Nucleoside transporters in PET imaging of proliferating cancer cells using 3 ꞌ-deoxy-3 ꞌ-[ 18 F ] fluoro-L-thymidine

The movement of physiologic nucleosides and nucleoside analogue drugs across biological membranes is mediated by nucleoside transport proteins. In cancer, nucleoside transporters have an important role in maintaining the hyperproliferative state of tumours and are important targets for diagnostic and therapeutic agents in the detection, treatment and monitoring of cancers. The nucleoside-based probe 3ꞌ-deoxy-3ꞌ-[F]fluoro-Lthymidine ([F]FLT) has been developed for PET imaging of proliferating cancer cells, which is less prone than 2deoxy-2-[F]fluoro-D-glucose ([F]FDG) to non-specific effects. [F]FLT enters proliferating cells through nucleoside transporters, then becomes phosphorylated and blocks DNA synthesis, whilst also becoming trapped inside the cell. Practicable and automated chemical syntheses of [F]FLT have been developed, for which the most widely used radiolabelling precursor is the thymidine derivative 3-N-boc-5ꞌ-O-dimethoxytrityl-3ꞌ-O-nosylthymidine. [F]FLT PET imaging has undergone feasibility studies and has been assessed in pre-clinical and clinical studies for the detection and diagnosis of cancers and in monitoring their response to treatments. The roles of nucleoside transporters, especially ENT1, in the cellular uptake of [F]FLT have been investigated.

in the treatment of cancer and viral diseases.In the case of cancer, nucleoside transport has an important role in maintaining the hyperproliferative state of most tumours and is therefore an important target for diagnostic and therapeutic agents in the detection, treatment and monitoring of cancers.Indeed, the clinical efficacy of anticancer nucleoside analogue drugs depends on a complex interdependence of transporters mediating entry of drugs into cells, efflux mechanisms that remove drugs from intracellular compartments and cellular metabolism to active metabolites [1][2][3][4][5][6].
In humans, two solute carrier gene families (SLC28 and SLC29) are foremost responsible for the uptake of nucleosides and nucleoside analogues into cells [7][8][9][10][11].The SLC28 human concentrative nucleoside transporter (hCNT) family contains three members that mediate unidirectional transport of nucleosides into cells against their concentration gradient driven by a downward sodium
The pyrimidine nucleoside analogue gemcitabine (2',2'difluorodeoxycytidine, trade name Gemzar) (1) is widely used as a first-line chemotherapeutic drug in the treatment of various cancers including bladder cancer, breast cancer, non-small cell lung cancer, ovarian cancer and pancreatic cancer.Unfortunately, there is often rapid development of either de novo or induced drug resistance, which significantly limits the effectiveness of gemcitabine chemotherapy.
Whilst the cytotoxic effects of gemcitabine are exerted following phosphorylation and then inhibition of DNA synthesis, it must first enter cells through nucleoside transporters, especially the ubiquitous hENT1.Hence, hENT1 expression and activity has been identified as an important prognostic biomarker in gemcitabine-treated cancers and therefore as a predictive biomarker of gemcitabine efficacy.This is particularly true of pancreatic cancer, where high expression of hENT1 is associated with increased overall survival and disease-free survival in patients treated with gemcitabine [26][27][28][29][30].It therefore follows that a deficiency in hENT1 confers resistance to the cytotoxicity of gemcitabine [31][32][33][34] and approaches have been explored to overcome hENT1 deficiency.For example, upregulation of hENT1 expression in pancreatic cell lines by indole-3-carbinol enhanced the efficacy of gemcitabine [35].Functionalised lipophilic nanoparticles have also been developed for delivery of gemcitabine into cells that bypass nucleoside transporters [36,37].In addition to gemcitabine, other nucleoside analogues have been used and explored as chemotherapeutic drugs [38][39][40][41].

