Noninvasive detection of enzyme activity in tumor models of human ovarian cancer using catalyCEST MRI

Abstract

Purpose: We proposed to detect the in vivo enzyme activity of γ-glutamyl transferase (GGT) within mouse models of human ovarian cancers using catalyCEST MRI with a diamagnetic CEST agent.

Methods: A CEST-FISP MRI protocol and a diamagnetic CEST agent were developed to detect GGT enzyme activity in biochemical solution. A quantitative Michaelis-Menten enzyme kinetics study was performed to confirm that catalyCEST MRI can measure enzyme activity. In vivo catalyCEST MRI studies generated pixel-wise activity maps of GGT activities. Ex vivo fluorescence imaging was performed for validation.

Results: CatalyCEST MRI selectively detected two CEST signals from a single CEST agent, whereby one CEST signal was responsive to GGT enzyme activity and the other CEST signal was an unresponsive control signal. The comparison of these CEST signals facilitated in vivo catalyCEST MRI studies that detected high GGT activity in OVCAR-8 tumors, low GGT activity in OVCAR-3 tumors, and low or no GGT activity in muscle tissues.

Conclusion: CatalyCEST MRI with a diamagnetic CEST agent can detect the level of GGT enzyme activity within in vivo tumor models of human ovarian cancers. Magn Reson Med 77:2005-2014, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Keywords: CEST MRI; enzyme activity; glutamyl transferase; molecular imaging; ovarian cancer.

Figure 1

Substrates for γ-glutamyl transferase (GGT). (a) The natural metabolite glutathione is cleaved by GGT to produce glutamate and a cysteine-glycine dipeptide. (b) A florescence agent, gGlu-HMRG, is activated after the glutamyl ligand is cleaved via GGT catalysis. (c) The proposed mechanism for GGT cleavage of the CEST MRI contrast agent, based on reference 16.

Figure 2

CatalyCEST MRI. (a) Selective saturation of the net coherent magnetization of the amide proton (red hydrogen atom) followed by chemical exchange of the proton to water, causes a decrease in the MR signal of water. (b) The GGT enzyme can cleave the glutamyl moiety of the CEST agent, causing a loss of CEST signal from the aryl amide proton. However, the salicylic acid moiety can still generate a CEST signal. (c) The CEST spectra of the agent before and 12 hours after addition of GGT enzyme showed a disappearance of the CEST signal at 4.8 ppm while the CEST signal at 9.2 ppm was unchanged. Dark blue: CEST spectrum of the substrate; light blue: CEST spectrum of the product; Thick red line: % CEST signals of the substrate; thin red line: % CEST signals of the product.

Figure 3

The CEST-FISP MRI pulse sequence with respiration gating. The respiration gating was adjusted to allow the start of acquisition in a 200 ms window (thick black line) that started 300 ms after inhalation. The series of saturation pulses typically ended within a respiration gating window, and the FISP acquisition was immediately started. Due to variable breathing rate, additional 600 ms saturation pulses was added to the saturation period until the total saturation period ended within a respiration gating window.

Figure 4

Michaelis-Menten enzyme kinetics. (a) The CEST spectrum showed a loss of the CEST at 4.8 ppm from 0.25 hours to 10 hours after adding enzyme to a 68 mM sample of the agent. (b) The % CEST at 4.8 ppm decreased when 0.4 units of GGT enzyme was added to each sample of the agent at different concentrations. The signal values were converted to concentrations (using the calibration shown in Sup. Fig. S4), and the initial reaction velocity, v₀, was determined from the loss in concentration during the first 2.5 hours after adding GGT enzyme to each sample. (c) A Hanes-Woolf plot was used to determine Michaelis-Menten kinetics parameters. (d) The k_cat rate of catalysis of the catalyCEST agent was comparable to the catalysis rate of a fluorescence agent. However, the K_M dissociation constant was higher for the catalyCEST agent, leading to a lower k_cat/K_M catalytic efficiency for the catalyCEST agent.

Figure 5

In vivo catalyCEST MRI. (a) The anatomical images show the region of the OVCAR-8 tumor, OVCAR-3 tumor, and muscle tissue that was analyzed. The red rectangle shows the regions displayed in the remainder of the figure. (b) The parametric maps of % CEST signal amplitudes for CEST signals at 9.2 and 4.8 ppm demonstrate good detection of both CEST effects in the tumor and muscle tissues. (c) The activity maps of the GGT enzyme show high activity in the OVCAR-8 tumor, low activity in the OVCAR-3 tumor, and no activity in the muscle tissue. (d) The CEST spectra (blue) and CEST signals (red) for the region of the tumor and muscle also show good detection sensitivity of the agent and demonstrate the relative ratios of both CEST signals.

Figure 6

Temporal catalyCEST MRI results. (a) The two CEST signals of the agent changed during the time course of the in vivo study, demonstrating that the agent experienced pharmacokinetic wash-out from the tumor as well as experiencing the effects of enzyme activity. Error bars represent the standard deviation of the measurements. (b) The temporal change of the ratio of the two CEST signals approximated a pseudo-first order reaction, as expected for enzyme kinetics with a high substrate concentration.

Figure 7

The effect of pH on the determination of the reaction coordinate. (a) The chemical exchange rate of the salicylic acid moiety and aryl amide moiety are each base catalyzed, and therefore generate higher % CEST signal amplitudes at higher pH. (b) The % CEST signal amplitudes were converted to concentrations, then used to estimate a reaction coordinate at each pH value, which were normalized to the reaction coordinate at pH 7.4. Linear trendlines were fit to data in each graph. These results demonstrate that the reaction coordinate is underestimated under acidic pH conditions, which can occur in tumor tissues.

Figure 8

Ex vivo fluorescence imaging with gGLU-HMRG dye confirmed that (a) the OVCAR-8 tumor had high GGT activity, (b) the OVCAR-3 tumor had GGT activity, and normal tissues had no detectable GGT activity.