Glutaminolysis is a metabolic pathway adapted by many aggressive cancers, including triple-negative breast cancers (TNBC), to utilize glutamine for survival and growth. In this study, we examined the utility of [¹⁸F](2S,4R)4-fluoroglutamine ([¹⁸F]4F-Gln) PET to measure tumor cellular glutamine pool size, whose change might reveal the pharmacodynamic (PD) effect of drugs targeting this cancer-specific metabolic pathway. High glutaminase (GLS) activity in TNBC tumors resulted in low cellular glutamine pool size assayed via high-resolution ¹H magnetic resonance spectroscopy (MRS). GLS inhibition significantly increased glutamine pool size in TNBC tumors. MCF-7 tumors, with inherently low GLS activity compared with TNBC, displayed a larger baseline glutamine pool size that did not change as much in response to GLS inhibition. The tumor-to-blood-activity ratios (T/B) obtained from [¹⁸F]4F-Gln PET images matched the distinct glutamine pool sizes of both tumor models at baseline. After a short course of GLS inhibitor treatment, the T/B values increased significantly in TNBC, but did not change in MCF-7 tumors. Across both tumor types and after GLS inhibitor or vehicle treatment, we observed a strong positive correlation between T/B values and tumor glutamine pool size measured using MRS (r² = 0.71). In conclusion, [¹⁸F]4F-Gln PET tracked cellular glutamine pool size in breast cancers with differential GLS activity and detected increases in cellular glutamine pool size induced by GLS inhibitors. This study accomplished the first necessary step toward validating [¹⁸F]4F-Gln PET as a PD marker for GLS-targeting drugs. Cancer Res; 77(6); 1476–84. ©2017 AACR.

Glutamate is an important metabolite of glutaminolysis, a metabolic pathway used by many aggressive cancers, including triple-negative breast cancer (TNBC). With the exception of the brain, in vivo detection of glutamate in tissues using 1H magnetic resonance spectroscopy (MRS) is challenging. Compared with MRS, glutamate-weighted chemical exchange saturation transfer MR imaging (GluCEST MRI) offers a more sensitive detection mechanism that is free of glutamine interference. Here, we developed a robust, highly repeatable GluCEST MRI protocol in mice bearing human TNBC xenografts and treated with a potent glutaminase inhibitor, CB-839. In paired studies, treatment with CB-839 for 2 days reduced the GluCEST asymmetry value compared with baseline (P < 0.05, n = 10). The absolute change of the GluCEST asymmetry value was −2.5 percent points after CB-839 treatment versus +0.3 after vehicle (P < 0.01). Correspondingly, treatment with CB-839 reduced tumor glutamate concentrations by 1.5 mmol/L, consistent with prior calibration between changes of the GluCEST value versus tissue glutamate concentration; CB-839, however, did not change tumor intracellular pH. These results demonstrate in a mouse model of breast cancer the utility of GluCEST MRI to detect the early response to glutaminase inhibition. Significance: A sensitive method enables noninvasive detection of tumor response to inhibitors of glutamine metabolism. Cancer Res; 78(19); 5521–6. ©2018 AACR.

Low-density lipoprotein (LDL)-mimetic lipid nanoparticles (LNPs), decorated with MRI contrast agents and fluorescent dyes, were prepared by the covalent attachment of apolipoprotein-mimetic peptide (P), Gd(III)-chelate (Gd), and sulforhodamine B (R) moieties on the LNP surface. The functionalized LNPs were prepared using the amide-forming potassium acyltrifluoroborate (KAT) ligation reaction. The KAT groups on the surface of LNPs were allowed to react with the corresponding hydroxylamine (HA) derivatives of P and Gd to provide bi-functionalized LNPs (PGd-LNP). The reaction proceeded with excellent yields, as observed by ICP-MS (for B and Gd amounts) and MALDI-TOF-MS data, and did not alter the morphology of the LNPs (mean diameter: ca. 50 nm), as shown by DLS and cryoTEM analyses. With the help of the efficient KAT ligation, a high payload of Gd(III)-chelate on the PGd-LNP surface (ca. 2800 Gd atoms per LNP) was successfully achieved and provided a high r₁ relaxivity (r₁ = 22.0 s⁻¹ mM⁻¹ at 1.4 T/60 MHz and 25 °C; r₁ = 8.2 s⁻¹ mM⁻¹ at 9.4 T/400 MHz and 37 °C). This bi-functionalized PGd-LNP was administered to three atherosclerotic apoE^−/− mice to reveal the clear enhancement of atherosclerotic plaques in the brachiocephalic artery (BA) by MRI, in good agreement with the high accumulation of Gd in the aortic arch as shown by ICP-MS. The parallel in vivo MRI and ex vivo studies of whole mouse cryo-imaging were performed using triply functionalized LNPs with P, Gd, and R (PGdR-LNP). The clear presence of atherosclerotic plaques in BA was observed by ex vivo bright field cryo-imaging, and they were also observed by high emission fluorescent imaging. These directly corresponded to the enhanced tissue in the in vivo MRI of the identical mouse.