Overview
Positron emission tomography (PET) is a non-invasive clinical nuclear medicine technique routinely used to produce two-or three-dimensional images of the body for diagnosing and monitoring a wide range of human diseases.The PET system detects pairs of gamma rays emitted indirectly by a short-lived positron emitting radionuclide (or radiotracer), which is introduced into the body on a biologically active molecule [42].Because PET images directly reflect in vivo tissue physiology and metabolism, one of their foremost uses is in the detection of proliferating cancer cells and monitoring their response to treatments.Indeed, the early metabolic changes associated with cancers can be detected by PET imaging before more advanced morphologic changes are detected by anatomic imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI).This allows earlier diagnosis and earlier intervention with appropriate treatments that are more likely to have a successful outcome.By far the most commonly used radiotracer in PET imaging is 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG) (2) [42,43].In the case of imaging cancers, however, [ 18 F]FDG is not necessarily the most appropriate radiotracer to use because it can accumulate nonspecifically to produce false-positive findings [44].For example, enhanced uptakes of [ 18 F]FDG also occur in infection and in inflamed cells and lesions as well as in necrotic cells [45,46].Alternative nucleoside-based probes that are less prone to non-specific effects have have therefore been developed for imaging tumour proliferation to use alongside [ 18 F]FDG [44,47], the most successful being 3ꞌ-deoxy-3ꞌ-[ 18 F]fluoro-L-thymidine ([ 18

Cellular trapping of [ 18 F]FLT
Like thymidine (4), [ 18 F]FLT is transported into cells by nucleoside transporters.Once inside the cell, [ 18 F]FLT is a substrate for thymidine kinase I (TK1) and is phosphorylated but is not incorporated into DNA.Phosphorylated [ 18 F]FLT cannot exit the cell and [ 18 F]FLT is not a substrate for thymidine phosphorylase and so is not significantly degraded in vivo and is retained inside the cell (Figure 1).TK1 is a key enzyme that is upregulated in cancer cells and, in agreement with separate studies [49,50], it is assumed that the concentration of [ 18 F]FLT inside cells is proportional to TK1 activity and therefore to cellular proliferation.One of the characteristics of tumour cells is an unchecked proliferation and it is important to measure the proliferation rate of cancer lesions to help differentiate benign from malignant tumours and to characterise malignant tumours amongst normal tissues.A further advantage of [ 18 F]FLT is that it is only a substrate for TK1 and not for mitochondrial TK2, making it a more specific radiotracer compared with other fluorinated nucleoside analogues for cellular proliferation.In the thymidine salvage pathway, both thymidine (4) and [ 18 F]FLT (3) undergo uptake into cells by nucleoside transporters (NT) and are initially phoshorylated by thymidine kinase 1 (TK1) and then further phosphorylated by thymidine monophosphate kinase (TMPK) and thymidine diphosphate kinase (TDPK).There is also a de novo synthesis of TMP by thymidylate synthase (TS) from deoxyuridine monophoshate (dUMP).Whilst phosphorylated thymidine is incorporated into DNA, phosphorylated [ 18 F]FLT is not a substrate for DNA polymerase (DP) or nucleoside transporters and therefore becomes trapped inside the cell.Similarly, [ 18 F]FLT is not a substrate for thymidine phosphorylase (TP) and so does not undergo significant degradation to thymine and ribose-1-phosphate (R1P).[ 18 F]FLT is ultimately metabolised to its glucuronide by glucuronyl transferase in the liver and excreted by the kidney.Some cancer drugs inhibit the glucuronosyl transferase reaction, however.

