Imaging thiol redox status in murine tumors in vivo with rapid-scan electron paramagnetic resonance

Abstract

Thiol redox status is an important physiologic parameter that affects the success or failure of cancer treatment. Rapid scan electron paramagnetic resonance (RS EPR) is a novel technique that has shown higher signal-to-noise ratio than conventional continuous-wave EPR in in vitro studies. Here we used RS EPR to acquire rapid three-dimensional images of the thiol redox status of tumors in living mice. This work presents, for the first time, in vivo RS EPR images of the kinetics of the reaction of ²H,¹⁵N-substituted disulfide-linked dinitroxide (PxSSPx) spin probe with intracellular glutathione. The cleavage rate is proportional to the intracellular glutathione concentration. Feasibility was demonstrated in a FSa fibrosarcoma tumor model in C3H mice. Similar to other in vivo and cell model studies, decreasing intracellular glutathione concentration by treating mice with l-buthionine sulfoximine (BSO) markedly altered the kinetic images.

Keywords: EPR imaging; Electron paramagnetic resonance; Glutathione; Rapid scan EPR; Thiol redox status.

Figure 1

Left. Schematic of the reaction of the disulfide-dinitroxide probe PxSSPx (1) with a thiol (RSH), leading to cleaved monomeric products, PxSH (2). Right. 251.1 MHz in vitro rapid-scan EPR spectra of PxSSPx (1) and PxSH (2) after cleavage of PxSSPx by RSH.

Figure 2

250.8 MHz rapid-scan spectra of PxSSPx after injection into a tumor in vivo. Relative amplitudes are preserved. A. Immediately after injection. Circles represent data; solid line is simulated spectrum with ¹⁵N hfi A = 2.04 mT and exchange coupling J = 110 MHz. B. At 175 s post-injection. C. At 1850 s post-injection. Solid line is simulated spectrum with 15N hfi A = 2.31 mT. D. Time dependence of the PxSSPx (triangles) and PxSH (circles) spin numbers; solid curves are simulations with $k_1^{\mathit{\text{obs}}}=1.85·{10}^{-3}{\mathrm{s}}^{-1},k_2^{\mathit{\text{obs}}}=6.65·{10}^{-3}{\mathrm{s}}^{-1}$ and $k_2^{\mathit{\text{clr}}}=0.39·{10}^{-3}{\mathrm{s}}^{-1}$ (see main text for details).

Figure 3

Sagittal slices and histograms of cleavage rate k^obs images in FSa tumor A. before; and B. 24 h after application of BSO. Magenta outline shows the tumor border as obtained from a registered MRI image. The histogram shows k^obs in all voxels of the tumor. Sagittal slices and histograms of clearance rate k^clr, images in FSa tumor C. before; and D. 24 h after application of BSO. Magenta outline shows the tumor border as obtained from a registered MRI image. The histogram shows k^obs, in all voxels of the tumor.