Resistance to chemotherapy is an important challenge in the clinical management of triple-negative breast cancer (TNBC). Utilization of the amino acid glutamine as a key nutrient is a metabolic signature of TNBC featuring high glutaminase (GLS) activity and a large pool of cellular glutamate, which mediates intracellular enrichment of cystine via xCT (SLC7A11) antiporter activity. To overcome chemo-resistant TNBC, we identified a strategy of dual metabolic inhibition of GLS and xCT to sensitize resistant TNBC cells to chemotherapy. We successfully tested this strategy in a human TNBC line and its chemoresistant variant in vitro and their xenograft models in vivo. Key findings of our study include: 1. Dual metabolic inhibition induced pronounced reductions of cellular glutathione accompanying significant increases of cellular superoxide level in both parent and resistant TNBC cells. While GLS and xCT inhibition did not directly kill cells via apoptosis, they potentiated doxorubicin (DOX) and cisplatin (CIS) to induce remarkably higher levels of apoptosis than DOX or CIS alone. 2. Although the resistant TNBC cells exhibited higher capacity to mitigate oxidative stress than the parent cells, their resistance was overcome by dual metabolic inhibition combined with DOX or CIS. 3. In vivo efficacy and safety of the triple combination (GLS and xCT inhibition plus DOX or CIS) were demonstrated in both chemo sensitive and resistant TNBC tumors in mice. In conclusion, GLS and xCT inhibition resulted in unmitigated oxidative stress due to depletion of glutathione, representing a promising strategy to overcome chemoresistance in glutamine-dependent TNBC.

Aggressive cancers such as triple-negative breast cancer (TNBC) avidly metabolize glutamine as a feature of their malignant phenotype. The conversion of glutamine to glutamate by the glutaminase (GLS) enzyme represents the first and rate-limiting step of this pathway, and a target for drug development. Indeed, a novel GLS inhibitor (GLSi) has been developed and tested in clinical trials, but with limited success, suggesting the potential for a biomarker to select patients that could benefit from this novel therapy. Here, we study a non-metabolized amino acid analog, ¹⁸F-Fluciclovine (Axumin®) as a PET imaging biomarker for detecting the pharmacodynamic response to GLSi. We show that glutamine transporters mediate the uptake of ¹⁸F-Fluciclovine into human breast cancer cells. To allow ¹⁸F-Fluciclovine PET to be performed in mice, citrate in the tracer formulation is replaced by PBS. Mice bearing TNBC (HCC38, HCC1806 and MBA-MD-231) and ER-positive (MCF-7) breast cancer xenografts were imaged with dynamic PET at baseline and after a 2-day treatment of GLSi (CB839 (Telaglenastat)) or vehicle. Kinetic analysis suggested reversible uptake of the tracer and the distribution volume (VD) of ¹⁸F-Fluciclovine was estimated by Logan plot analysis. A significant increase of VD was observed after CB839 treatment in TNBC models exhibiting high GLS activity (HCC38 and HCC1806), but not in TNBC or MCF-7 exhibiting low GLS. Changes of VD were corroborated with changes in GLS activity measured in CB839- versus vehicle-treated tumors, as well as with changes of VD of [¹⁸F]-(2S,R4)-fluoroglutamine, which we previously validated as a measure of cellular glutamine pool size. A moderate, albeit significant decrease of [¹⁸F]fluorodeoxyglucose (FDG) PET signal was observed in HCC1806 tumors after CB839 treatment. In conclusion, ¹⁸F-Fluciclovine PET has potential to serve as a clinical translatable, pharmacodynamic biomarker of GLSi.

Objectives: Evaluate the effects of pressure and duration of intracoronary (IC) infusion of mesenchymal stem cells (MSCs) on delivery efficiency and safety after myocardial infarction (MI). Background: Standard IC delivery of MSCs can lead to intravascular plugging and reduced coronary blood flow. The optimal delivery pressure and duration is unknown. Methods: Immediately after MI pigs were randomized to 1 of 3 delivery protocols of 5 × 10⁷ iron‐fluorescent microspheres labeled MSCs, control received 2 ml infusions at 1 ml/min (five times), very high flow rate (VHFR) a single 10 ml infusion at 60 ml/min and the high flow rate (HFR) group a single 10 ml infusion at 20 ml/min. TIMI grade flow was assessed throughout the procedure and at sacrifice (day 14). MSCs distribution was analyzed in isolated hearts by 4.7T MRI. Delivery efficiency was quantified via fluorescent microsphere recovery using a magnetic separation technique and by light microscopy. Results: TIMI grade flow did not change following MI (all groups TIMI 3). However, following MSCs delivery only 18% (2/11) of control animals had TIMI 3 blood flow vs. 56% (5/9) in VHFR and 67% (4/6) in HFR (P = 0.03). As a consequence, 63% of control animals died within 24 hr, 33% in VHFR and none in HFR (P = 0.02). MSCs delivery in the infarct tissue did not differ between the groups (P = 0.06). Conclusions: A single MSCs infusion at 20 ml/min resulted in improved coronary blood flow and decreased mortality, without sacrificing delivery efficiency.