Synthesis and quality control of [ 18 F]FLT
Radiosynthesis of [ 18 F]FLT was first reported by Wilson et al. [51] using a thymidine precursor (5) with trityl and mesyl protecting groups at the 5ꞌ-and 3ꞌ-hydroxyl positions, respectively, and this was treated with [ 18 F]potassium fluoride (Scheme 1).Significant developments towards a more practicable method to produce [ 18 F]FLT for clinical PET imaging were later made by Grierson and Shields [52][53][54][55].Their improved method made minimal use of specialised materials and apparatus and included a threestep radiosynthesis producing [ 18 F]FLT with a radiochemical yield (at end of bombardment) of 13% and an end of synthesis yield of 7% over 94 minutes [56].The method used a nosylate (4-nitrobenzenesulphonate) ester as the radiolabelling precursor ) that was synthesised in seven steps from thymidine (4) in an overall yield of 17% (Scheme 2).Nucleophilic displacement of ( 12) with [ 18 F]fluoride was followed by deprotection with ceramic ammonium nitrate (CAN) and then product isolation by C-18 preparative HPLC.Use of CAN for deprotection resulted in formation of precipitates such that filtration was required before HPLC, which is not conducive with synthesis automation.An alternative approach using an anhydro derivative as the radiolabelling precursor [2,3′-anhydro-5′-O-(4,4′-dimethoxytrityl)thymidine] ( 14) achieved an end of synthesis [ 18 F]FLT yield of 5.6 ± 1.4% over 90-140 minutes [57].Whilst this method had a simpler precursor synthesis, radiolabelling reaction and workup of [ 18 F]FLT, the requirement for a high boiling point reaction solvent (DMSO) compromised HPLC isolation of [ 18 F]FLT.Using the same anhydro precursor ( 14) and single neutral alumina column purification, a fully automated and simplified synthesis of [ 18 F]FLT produced an uncorrected radiochemical yield of 8.48 ± 0.93% (n = 5) in a total time of 68 ± 3 minutes and a radiochemical purity of >95% [58].This method obviated the need for HPLC purification and the product was tested for safe levels of residual aluminium and DMSO.
The nosylated precursors proved most successful for radiolabelling with 18 F and best results were obtained using 3-N-boc-5′-O-dimethoxytrityl-3′-O-nosyl-thymidine (15) with an [ 18 F]FLT yield (at end of bombardment) of 19.8% and an end of synthesis yield of 11.7% over 85 minutes.Using the same radiolabelling precursor (15), various [ 18 F]fluorination and purification conditions were assessed for achieving a higher radiochemical yield of [ 18 F]FLT [60].Purification of the reaction mixture using an Alumina N Sep-Pak cartridge before HPLC application significantly increased the radiochemical yield to 42 ± 5.4% (decaycorrected) in under 60 min with a radiochemical purity of >97%.Again using precursor 15, a fully automated method for synthesis of [ 18 F]FLT was developed by modifying a commercial synthesiser for [ 18 F]FDG that uses disposable cassettes [61].[ 18 F]FLT yields (decay corrected) of 50.5 ± 5.2% (n = 28) and 48.7 ± 5.6% (n = 10) were obtained using 3.7 and 37.0 GBq of [ 18 F]fluoride starting activity, respectively, in 60.0 ± 5.4 minutes including HPLC isolation.A simplified and fully automated synthesis of [ 18 F]FLT was developed using a PET-MF-2V-IT-I [ 18 F]FDG synthesis module by a one-pot two-step reaction procedure.The method included nucleophilic fluorination of 15 with [ 18 F]fluoride, followed by hydrolysis of the protecting group with 1.0 M HCl in the same reaction vessel and purification with SEP PAK cartridges instead of HPLC [62].The corrected [ 18 F]FLT radiochemical yield was 23.2 ± 2.6% (n = 6) and the radiochemical purity was >97% obtained in a total time of 35 minutes.It was also discovered that nucleophilic fluorination of 15 using a protic solvent produced an improved radiochemical yield of [ 18 F]FLT.Reaction in t-butanol using an automated synthesis module led to an [ 18 F]FLT radiochemical yield of 60.2 ± 5.2% after HPLC purification [63].
The 3-N-Boc-protected compound 15 remains the most commonly used radiolabelling precursor for [ 18 F]FLT synthesis and is commercially available at GMP grade.Indeed, [ 18 F]FLT suitable for microPET studies has been efficiently synthesised from 15 using an electrowetting-ondielectric digital microfluidic chip [64] and an automated and efficient radiosynthesis of [ 18 F]FLT using a low amount of 15 (5 mg) has been developed, achieving a corrected radiochemical yield of 54% in a time of 52 minutes [65].
It is clear that the radiosynthesis of [ 18 F]FLT can lead to many complex and potentially toxic side-products.According to the Society of Nuclear Medicine and Molecular Imaging (SNMMI), the NIH requires an [ 18 F]FLT radiochemical purity of no less than 95% and no more than 5 mcg of nonradioactive FLT and no more than 5 mcg of other UV-absorbing impurities http://interactive.snm.org/docs/PET_PROS/FLT_07-11-12%20Final.pdf.[ 18 F]FLT is therefore subject to stringent tests of quality control and biological assessment [66,67].

