Detection of inflammatory cell function using 13C MRS of hyperpolarized 13C-labeled arginine
Chloe Najac1, Myriam M Chaumeil1, Gary Kohanbash2, Caroline Guglielmetti3, Jeremy Gordon1, Hideho Okada2, and Sabrina M Ronen1

1Radiology and Biomedical Imaging, University of California San Francisco, San Francisco, CA, United States, 2Neurological Surgery, University of California San Francisco, San Francisco, CA, United States, 3Bio-Imaging Lab, University of Antwerp, Antwerpen, Belgium

Synopsis

Myeloid-derived suppressor cells (MDSCs) are inflammatory cells in the tumor microenvironment that inhibit T-cell-mediated immunosuppression by expressing high levels of arginase. Arginase catalyzes the breakdown of arginine into urea. To monitor the enzymatic conversion, we developed a new hyperpolarized (HP) probe, namely [guanido-13C]-arginine. We first characterized the probe and confirmed the production of HP 13C urea in solution with different arginase concentrations. Then, we demonstrated its potential to probe the increase in arginase activity in MDSCs. This new HP probe could serve as a readout of MDSC function in tumor and its inhibition following MDSC-targeted immunotherapies.

Introduction

Myeloid-derived suppressor cells (MDSCs) are highly prevalent inflammatory cells that play a key role in tumor development1,2 and are considered therapeutic targets3. MDSCs promote tumor growth by blocking T-cell-mediated immunosuppression through depletion of the extracellular arginine pool essential for T-cell proliferation2,4. To deplete arginine, MDSCs express high levels of the enzyme arginase, which catalyzes the breakdown of arginine into urea and ornithine (Figure 1). Based on this knowledge, we developed and characterized 13C-labeled arginine ([guanido-13C]-arginine) as a new hyperpolarized probe. We show that [guanido-13C]-arginine can be hyperpolarized and can be used to detect arginase activity in solution, and in MDSCs, demonstrating its potential for imaging MDSCs and their inhibition in cancer.

Material & Methods

Hyperpolarized 13C arginine preparation and characterization 3.4M [guanido-13C]-arginine was prepared in 7.5μM Trizma solution in water with 15mM OX63 and 1.5mM Dotarem, and polarized using a Hypersense DNP polarizer (Oxford Instruments) for 90 minutes. After dissolution in a Tris-based buffer (40mM Tris, 3μM EDTA, pH~7.8), single-pulse 13C spectra were acquired using an 11.7T INOVA spectrometer (Agilent Technologies) to evaluate T1 and signal-to-noise ratio (SNR) enhancement. T1 was also evaluated on a 3T GE clinical scanner (GE Healthcare). Spectra were quantified by peak integration using MestRenova (Mestrelab).

Enzyme studies Following polarization and dissolution, 3mL of hyperpolarized [guanido-13C]-arginine was injected into an NMR tube containing different concentrations of arginase (0 to 2000U/L; n=3/concentration). Dynamic sets of hyperpolarized 13C spectra (TR=3s, flip angle=10deg, number of transients=50, spectral width=20kHz, 20000 points), and thermal equilibrium 13C spectra at the end of the study (90deg pulse, TR=80s, NT=16), were acquired on the 11.7T scanner and analyzed as above except that for hyperpolarized data contaminants were subtracted.

MDSC studies To generate MDSCs, bone marrow cells isolated from Balb/c mice were treated with colony-stimulating factors (0.1μg/mL G-CSF; 250U/mL GM-CSF, Peprotech) then exposed to interleukin-13 (+IL13, 80ng/mL) to induce arginase expression. Controls were not exposed to IL13 (-IL13)2. To probe for arginase activity, 3mL of hyperpolarized [guanido-13C]-arginine were injected into an NMR tube containing: (1) Cells in 500μL fresh medium ­– to assess intracellular arginase activity (~1e7 cells, n=3 +IL13/-IL13), or (2) 500μL of growth medium previously exposed to MDSCs ­– to assess extracellular arginase activity (n=3 +IL13/-IL13). Dynamic hyperpolarized spectra followed by thermal equilibrium spectra were recorded and analyzed as for enzyme. Extracellular and intracellular arginase activities were determined using a colorimetric assay (QuantiChrom Arginase Assay Kit, BioAssays, n=3).

