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 development
1,2 and are considered therapeutic
targets
3. MDSCs promote tumor growth by blocking T-cell-mediated
immunosuppression through depletion of the extracellular arginine pool
essential for T-cell proliferation
2,4. 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 P41EB013598References
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