Feasibility studies for measuring tumour proliferation using [ 18 F]FLT
Early prospective and feasibility studies were highly supportive of [ 18 F]FLT as a PET radiotracer for measuring tumour proliferation, many of which performed direct comparisons of [ 18 F]FLT and [ 18 F]FDG uptake and correlations with immunohistochemistry results.For example, in a validation study for [ 18 F]FLT PET imaging of proliferation in early stage non-small cell lung cancer, there was excellent correlation of [ 18 F]FLT uptake with immunohistochemistry marker of cell proliferation Ki-67 values and flow cytometry results [68].In a separate prospective PET study of newly diagnosed lung nodules, [ 18 F]FLT uptake correlated significantly better with proliferation of lung tumours than did uptake of [ 18 F]FDG, suggesting that it might be more useful as a selective biomarker for tumour proliferation [69].PET imaging of cell proliferation in colorectal cancer using [ 18 F]FLT and [ 18 F]FDG showed a statistically significant positive correlation between SUVs of tumours visualised with [ 18 F]FLT and the corresponding immunohistochemistry results, whilst no such correlation was demonstrated with [ 18 F]FDG avid lesions [70].In a study investigating the feasibility of [ 18 F]FLT PET imaging for detection and grading of soft tissue sarcoma at the extremities, the method was successful in visualising cell proliferation and in differentiating between low-grade and high-grade lesions (Figure 2).The uptake of [ 18 F]FLT correlated with the proliferation of soft tissue sarcoma [71].In a comparative study for imaging laryngeal cancer with [ 18 F]FLT and [ 18 F]FDG, the numbers of cancers detected with both tracers were equal and the uptake of [ 18 F]FDG was higher than that of [ 18 F]FLT [72].In a study that directly compared [ 18 F]FLT and [ 18 F]FDG for imaging proliferation in brain tumours of the same patients, [ 18 F]FLT was more sensitive than [ 18 F]FDG for imaging recurrent high-grade tumours (Figure 3), it correlated better with immunohistochemistry Ki-67 values and was a more powerful predictor of tumour progression and survival [73].More recently, a study investigating the performance of cellular metabolism imaging with [ 18 F]FDG versus cellular proliferation imaging with [ 18 F]FLT for detecting cervical lymph node metastases in oral/head and neck cancer was performed.
Whilst [ 18 F]FLT showed better overall performance for detecting lymphadenopathy on qualitative assessment within the total nodal population, [ 18

