We compared overexpression of the magnetotactic bacterial gene MagA with the modified mammalian ferritin genes HF+LF, in which both heavy and light subunits lack iron response elements. Whereas both expression systems have been proposed for use in non-invasive, magnetic resonance (MR) reporter gene expression, limited information is available regarding their relative potential for providing gene-based contrast. Measurements of MR relaxation rates in these expression systems are important for optimizing cell detection and specificity, for developing quantification methods, and for refinement of gene-based iron contrast using magnetosome associated genes. We measured the total transverse relaxation rate (R2*), its irreversible and reversible components (R2 and R2′, respectively) and the longitudinal relaxation rate (R1) in MDA-MB-435 tumor cells. Clonal lines overexpressing MagA and HF+LF were cultured in the presence and absence of iron supplementation, and mounted in a spherical phantom for relaxation mapping at 3 Tesla. In addition to MR measures, cellular changes in iron and zinc were evaluated by inductively-coupled plasma mass spectrometry, in ATP by luciferase bioluminescence and in transferrin receptor by Western blot. Only transverse relaxation rates were significantly higher in iron-supplemented, MagA- and HF+LF-expressing cells compared to non-supplemented cells and the parental control. R2* provided the greatest absolute difference and R2′ showed the greatest relative difference, consistent with the notion that R2′ may be a more specific indicator of iron-based contrast than R2, as observed in brain tissue. Iron supplementation of MagA- and HF+LF-expressing cells increased the iron/zinc ratio approximately 20-fold, while transferrin receptor expression decreased approx. 10-fold. Level of ATP was similar across all cell types and culture conditions. These results highlight the potential of magnetotactic bacterial gene expression for improving MR contrast.

Magnetic resonance imaging (MRI) is a non-invasive imaging modality used in longitudinal cell tracking. Previous studies suggest that MagA, a putative iron transport protein from magnetotactic bacteria, is a useful gene-based magnetic resonance contrast agent. Hemagglutinin-tagged MagA was stably expressed in undifferentiated embryonic mouse teratocarcinoma, multipotent P19 cells to provide a suitable model for tracking these cells during differentiation. Western blot and immunocytochemistry confirmed the expression and membrane localization of MagA in P19 cells. Surprisingly, elemental iron analysis using inductively-coupled plasma mass spectrometry revealed significant iron uptake in both parental and MagA-expressing P19 cells, cultured in the presence of iron-supplemented medium. Withdrawal of this extracellular iron supplement revealed unexpected iron export activity in P19 cells, which MagA expression attenuated. The influence of iron supplementation on parental and MagA-expressing cells was not reflected by longitudinal relaxation rates. Measurement of transverse relaxation rates (R₂* and R₂) reflected changes in total cellular iron content but did not clearly distinguish MagA-expressing cells from the parental cell type, despite significant differences in the uptake and retention of total cellular iron. Unlike other cell types, the reversible component R₂′ (R₂* ‒ R₂) provided only a moderately strong correlation to amount of cellular iron, normalized to amount of protein. This is the first report to characterize MagA expression in a previously unrecognized iron exporting cell type. The interplay between contrast gene expression and systemic iron metabolism substantiates the potential for diverting cellular iron toward the formation of a novel iron compartment, however rudimentary when using a single magnetotactic bacterial gene expression system like magA. Since relatively few mammalian cells export iron, the P19 cell line provides a tractable model of ferroportin activity, suitable for magnetic resonance analysis of key iron-handling activities and their influence on gene-based MRI contrast.

The Interface Between Iron Metabolism and Gene-Based Iron Contrast for MRI Supplementary Issue: New Concepts in Magnetic Resonance as Applied to Cellular and In Vivo Applications