Biophysical features of MagA expression in mammalian cells: implications for MRI contrast

Abstract

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 approximately 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.

Keywords: MagA; cancer cells; iron; magnetic resonance imaging; modified ferritin subunits; relaxation rates.

FIGURE 1

Relaxation rate measurement in a spherical phantom. Representative data indicate the influence of echo time (TE) on signal decay. (A) Single-echo SE images show sample wells in cross section at three different TE values (13, 100, and 300 ms): 1, parental (P); 2, MagA (A); 3, iron-supplemented MagA (A + Fe); 4, iron-supplemented parental (P + Fe); and 5 polystyrene marker for reference. Samples along the bottom row are combinations of MagA-expressing and parental cells. (B) R2 relaxation curves are shown for iron-supplemented samples. Symbols indicate the mean signal intensity within a region of interest (ROI) at each TE. Curves represent the best fit to an exponential decay.

FIGURE 2

Transverse relaxation rate mapping. Representative maps are shown for (A) R2, (B) R2^* and (C) R2’. The first two maps were obtained using voxel by voxel curve fitting with an exponential decay function and the R2’ map was obtained by subtraction (R2^*–R2). The units of the scale bar are sec^-1. Images show sample wells in the phantom, in cross section. From left to right across the top row are: parental (P); MagA (A); iron-supplemented MagA (A + Fe); and iron-supplemented parental (P + Fe). Along the bottom row, from left to right, there is a polystyrene marker for reference and samples with combinations of iron-supplemented, MagA-expressing and parental cells. These samples decrease from 70% A + Fe to 50% and 30% and were not further evaluated. Note that pixel values for all three rates are highest for A + Fe. Maps are provided for display only; relaxation rates (Table 2) were determined as outlined in methods.

FIGURE 3

Cellular ATP content before and after MR scanning. ATP was quantified in each cell type using a luciferase bioluminescence assay and normalized to protein content (black bars). In parental and MagA-expressing cells, ATP content was not statistically different in the presence and absence of iron supplementation. In cells mounted in a gelatin phantom (open bars), the ATP content decreased variably within 24 h of harvest and scanning. Error bars represent SEM where n = 3–4; both values are shown where n = 2.

FIGURE 4

Decrease in transferrin receptor expression upon iron supplementation. Tumor cells were cultured in the presence (+) or absence (-) of iron supplementation, lysed, and analyzed by Western blot using a primary antibody to transferrin receptor. Both MagA- and HF + LF-expressing cells showed greater immunoreactivity toward the soluble form of transferrin receptor (arrow) in cells cultured in the absence of iron supplementation. Under reducing conditions, soluble transferrin receptor migrates at a M.W. of approximately 95K. Protein M.W. standards are indicated on the right margin.