled choline as an oncologic tracer for positron emission tomography: initial findings in prostate cancer
.
Cancer Res
2001
;
61
:
110
7
.
32.
Wirthensohn
G
,
Vandewalle
A
,
Guder
WG
. 
Choline kinase activity along the rabbit nephron
.
Kidney Int
1982
;
21
:
877
9
.
33.
Roivainen
A
,
Forsback
S
,
Gronroos
T
,
Lehikoinen
P
,
Kahkonen
M
,
Sutinen
E
, et al
Blood metabolism of [methyl-11C]choline; implications for in vivo imaging with positron emission tomography
.
Eur J Nucl Med
2000
;
27
:
25
32
.
34.
Sumimoto
H
,
Imabayashi
F
,
Iwata
T
,
Kawakami
Y
. 
The BRAF-MAPK signaling pathway is essential for cancer-immune evasion in human melanoma cells
.
J Exp Med
2006
;
203
:
1651
6
.
35.
Ratnam
S
,
Kent
C
. 
Early increase in choline kinase activity upon induction of the H-ras oncogene in mouse fibroblast cell lines
.
Arch Biochem Biophys
1995
;
323
:
313
22
.
36.
Huang
SC
,
Yu
DC
,
Barrio
JR
,
Grafton
S
,
Melega
WP
,
Hoffman
JM
, et al
Kinetics and modeling of L-6-[18F]fluoro-dopa in human positron emission tomographic studies
.
J Cereb Blood Flow Metab
1991
;
11
:
898
913
.
37.
Tomasi
G
,
Bertoldo
A
,
Bishu
S
,
Unterman
A
,
Smith
CB
,
Schmidt
KC
. 
Voxel-based estimation of kinetic model parameters of the L-[1-(11)C]leucine PET method for determination of regional rates of cerebral protein synthesis: validation and comparison with region-of-interest-based methods
.
J Cereb Blood Flow Metab
2009
;
29
:
1317
31
.