Development and Validation of [18f](2 S,4 R)-4-Fluoroglutamine in Multiple Myeloma Mouse Models
Authors: Toscani Denise , Valtorta Silvia , Chiu Martina , Sartori Andrea , Coliva Angela , Brevi Arianna , Taurino Giuseppe , Grioni Matteo , Raimondi Vincenzo , Burroughs-Garcia Jessica , Storti Paola , Ruffini Livia , Vacondio Federica , Zanardi Franca , Bellone Matteo , Moresco Rosa Maria , Bussolati Ovidio , Giuliani Nicola 
Published: November 2021

Abstract
Glutamine (Gln) addiction has been recently described as a typical metabolic feature of MM by our group. In order to sustain high Gln demand, MM cells upregulate the expression of the Gln transporters ASCT2, SNAT1 and LAT1 and are characterized by fast Gln uptake. Currently, 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) is the standard tracer for the positron emission tomography/computed tomography (PET/CT) scan to detect both medullary and extramedullary disease in MM patients. However, FDG-PET has some limitations, since there is a portion of MM patients who are false-negative. For these reasons, the aim of our study is to characterize Gln metabolism in vivo in different MM mouse models and investigate the possible use of Gln as a PET tracer in MM.
To this purpose, we have firstly synthesized enantiopure (2 S,4 R)-4-fluoroglutamine (4-FGln) and validated it in human MM cell lines (RPMI8226 and JJN3) comparing its uptake with that of 3H-labelled Gln. Both Gln and 4-FGln were actively accumulated by MM cells. Inhibition analysis revealed that ASCT2 was the major entry route of both compounds.
We then carried out the radiosynthesis of [18F]4-FGln and tested its uptake for MM detection by PET in comparison with [18F]FDG. Firstly, we characterized [18F]4-FGln kinetics using immunodeficient NOD/SCID mice subcutaneously injected with JJN3. In this xenograft model, [18F]FDG- and [18F]4-FGln-PET were performed after plasmacytomas became palpable. In tumor, [18F]4-FGln uptake peaked at 25 min slowly declining thereafter. The Logan plot confirmed linearity starting at 15 min from injection, consistent with largely reversible tracer exchange.
Thereafter, [18F]FDG and [18F]4-FGln distribution were assessed in both syngeneic (murine Vk12598 cells) and xenograft ( human JJN3 cells) MM models.
C57BL/6 mice were injected intravenously with Vk12598 cells obtained from transgenic Vk*MYC mice. Mice were monitored weekly for M-spike and MM progression by retro-orbital bleeding. At weeks 3, 4 and 5, mice underwent PET with [18F]FDG and [18F]4-FGln on consecutive days. The expression of ASCT2, checked in the femur of Vk12598 MM bearing mice, increased along with MM progression. Uptake of both tracers was measurable in the spleen of MM mice by week 3, reached a peak at week 4 and declined thereafter. In JJN3 model, all the tumors were positive for [18F]FDG and displayed [18F]4-FGln uptake (18F]4-FGln: tumor to muscle ratios (T/M): 1.6 ± 0.1; [18F]FDG: T/M: 3.5 ± 1.1). Tumor volume (896.8 ±349.00 mm3), [18F]4-FGln uptake and [18F]FDG uptake (T/M: 2.3 ± 0.3 and 7.1 ± 2.6, respectively) increased after 1 week ([18F]4-FGln p = 0.0156; [18F]FDG p = 0.0167).
Thereafter, the effect of bortezomib (BOR) was investigated to evaluate the potential use of [18F]4-FGln to monitor anti-MM treatment. JJN3-bearing mice were treated with BOR (1mg/kg) by intravenous injections twice weekly. Before and after treatment, animals performed the PET acquisitions with [18F]FDG and [18F]4-FGln on consecutive days and were sacrificed for post-mortem analysis on day 7. As expected, bortezomib treatment reduced tumor size compared with vehicles (172.0 ± 86.6 vs 687.9 ± 286.5 mm3; p = 0.0006).
PET analysis performed after BOR treatment showed that BOR significantly reduced the uptake of both radiopharmaceuticals in comparison with vehicles (p&lt; 0.05). BOR-treated mice were classified as responders and non responders according to the adapted RECIST score. Interestingly, responder mice showed a reduction of [18F]4-FGln T/M ratio and [18F]4-FGln-related tumor volume. On the contrary, all mice displayed increased [18F]FDG parameters independently from the response. Lastly, tracer overlap analysis showed that the area of relative exclusive uptake of [18F]4-FGln decreased in responders compared to non responders (9% versus 32%, respectively, p=0.04), while that of [18F]FDG increased in responders compared to non responders (82% versus 23%, respectively, p=0.04).
Our data indicate that [18F](2 S,4 R)-4-FGln is a new PET tracer in pre-clinical MM models, providing the rationale to design studies in MM patients.

Disclosures
Giuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: congress, Research Funding; Millenium Pharmaceutical: Other: clincial studies; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical studies, congress, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Other: clinical studies; Bristol Mayers Squibb: Other: congress.