NUCLEOSIDE TRANSPORTERS IN [ 18 F]FLT PET IMAGING OF CANCER
A number of studies have investigated the roles of nucleoside transporters, especially hENT1, in measuring cell proliferation of cancers using [ 18 F]FLT PET.One of the first studies implicating a role for ENT1 in uptake of [ 18 F]FLT into cancer cells was an assessment of [ 18 F]FLT PET imaging for early measurement of thymidylate synthase inhibition in tumours [81].Radiation-induced fibrosarcoma-1 tumor-bearing mice, injected with the thymidylate synthase inhibitor 5-fluorouracil, were imaged by [ 18 F]FLT PET 1 to 2 hours after treatment (Figure 4).Parallel measurements included whole-cell assays implicating a functional role for ENT1, in which there was an increase in ENT1-binding sites per cell from 49,110 in untreated cells to 73,142 (P = 0.03) in cells treated with 5fluorouracil (10 g/ml, 2 hours), without a change in transporter affinity (P = 0.41).It was concluded that [ 18 F]FLT PET can measure thymidylate synthase inhibition as early as 1 to 2 hours after treatment with 5-fluorouracil by a mechanism involving redistribution of ENT1to the plasma membrane [81].
In a study specifically designed to investigate roles of human nucleoside transporters in uptake of FLT [82], binding of FLT to transporters was initially monitored by its inhibitory effects on [ 3 H]uridine (1 M) uptake in yeast cells producing recombinant transporters.The lowest FLT Ki value for inhibition of [ 3 H]uridine uptake was produced by hCNT1, followed by hCNT3, hENT2, hENT1 and hCNT2.Transport of [ 3 H]FLT (20 M) into Xenopus laevis oocytes individually producing recombinant nucleoside transporters produced uptake values of 48 ± 8, 32 ± 5, 12 ± 1, 11 ± 0.8 and 2.0 ± 0.2 pmol/oocyte/30 min for hCNT1, hCNT3, hENT2, hENT1 and hCNT2, respectively (Figure 5A).Transport of [ 3 H]FLT by hENT1, hENT2, hCNT1 and hCNT3 was concentration-dependent and conformed to Michaelis-Menten kinetics (Figure 5B).hENT1 and hENT2 produced higher transport capacities and lower apparent affinities than hCNT1 and hCNT3.The transport efficiency (Vmax/Km) was approximately 6-fold greater for hCNT1 and hCNT3 than for hENT1 and hENT2, suggesting that hCNT1 and hCNT3 transport [ 3 H]FLT more efficiently than hENT1 and hENT2 at lower (micromolar) concentrations [82].[ 3 H]FLT uptake in six different cancer cell lines was inhibited at least 50% by the hENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR) and, according to real-time polymerase chain reactions, hENT1 and hENT2 had the most abundant nucleoside transporter transcripts in all cell lines.
Further binding assays demonstrated a strong correlation between extracellular NBMPR binding sites/cell and [ 3 H]FLT uptake for all but one of the cell lines, consistent with plasma membrane nucleoside transporters (especially hENT1) having important roles in cellular FLT uptake [82].A subsequent study investigated the importance of ENT1 for [ 18 F]FLT uptake in normal tissues and tumours [83].ENT1-knockout (ENT1(-/-)) mice were compared with wild-type (ENT1(+/+)) mice using small-animal [ 18 F]FLT PET in absence and presence of NBMPRphosphate (Figure 6).
A parallel study was performed to determine if FLT uptake is a predictor of gemcitabine uptake and/or toxicity in a panel of six different human pancreatic cancer cell lines (Capan-2, AsPC-1, BxPC-3, PL45, MIA PaCa-2 and PANC-1) [84].Capan-2 cells displayed the lowest levels of extracellular NBMPR binding, FLT and gemcitabine uptake during short (1-45 seconds) and prolonged (1 hour) periods, and gemcitabine sensitivity.Exposure to NBMPR (inhibits only hENT1) or dilazep (inhibits hENT1 and hENT2) reduced FLT and gemcitabine uptake and gemcitabine sensitivity, with dilazep having greater effects than NBMPR.Gemcitabine permeation was primarily mediated by hENT1, and to a lesser extent by hENT2, whilst FLT permeation included a substantial component of passive diffusion [84].In five out of six cell lines, correlations were observed between FLT and gemcitabine initial rates of uptake, gemcitabine uptake and gemcitabine toxicity, FLT uptake and gemcitabine toxicity, and ribonucleotide reductase subunit M1 expression and gemcitabine toxicity.Uptakes of FLT and gemcitabine were comparable for predicting gemcitabine toxicity in the tested pancreatic cancer cell lines, it was therefore concluded that [ 18 F]FLT may provide clinically useful information about tumour gemcitabine transport capacity and sensitivity [84].
In a study investigating the correlation of [ 18 F]FLT uptake with mRNA expressions of hENT1 and TK1 in tissue samples from newly diagnosed gastrointestinal cancers, of all lesions tested only one gastric cancer showed focally increased uptake of [ 18 F]FLT.The mean [ 18 F]FLT SUV in gastrointestinal cancer was 5.48 ± 1.87.No significant correlation was observed between [ 18 F]FLT SUV and hENT1 mRNA expression (P = 0.90), whilst there was a significant correlation between [ 18 F]FLT SUV and TK1 mRNA expression (P <0.05) [85].