Results & Discussion

Characterization Following polarization, the resonance of hyperpolarized [guanido-13C]-arginine was detected at 159.7ppm with an SNR enhancement of 5018±412 compared to the thermal spectrum (n=3; Figure 2). Additional resonances detected at 177.1, 165.5 and 164.2ppm were attributed, respectively, to the natural abundance 13C carbonyl of arginine and to 13C-urea and [6-13C]-citrulline contaminations. The T1 of hyperpolarized [guanido-13C]-arginine was 9.9±0.1s at 11.7T (n=3) and 12.3±0.8s at 3T (n=2), indicating little T1 dependence on magnetic field, in contrast to carbonyls5.

Enzyme studies Exposure of hyperpolarized arginine to arginase at or above 300U/L resulted in a detectable build-up of hyperpolarized 13C-urea at 165.5ppm with a maximum at 14±2s post-maximum arginine signal (Figure 3A). Furthermore, hyperpolarized 13C-urea production increased linearly with enzyme concentration (Figure 3B), but no build-up in [6-13C]-citrulline could be detected. Thermal 13C spectra acquired post-hyperpolarized signal decay confirmed continued production of urea (Figure 3C/3D).

MDSC studies Following injection of hyperpolarized arginine, a build-up in hyperpolarized 13C-urea was observed in +IL13 MDSCs but not in –IL13 cells (Figure 4A/4B) resulting in a significantly higher hyperpolarized 13C urea-to-arginine signal (Figure 4C), and consistent with the expected induction of arginase expression only in cells exposed to IL13. No hyperpolarized 13C-urea was observed in the growth media, indicating that extracellular arginase was below detection (Figure 4C). No build-up in hyperpolarized 13C citrulline was observed consistent with the expected absence of iNOS activation. Production of urea only in the +IL13 MDSCs was confirmed on thermal 13C spectra. To validate our hyperpolarized findings, we used a colorimetric enzyme assay to determined arginase activity. As expected, the extracellular arginase activity, as well as the intra-cellular arginase activity in –IL13 cells, were below detection level of the hyperpolarized 13C method (<260U/L in –IL13 cells and <25U/L in +IL13/-IL13 growth media), whereas the up-regulated intracellular arginase activity in +IL13 MDSCs (576±67U/L) was readily detectable by our hyperpolarized probe and significantly higher than in –IL13 cells (unpaired t-test, p-value=0.004) (Figure 4D).

Conclusion

We demonstrate, to our knowledge for the first time, the feasibility of using [guanido-13C]-arginine to monitor arginase activity. This probe could serve as a readout of MDSC function in tumor development and its inhibition following MDSC-targeted immunotherapies.

Acknowledgements

Work supported by NIH grants R01CA172845, Cal-BRAIN349087, R21CA201453 and center grant P41EB013598

References

1 Hanahan D et al., Hallmarks of cancer: the next generation, Cell (2011). 2 Kohanbash G et al., Myeloid-derived suppressor cells (MDSCs) in gliomas and glioma-development, Immunological investigations (2012). 3 Yang et al., Polarization and reprogramming of myeloid-derived suppressor cells, Journal of Molecular Cell Biology (2013). 4 Thaci B et al., Depletion of myeloid-derived suppressor cells during interleukin-12 immunogene therapy does not confer survival advantage in experimental malignant glioma, Cancer Gene Therapy (2014). 5 Chaumeil MM et al, Studies of metabolism using (13)C MRS of hyperpolarized probes, Methods Enzymology (2015).

Figures

Figure 1: Metabolism of [guanido-13C]-arginine, showing conversion into 13C-urea via arginase enzyme, and into [6-13C]-citrulline via nitric oxide synthase (iNOS).

Figure 2: (A)[Guanido-13C]-arginine (159.7ppm) thermal equilibrium spectrum and (B)hyperpolarized spectrum. The DNP method increases SNR by a factor of ~5000.

Figure 3: (A)Build-up of HP 13C-urea normalized to maximum [guanido-13C]-arginine over time illustrating urea production. (B)Area under the curve (AUC) of build-up as a function of arginase concentration, showing a correlation between 13C-urea production and enzyme concentration. (C)Stackplot of thermal spectra and (D)13C-urea from thermal spectra, confirming HP 13C-urea detection.

Figure 4: (A)Build-up of HP 13C-urea normalized to maximum [guanido-13C]-arginine over time and (B)Stackplot of HP 13C spectra in +IL13 cells. (C)Area under the curve (AUC) of HP 13C-urea in +IL13/-IL13 cells and growth media. (D)Enzyme activity in cells and growth media as measured by enzyme activity assay.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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