Background[18F]BF4−, the first 18F-labelled PET imaging agent for the sodium/iodide symporter (NIS), was produced by isotopic exchange yielding a product with limited specific activity (SA, ca. 1 GBq/μmol) posing a risk of sub-optimal target-to-background ratios (TBR) in PET images due to saturation of NIS in vivo. We sought to quantify this risk and to develop a method of production of [18F]BF4− with higher SA.MethodsA new radiosynthesis of [18F]BF4− was developed, involving reaction of [18F]F− with boron trifluoride diethyl etherate under anhydrous conditions, guided by 11B and 19F NMR studies of equilibria involving BF4− and BF3. The SA of the product was determined by ion chromatography. The IC50 of [19F]BF4− as an inhibitor of [18F]BF4− uptake was determined in vitro using HCT116-C19 human colon cancer cells expressing the human form of NIS (hNIS). The influence of [19F]BF4− dose on biodistribution in vivo was evaluated in normal mice by nanoPET imaging and ex vivo tissue counting.ResultsAn IC50 of 4.8 μΜ was found in vitro indicating a significant risk of in vivo NIS saturation at SA achieved by the isotopic exchange labelling method. In vivo thyroid and salivary gland uptake decreased significantly with [19F]BF4− doses above ca. 10 μg/kg. The new radiosynthesis gave high radiochemical purity (>99 %) and moderate yield (15 %) and improved SA (>5 GBq/μmol) from a starting activity of only 1.5 GBq.Conclusions[18F]BF4− produced at previously reported levels of SA (1 GBq/μmol) can lead to reduced uptake in NIS-expressing tissues in mice. This is much less likely in humans. The synthetic approach described provides an alternative for production of [18F]BF4− at higher SA with sufficient yield and without need for unusually high starting activity of [18F]fluoride, removing the risk of NIS saturation in vivo even in mice.Trial registrationISRCTN75827286.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-016-0188-5) contains supplementary material, which is available to authorized users.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

Anion transport by the human sodium-iodide symporter (hNIS) is an established target for molecular imaging and radionuclide therapy. Current radiotracers for PET of hNIS expression are limited to 124 I 2 and 18 F-BF 4 2 . We sought new 18 F-labeled hNIS substrates offering higher specific activity, higher affinity, and simpler radiochemical synthesis than 18 F-BF 4 2 . Methods: The ability of a range of anions, some containing fluorine, to block 99m TcO 4 2 uptake in hNIS-expressing cells was measured. SO 3 F 2 emerged as a promising candidate. 18 F-SO 3 F 2 was synthesized by reaction of 18 F 2 with SO 3 -pyridine complex in MeCN and purified using alumina and quaternary methyl ammonium solid-phase extraction cartridges. Chemical and radiochemical purity and serum stability were determined by radiochromatography. Radiotracer uptake and efflux in hNIS-transduced HCT116-C19 cells and the hNIS-negative parent cell line were evaluated in vitro in the presence and absence of a known competitive inhibitor (NaClO 4 ). PET/CT imaging and ex vivo biodistribution measurement were conducted on BALB/c mice, with and without NaClO 4 inhibition. Results: Fluorosulfate was identified as a potent inhibitor of 99m TcO 4 2 uptake via hNIS in vitro (half-maximal inhibitory concentration, 0.55-0.56 mM (in comparison with 0.29-4.5 mM for BF 4 2 , 0.07 mM for TcO 4 2 , and 2.7-4.7 mM for I 2 ). Radiolabeling to produce 18 F-SO 3 F 2 was simple and afforded high radiochemical purity suitable for biologic evaluation (radiochemical purity . 95%, decay-corrected radiochemical yield 5 31.6%, specific activity $ 48.5 GBq/mmol). Specific, blockable hNIS-mediated uptake in HCT116-C19 cells was observed in vitro, and PET/CT imaging of normal mice showed uptake in thyroid, salivary glands (percentage injected dose/g at 30 min, 563 6 140 and 32 6 9, respectively), and stomach (percentage injected dose/g at 90 min, 68 6 21). Conclusion: Fluorosulfate is a high-affinity hNIS substrate. 18 F-SO 3 F 2 is easily synthesized in high yield and very high specific activity and is a promising candidate for preclinical and clinical PET imaging of hNIS expression and thyroid-related disease; it is the first example of in vivo PET imaging with a tracer containing an S-18 F bond.

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