In Vivo Imaging of the Tumor-Associated Enzyme NCEH1 with a Covalent PET Probe

Abstract

Herein, we report the development of an ¹⁸ F-labeled, activity-based small-molecule probe targeting the cancer-associated serine hydrolase NCEH1. We undertook a focused medicinal chemistry campaign to simultaneously preserve potent and specific NCEH1 labeling in live cells and animals, while permitting facile ¹⁸ F radionuclide incorporation required for PET imaging. The resulting molecule, [¹⁸ F]JW199, labels active NCEH1 in live cells at nanomolar concentrations and greater than 1000-fold selectivity relative to other serine hydrolases. [¹⁸ F]JW199 displays rapid, NCEH1-dependent accumulation in mouse tissues. Finally, we demonstrate that [¹⁸ F]JW199 labels aggressive cancer tumor cells in vivo, which uncovered localized NCEH1 activity at the leading edge of triple-negative breast cancer tumors, suggesting roles for NCEH1 in tumor aggressiveness and metastasis.

Keywords: NCEH1; activity-based probes; imaging; positron emission tomography; radiotracers.

Figure 1.

JW199 is a potent and specific inhibitor of NCEH1. A. Chemical structure of JW199. B. Gel-based profiling of active serine hydrolases in breast MDA-MB231 cells (membrane fraction) treated with JW199 in vitro (left) and in situ (right). Bands mark active serine hydrolases labeled fluorophosphonate-rhodamine. C. Quantitation of JW199-dependent NCEH1 inhibition in MDA-MB231 cells in vitro (red) and in situ (black). Data shown represent the mean and 95% confidence interval (95% C.I.) from n = 3 biological replicates. D. Radiosynthesis of [¹⁸F]JW199. E. Absorbance (black) and crude radioactivity (red) chromatograms of pure TsO-JW199 starting material (1, left), ¹⁸F-labeled, crude JW199 (middle), and purified [¹⁸F]JW199 (right). Arrowheads indicate peaks corresponding to the [¹⁸F]JW199 radiosynthetic product. F. Stability of [¹⁸F]JW199 in phosphate buffered saline containing 10% ethanol over time, as measured by radioactive HPLC.

Figure 2.

NCEH1-dependent imaging with [¹⁸F]JW199. A. Treatment and imaging timeline. B. Whole-body PET-CT scan of wild-type mice following intravenous administration of [¹⁸F]JW199. Arrowhead marks radiosignal in the kidney. Image is representative of n = 3 mice. C. Correlation between Nceh1 mRNA abundance in mouse tissues (obtained from BioGPS.org) and [¹⁸F]JW199 accumulation, quantified as the percentage of initial dose per volume of bodyweight (%ID/cc). D. [¹⁸F]JW199 radiosignal kinetics in indicated organs following tracer injection. E. Timeline of JW480 competition experiment (top) and PET-CT images of two representative mice (bottom). F. Radiotracer signal quantification in tissues from mice in the [¹⁸F]JW199 (red) and competition (blue) experimental groups, 3 hours post injection of [¹⁸F]JW199. All images are representative of n = 3 mice per group and data quantitation represent mean ± standard deviation. ns, not significant, *p < 0.05, **p < 0.01, as determined by Student’s t-test.

Figure 3.

[¹⁸F]JW199-mediated labelling of breast cancer tumor xenografts. A. Representative whole-body PET-CT scan of a MDA-MB231 tumor-bearing mouse following administration of [¹⁸F]JW199. Tumor boundary is delineated in the whole-body image and magnified in inset. Image is representative of n = 3 mice. B–C. NCEH1 activity profiling in the edge and core areas of MDA-MB231 tumor xenografts, as measured via gel-based profiling with JW576 (B) and the soluble activity-dependent proximity ligation (sADPL) profiling (C). D. Western blot of NCEH1 protein level in the edge (red) and core (blue) areas of tumor xenografts. E. Representative NCEH1 immunofluorescence of xenograft sections (5 µm thick). Blue channel is DAPI-stained nuclei, while red channel indicates anti-NCEH1. Division between the edge and inside areas are indicated with a dashed line. Scale bar = 5 µm. Data shown represent the mean ± standard deviation from n = 3 mice (C, E) and triplicate samples from n = 3 mice (D). ***p < 0.001, ****p < 0.0001 as determined by Student’s t-test.