Abstract View

Objectives: Triple-negative breast cancer (TNBC) cells have increased amino acid (AA) uptake and are typically glutamine dependent. This metabolic phenotype creates new opportunities for imaging and targeted therapy. Understanding AA transport mechanisms in TNBC is valuable for developing transporter‑specific imaging and metabolic targeting approaches. (R)-3-[¹⁸F]fluoro-2-methyl-2-N-(methylamino) propanoic-acid ((R)-[¹⁸F]MeFAMP) is an analog of MeAIB, a selective system A inhibitor, while (2S,4R)4-[¹⁸F]Fluoroglutamine ([¹⁸F]FGln) is a glutamine analog potentially transported via multiple systems (e.g. systems A, L, ASC)¹. Both AA-targeted tracers have shown promising imaging properties in preclinical brain and breast tumor models. Here, we performed automated radiosynthesis and investigated in vitro time‑dependent uptake and transport of these tracers in a syngeneic murine TNBC cell, E0771. 

Methods: Radiosynthesis of (R)-[¹⁸F]MeFAMP and [¹⁸F]FGln was adapted from published procedures for Synthra module. Briefly, nucleophilic ring-opening of a cyclic sulfamidate precursor by [¹⁸F]fluoride and acid hydrolysis yielded (R)-[¹⁸F]MeFAMP². [¹⁸F]FGln was achieved by [¹⁸F]fluoride substitution of a (2S,4S)-tosylated precursor followed by acid hydrolysis of the resulting intermediate³. (R)-[¹⁸F]MeFAMP and [¹⁸F]FGln were purified using HPLC, Chirobiotic-TAG (0.2% HCOOH in water) and Luna-C18 (73.5% MeOH/26.5% H2O in 0.1% HCOOH), respectively.  In vitro cell uptake and transporter-specific studies were performed in E0771 cells at 1,5,30 and 60 min post-tracer addition. Competitive inhibition studies using AA transport inhibitors/substrates (10 mM): MeAIB for system A, BCH for system L and Ala/Ser/Cys for system ASC. Sodium containing and sodium free buffers assessed the contribution of sodium-dependent transporters (systems A and ASC) for [¹⁸F]FGln uptake.

Results: (R)-[¹⁸F]MeFAMP and [¹⁸F]FGln were synthesized with decay-corrected yield 52% and 12%, radiochemical and enantiomeric purity>96% and molar activities of >80 GBq/μmol and 4.1 GBq/μmol, respectively. (R)-[¹⁸F]MeFAMP uptake showed a time‑dependent increase, while [¹⁸F]FGln reached maximal uptake by 30 min and maintained uptake levels through 60 min. [¹⁸F]FGln had two-fold higher uptake at 60 min compared to (R)-[¹⁸F]MeFAMP (30.26 vs 12.23 %/mg protein). Two-way ANOVA revealed significant effects of time, tracer, and tracer-time interaction (p<0.0001). Transporter-inhibition studies showed (R)-[¹⁸F]MeFAMP uptake was significantly reduced by MeAIB (p<0.001), Ala/Ser/Cys (p<0.001) whereas BCH had no effect. [¹⁸F]FGln uptake was sodium-independent and insensitive to MeAIB while BCH (p<0.01) and ASC (p<0.001) induced significant reduction observed at 30 min. 

Conclusions: (R)-[¹⁸F]MeFAMP and [¹⁸F]FGln were synthesized in reasonable yields and high purity for biologicalapplications. Cell uptake demonstrated significant different in uptake kinetics, with (R)-[¹⁸F]MeFAMP showing a slower uptake with time‑dependent increase, while [¹⁸F]FGln exhibited rapid uptake and early stabilization. The uptake of (R)-[¹⁸F]MeFAMP was primarily mediated by system A in E0771 cells, whereas [¹⁸F]FGln was sodium‑independent and predominantly mediated by L transporter. These findings provide a mechanistic basis for transporter‑specific AA PET imaging in TNBC.

Acknowledgments: Radiological Society of North America, SNMMI Molecular Imaging Research Grant, UAB Radiology, School of Medicine, O’Neal Comprehensive Cancer Center, UAB small imaging facility (P30CA013148) and UAB cyclotron facility.

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