Saidijam et al.
Isolated human B-lymphobast cells, either proficient or deficient in TK1, were studied to show how metabolism and nucleoside transport influence uptake and retention of FLT [86].Both influx and efflux of FLT were measured under conditions where concentrative and equilibrative transport could be distinguished.Whilst initial rates of FLT uptake were a function of both concentrative and equilibrative transporters, concentrative FLT transport dominated over equilibrative transport.Inhibition of hENT1 reduced FLT uptake, but there were no correlations between clonal variations in hENT1 levels and FLT uptake.TK1 was mandatory for the cellular concentration of FLT and uptake peaked after 60 minutes of incubation with FLT, followed by a decline in intracellular levels of FLT and its metbolites.Efflux was rapid and was associated with reductions in FLT and its metabolites [86].
In a study examining the extent to which ENT1 levels vary in a proliferation-dependent manner in human A549 tumor cells grown as tumor xenografts in nude mice, [ 18 F]FLT uptake was measured in vivo using small animal PET and further examined ex vivo using autoradiography [87].[ 18 F]FLT uptake patterns were also compared to immunohistochemical analysis of ENT1 and the proliferation markers Ki67 and BrdU.ENT1 levels were approximately twice as high in actively proliferating regions of tumours grown in vivo.Proliferating regions showed increased [ 18 F]FLT uptake compared with nonproliferating tumour regions, hence confirming the role of hENT1 in [ 18 F]FLT uptake and strengthening the case for using [ 18 F]FLT as a tracer for both cell proliferation and relative ENT1 levels [87].
A later study investigated whether uptake of [ 18 F]FLT in newly diagnosed gliomas correlates with ENT1 mRNA expression, microvascular density (assessed by CD34 immunohistochemistry) and blood-brain barrier permeability [88].In tumour lesions identified by increased [ 18 F]FLT uptake, dynamic analysis revealed correlations between the phosphorylation rate constant k3 and ENT1 expression, but there was no correlation between the kinetic parameters and CD34 score.
Good correlation was observed between the gadolinium (Gd) enhancement score (evaluating blood-brain barrier breakdown) and ENT1 expression, CD34 score and Ki-67 index.It was therefore concluded that ENT1 expression might not reflect accumulation of [ 18 F]FLT in vivo due to blood-brain barrier permeability in glioma [88].
TAS-102 is a recently developed orally administered combination chemotherapy drug composed of α,α,αtrifluorothymidine (TFT) and a thymidine phosphorylase inhibitor (tipiracil hydrochloride, TPI) in a 1:0.5 ratio.TAS-102 has especially been targeted at metastatic colorectal cancer [89][90][91][92][93][94].In the mechanism of action of TAS-102, TFT is intracellularly phosphorylated and then incorporated into DNA, which leads to DNA damage and cell cycle arrest.TPI is an inhibitor of thymidine phosphorylase that metabolises TFT, therefore increasing the bioavailability of TFT, and TPI is also an inhibitor of angiogenesis.hCNT1 has a major role in intestinal absorption of TFT and, when expressed in Xenopus laevis oocytes, uptake of TFT by hCNT1 has Km and Vmax values of 69.0 μM and 516 pmol/oocyte/30 min, respectively [95].
In human colon cancer xenografts in mice, administration of TAS-102 imparted a decrease in cell viability and an increase in [ 18 F]FLT uptake.
Early after TAS-102 administration there may be decreased dephosphorylation of [ 18 F]FLT and, at a later time, increased TK1 expression and/or nucleoside transporter activity may be related to increased [ 18 F]FLT uptake.Hence, [ 18 F]FLT PET is potentially useful for assessing the pharmacodynamics of TAS-102 in cancer patients [96].

CONCLUSIONS
The nucleoside analogue [ 18 F]FLT is emerging as a feasible radiotracer for routine PET imaging, especially in the detection and monitoring of cancers.The important advantage of [ 18 F]FLT is that it suffers from a lower nonspecific background uptake than the established and widely used radiotracer [ 18 F]FDG.Practicable and automated chemical syntheses of [ 18 F]FLT have been developed, for which the most widely used radiolabelling precursor is the thymidine derivative 3-N-boc-5ꞌ-O-dimethoxytrityl-3ꞌ-Onosyl-thymidine.
[ 18 F]FLT enters proliferating cells through nucleoside transporters, which are also routes of entry into cells for anti-cancer and anti-viral nucleoside analogue drugs.The roles of nucleoside transporters, especially ENT1, in the cellular uptake of [ 18 F]FLT have been investigated.Further studies on structure-activity relationships and regulation of nucleoside transporters are necessary for improving the design and delivery of nucleoside analogue drugs and for ongoing developments in PET imaging of cancers and other diseases.

CONFLICTS OF INTEREST
The authors report no conflicts of interest.

