Development and Application of CEST MRI Contrast Agents that Evaluate Tumor Acidosis and Enzyme Activity

Abstract

The extracellular tumor microenvironments of many solid tumors have high acidosis and high protease activity. The detection of tumor acidosis and protease activity can potentially impact the cancer diagnosis. Noninvasive imaging methods have previously been developed that measure extracellular pH or can detect enzyme activity using Chemical Exchange Saturation Transfer Magnetic Resonance Imaging (CEST MRI). CEST MRI is an inherently insensitive MRI modality which requires a high concentration of small molecule agent to be delivered to the tumor. Therefore, nanosized molecules have received increased attention to be used in CEST imaging to improve the sensitivity. Herein, we developed a nanoscale CEST agent that can measure pH using acidoCEST MRI, which may decrease the requirement for high delivery concentrations of agent. We also developed a monomer agent for comparison to the polymer. After optimizing CEST experimental conditions, we determined that the polymer agent could be used during acidoCEST MRI studies at 125-fold and 488-fold lower concentration than the monomer agent and iopamidol, respectively. In vivo acidoCEST MRI studies using the three agents were performed to study a xenograft MDA-MB-231 model of mammary carcinoma. The tumor pHe measurements were 6.33 ± 0.12, 6.70 ± 0.15, and 6.85 ± 0.15 units with iopamidol, the monomer agent and polymer agent, respectively. The higher pHe measurements with the new agents was attributed to the concentration dependence of these agents. This study demonstrated that nanoscale agents have merit for CEST MRI studies, but consideration should be given to the dependence of CEST contrast on the concentration of these agents. We also investigated the development and application of a single hybrid CEST agent that can simultaneously measure pH and evaluate protease activity using a combination of dual-power acidoCEST MRI and catalyCEST MRI. Simultaneously assessing both characteristics may improve diagnostic evaluations of aggressive tumors and the effects of anti-cancer treatments. Our agent showed CEST signals at 9.2 ppm from a salicylic acid moiety and at 5.0 ppm from an aryl amide. The CEST signal at 9.2 ppm could be measured after selective saturation was applied at 1 and 4 T, and these measurements could be used with a ratiometric analysis to determine pH. The CEST signal at 5.0 ppm from the aryl amide disappeared after the agent was treated with cathepsin-B, while the CEST signal at 9.2 ppm remained, indicating that the agent could detect protease activity through amide bond clevage. Michaelis-Menton kinetics studies with catalyCEST MRI demonstrated that the binding affinity (as shown with the Michaelis constant KM), the catalytic turnover rate (kcat), and catalytic efficiency (kcat/kM), were each higher for cathepsin B at lower pH. The kcat rates measured with catalyCEST MRI were lower than the comparable rates measured with LC-MS, which reflected a limitation of inherently noisy and relatively insensitive CEST MRI analyses. Although this level of precision limited catalyCEST MRI to semi-quantitative evaluations, these semi-quantitative assessments of high and low protease activity still had value by demonstrating that high acidosis and high protease activity can be used as synergistic, multi-parametric biomarkers.