Noninvasive quantitation of cytosine deaminase transgene expression in human tumor xenografts with in vivo magnetic resonance spectroscopy

Abstract

Analysis of transgene expression in vivo currently requires destructive and invasive molecular assays of tissue specimens. Noninvasive methodology for assessing the location, magnitude, and duration of transgene expression in vivo will facilitate subject-by-subject correlation of therapeutic outcomes with transgene expression and will be useful in vector development. Cytosine deaminase (CD) is a microbial gene undergoing clinical trials in gene-directed enzyme prodrug gene therapy. We hypothesized that in vivo magnetic resonance spectroscopy could be used to measure CD transgene expression in genetically modified tumors by directly observing the CD-catalyzed conversion of the 5-fluorocytosine (5-FC) prodrug to the chemotherapeutic agent 5-fluorouracil (5-FU). The feasibility of this approach is demonstrated in subcutaneous human colorectal carcinoma xenografts in nude mice by using yeast CD (yCD). A three-compartment model was used to analyze the metabolic fluxes of 5-FC and its metabolites. The rate constants for yCD-catalyzed prodrug conversion (k(1)(app)), 5-FU efflux from the observable tumor volume (k(2)(app)), and formation of cytotoxic fluorinated nucleotides from 5-FU (k(3)(app)) were 0.49 +/- 0.27 min(-1), 0.766 +/- 0.006 min(-1), and 0.0023 +/- 0.0007 min(-1), respectively. The best fits of the 5-FU concentration data assumed first-order kinetics, suggesting that yCD was not saturated in vivo in the presence of measured intratumoral 5-FC concentrations well above the in vitro K(m). These results demonstrate the feasibility of using magnetic resonance spectroscopy to noninvasively monitor therapeutic transgene expression in tumors. This capability provides an approach for measuring gene expression that will be useful in clinical gene therapy trials.

Figure 1

Schematic representation of the 3-compartment model used in the pharmacokinetic analysis of 5-FC metabolism in yCD-transduced human tumor xenografts. The compartmental transfer constants are k₁^app and k₃^app; k₂^app is an elimination rate constant.

Figure 2

Serial ¹⁹F spectra of a subcutaneous HT29/yCD tumor. Acquisition began 20 min after i.p. administration of 1 g/kg 5-FC. Each spectrum is the average of 277 free induction decays collected in a 20-min interval. The time from 5-FC injection to the beginning of the acquisition interval are shown beside the spectra. The chemical shift assignments (in ppm) of the metabolite peaks relative to NaF are: FNuc, −45.0; 5-FC, −48.5; and 5-FU, −49.6.

Figure 3

Representative ¹⁹F spectra from subcutaneous tumors obtained 120–150 min after i.p. injection of 1 g/kg 5-FC. (A) Untransduced HT29 carcinoma. (B) HT29/yCD carcinoma. The chemical shift of FβAL relative to NaF is −68.2 ppm.

Figure 4

Pharmacokinetic analysis of 5-FC anabolites in a representative HT29/yCD tumor. The MRS-determined concentrations of 5-FU (A) and FNuc (B) were fit by using two independent pharmacokinetic models. The first (broken lines) assumes saturation kinetics and the second (solid lines) assumes first-order kinetics.

Figure 5

Patlak analysis of a representative animal. The net accumulation of FNuc in the tumor (moles FNuc⋅min⁻¹⋅mole⁻¹ 5-FC) is determined graphically by the slope of the time-dependence of the linearized 5-FC and FNuc tissue concentrations.