Figure 1 .
Figure1.Cellular uptake and fate of thymidine and [ 18 F]FLT.In the thymidine salvage pathway, both thymidine (4) and [ 18 F]FLT (3) undergo uptake into cells by nucleoside transporters (NT) and are initially phoshorylated by thymidine kinase 1 (TK1) and then further phosphorylated by thymidine monophosphate kinase (TMPK) and thymidine diphosphate kinase (TDPK).There is also a de novo synthesis of TMP by thymidylate synthase (TS) from deoxyuridine monophoshate (dUMP).Whilst phosphorylated thymidine is incorporated into DNA, phosphorylated [ 18 F]FLT is not a substrate for DNA polymerase (DP) or nucleoside transporters and therefore becomes trapped inside the cell.Similarly, [ 18 F]FLT is not a substrate for thymidine phosphorylase (TP) and so does not undergo significant degradation to thymine and ribose-1-phosphate (R1P).[ 18 F]FLT is ultimately metabolised to its glucuronide by glucuronyl transferase in the liver and excreted by the kidney.Some cancer drugs inhibit the glucuronosyl transferase reaction, however.

Figure 2 .
Figure 2. Images of soft tissue carcinomas. A. MRI (A1) and [ 18 F]FLT (A2) images of a low-grade soft tissue sarcoma.B. MRI (B1) and [ 18 F]FLT (B2) images of a high-grade soft tissue sarcoma.The MRI images of both patients demonstrate a heterogeneous tumour.[ 18 F]FLT uptake in the high-grade soft tissue sarcoma is higher than in the low-grade soft tissue sarcoma, however.This figure was reproduced with permission from Cobben et al. (2004) [71]; copyright © 2004 by American Association for Cancer Research.

Figure 3 .
Figure 3. Images of a newly diagnosed glioblastoma.A. MRI image (contrast-enhanced T1-weighted image) showing a large area of contrast enhancement in the right frontal lobe.Both [ 18 F]FDG PET (B) and [ 18 F]FLT PET (C) show increased uptake in same area.This figure was reproduced with permission from Chen et al. (2005) [73]; copyright © 2005 by Society of Nuclear Medicine and Molecular Imaging.

F]FDG 15 =
Saidijam et al. performed better for pathologic discrimination within the visible lymph nodes [74].[ 18 F]FLT PET imaging has been assessed in a range of further pre-clinical and clinical studies for the detection and diagnosis of cancers and in monitoring their response to treatments.A comprehensive overview of these studies is beyond the scope of this work, so the reader is referred to recent review articles on this theme [75-80].

Figure 4 .
Figure 4. [ 18 F]FLT PET imaging of thymidylate synthase inhibition in tumours.Typical 0.5-mm transverse [ 18 F]FLT PET slices through the thoracic region at the level of the maximum tumour diameter of a RIF-1 tumour-bearing mouse treated with PBS (control; A) and a RIF-1 tumourbearing mouse treated with 5-fluorouracil (B).Arrows = tumour.C. Summary of [ 18 F]FLT kinetics in control (•) and 5-fluorouracil-treated (○) RIF-1 tumours.Tumour-bearing mice were treated with PBS or 5fluorouracil at a dose of 165 mg/kg i.p. and scanned at 1 to 2 hours after injection.For each mouse, tumour/heart radioactivity ratios from five slices were averaged at each of the 19 time points.Data points represent mean tumour/heart ratios from eight control mice and five 5-fluorouraciltreated mice; error bars represent standard errors.This figure was reproduced with permission from Perumal et al. (2006) [81]; copyright © 2006 by American Association for Cancer Research.

Figure 5 .
Figure 5. Uptake of FLT by human nucleoside transporters.A. Uptake of [ 3 H]thymidine and [ 3 H]FLT (20 M) in Xenopus laevis oocytes producing different recombinant human nucleoside transport proteins.B. Concentration-dependent influx of [ 3 H]FLT in oocytes producing human nucleoside transport proteins with inset kinetic parameters.All experiments were performed with 12 oocytes per group and data are expressed as mean S.E.M. Error bars are not shown if the S.E.M. values were smaller than the size of the symbol.Values are for mediated uptake (uptake in RNA transcript-injected oocytes minus uptake in control oocytes injected with water alone).Pictures were reproduced with permission from Paproski et al. (2008) [82]; copyright © 2008 by American Society for Pharmacology and Experimental Therapeutics.