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. 2017 Apr 11;8(15):24415-24428.
doi: 10.18632/oncotarget.14705.

Radiosynthesis and validation of (±)-[18F]-3-fluoro-2-hydroxypropionate ([18F]-FLac) as a PET tracer of lactate to monitor MCT1-dependent lactate uptake in tumors

Vincent F Van Hée  1 , Daniel Labar  2 , Gwenaël Dehon  3 , Debora Grasso  1 , Vincent Grégoire  2 , Giulio G Muccioli  3 , Raphaël Frédérick  3 , Pierre Sonveaux  1
Affiliations

Radiosynthesis and validation of (±)-[18F]-3-fluoro-2-hydroxypropionate ([18F]-FLac) as a PET tracer of lactate to monitor MCT1-dependent lactate uptake in tumors

Vincent F Van Hée et al. Oncotarget. .

Abstract

Cancers develop metabolic strategies to cope with their microenvironment often characterized by hypoxia, limited nutrient bioavailability and exposure to anticancer treatments. Among these strategies, the metabolic symbiosis based on the exchange of lactate between hypoxic/glycolytic cancer cells that convert glucose to lactate and oxidative cancer cells that preferentially use lactate as an oxidative fuel optimizes the bioavailability of glucose to hypoxic cancer cells. This metabolic cooperation has been described in various human cancers and can provide resistance to anti-angiogenic therapies. It depends on the expression and activity of monocarboxylate transporters (MCTs) at the cell membrane. MCT4 is the main facilitator of lactate export by glycolytic cancer cells, and MCT1 is adapted for lactate uptake by oxidative cancer cells. While MCT1 inhibitor AZD3965 is currently tested in phase I clinical trials and other inhibitors of lactate metabolism have been developed for anticancer therapy, predicting and monitoring a response to the inhibition of lactate uptake is still an unmet clinical need. Here, we report the synthesis, evaluation and in vivo validation of (±)-[18F]-3-fluoro-2-hydroxypropionate ([18F]-FLac) as a tracer of lactate for positron emission tomography. [18F]-FLac offers the possibility to monitor MCT1-dependent lactate uptake and inhibition in tumors in vivo.

Keywords: MCT inhibitors; cancer metabolism; metabolic symbiosis; monocarboxylate transporter 1 (MCT1); positron emission tomography (PET).

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Conflict of interest statement

CONFLICTS OF INTEREST

European patent application No EP 16188093.5 has been submitted based on the results described in this manuscript. The authors declare that they have no other conflicts of interest.

Figures

Figure 1
Figure 1. Oxidative human cancer cells do not trap (±)-[18F]-2-fluoropropionate in a MCT1-dependent manner
A. Potential analogues of lactate considered for the development of a tracer of lactate uptake. B. Scheme for the radiosynthesis of (±)-[18F]-2-fluoropropionate. C. In vitro assay for the uptake of (±)-[18F]-2-fluoropropionate by oxidative SiHa (left panel) and oxidative SQD9 (right panel) cancer cells. Cells were pretreated during 1 h by vehicle or MCT1 inhibitor AR-C155858 (10 μM) in DMEM containing 10% of dialyzed FBS and 10 mM of L-lactate. This was followed by a 6 min incubation in a modified KREBS solution containing 10 mM of L-lactate, (±)-[18F]-2-fluoropropionate (45 μCi/mL) and 10 mM of L-lactate ± AR C155858 (10 μM) (*** p < 0.005; N = 2, n = 8). D. Same as C, but using MCT1 inhibitor AZD3965 (10 μM) instead of AR-C155858 (*** p < 0.001; N = 2, n = 8).
Figure 2
Figure 2. Synthesis of (±)-[18F]-3-fluoro-2-hydroxypropionate ([18F]-FLac)
A. Scheme for the radiosynthesis of (±)-[18F]-3-fluoro-2-hydroxypropionate. B. Co-elution spectra of (±)-[18F]-benzyl 3-fluoro-2-hydroxypropionate and nonradioactive benzyl 3-fluoro-2-hydroxypropionate, and (±)-[18F]-2-fluoro-3-hydroxybenzylacrylate and nonradioactive 2-fluoro-3-hydroxybenzylacrylate on a Supelco Discovery C18 HPLC column equipped with UV and NaI γ-ray detectors. C. Elution spectrum of (±)-[18F]-3-fluoro-2-hydroxypropionate ([18F]-FLac) on a IonPac AS15 Dionex HPLC column equipped with a NaI γ-ray detector.
Figure 3
Figure 3. Oxidative human cancer cells trap [18F]-FLac in a MCT1-dependent manner
A. Representative western blots showing MCT1 and MCT4 expression in SQD9, SiHa and SiHa-CRISPR-MCT1 human cancer cells. B. Oxygen consumption rate (OCR) of SiHa and SQD9 cells on a Seahorse bioanalyzer. Cells received either glucose (25 mM) + L-lactate (10 mM) (black bars) or only L-lactate (10 mM) (white bars) as oxidative fuels in DMEM containing 10% of dialyzed FBS (* p < 0.05, *** p < 0.005; n = 8). C. Cancer cells (or empty wells; blanks) were pretreated for 1 h with vehicle or AR-C155858 (10 μM) in DMEM containing 10% of dialyzed FBS and 10 mM of L-lactate, then incubated during 6 min in a modified KREBS solution containing 10 mM of L-lactate in the presence of (±)-[18F]-3-fluoro-2-hydroxypropionate ([18F]-FLac; 45 μCi/mL) with or without AR-C155858, washed, and intracellular 18F activity was measured using a Wiper Gold γ-counter (* p < 0.05, *** p < 0.001; N = 2, n = 8). D. As in C, except using SiHa-WT and SiHa-CRISPR-MCT1 cells without pretreatment (*** p < 0.001; N = 2, n = 8). E. As in C, but using AZD3965 (10 μM) instead of AR-C155858 (ns, p > 0.05; *** p < 0.001; N = 2, n = 8).
Figure 4
Figure 4. MCT1 inhibitor AR-C155858 blocks the in vivo uptake of [18F]-FLac by human SiHa tumors in mice
Mice were bearing 2 SiHa tumors expressing a control shRNA (shCTR) or a shRNA against MCT1 (shMCT1). A. Representative images of vehicle-pretreated mice showing the physiological distribution of (±)-[18F]-3-fluoro-2-hydroxypropionate ([18F]-FLac) 10, 30 and 60 min after tail vein injection (215 μCi in 100 μL). Color scale is normalized for the injected dose and animal weight. B. Western blot showing MCT1, MCT4, CD147/basigin, β-actin and Hsp90 expression in SiHa cells infected with shCTR or shMCT1 (Representative of n = 3). C. Same as in A, except that mice were pretreated with AR-C155858 (5 mg/Kg IV 10 min before tracer injection). The representative image shows the exact same mouse as in A (30 min tracer image), assessed one day after. The bladder is indicated. D. Quantification of tracer uptake (30 min after tracer injection) based on PET scan images. A same group of mice was treated with vehicle (white bars) on one day and, on the day after, with AR-C155858 (5 mg/Kg IV 10 min before tracer injection) (black bars) (*** p < 0.001; N = 2, n = 6-7).
Figure 5
Figure 5. MCT1 inhibitors AR-C155858 and AZD3965 block the in vivo uptake of [18F]-FLac by human SQD9 tumors in mice
Mice were bearing a single SQD9 tumor. A. Top left panel: representative PET image of a mouse that was pretreated for 10 min with vehicle, then imaged 30 min after a tail vein injection of (±)-[18F]-3-fluoro-2-hydroxypropionate ([18F]-FLac) (215 μCi in 100 μL). Top right panel: two days after, representative image of the exact same mouse that was pretreated for 10 min with AZD3965 (5 mg/Kg), then imaged 30 min after a tail vein injection of [18F]-FLac (215 μCi in 100 μL). Color scale is normalized for the injected dose and animal weight. The bottom graph shows quantification of tracer uptake based on PET scan images, where white bars correspond to vehicle treatment and black bars to AR-C155858 treatment (*** p < 0.001: N = 1, n = 6). B. Top left panel: representative PET image of a mouse that was pretreated for 10 min with vehicle, then imaged 30 min after a tail vein injection of [18F]-FLac (215 μCi in 100 μL). Top right panel: two days after, representative image of the exact same mouse that was pretreated for 10 min with AZD3965 (5 mg/Kg), then imaged 30 min after a tail vein injection of [18F]-FLac (215 μCi in 100 μL). Color scale is normalized for the injected dose and animal weight. The bottom graph shows quantification of tracer uptake based on PET scan images, where white bars correspond to vehicle treatment and black bars to AZD3965 treatment (*** p < 0.001: N = 1, n = 6).
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