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DOI: 10.1148/radiol.2211001784
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(Radiology. 2001;221:244-250.)
© RSNA, 2001

Experimental Studies

Human Transferrin Receptor Gene as a Marker Gene for MR Imaging1

Anna Moore, PhD, Lee Josephson, PhD, Rajeev M. Bhorade, MD, James P. Basilion, PhD and Ralph Weissleder, MD, PhD

1 From the Center for Molecular Imaging Research, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Bldg 149, 13th St, Rm 5419, Charlestown, MA 02129. Received November 10, 2000; revision requested December 23; revision received February 22, 2001; accepted March 30. Address correspondence to A.M. (e-mail: amoore@helix.mgh.harvard.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 STATISTICAL CONSULTANT...
 REFERENCES
 
PURPOSE: To quantitate and characterize the expression of an engineered human transferrin receptor (ETR) as a marker gene by using magnetic resonance (MR) imaging.

MATERIALS AND METHODS: Rat gliosarcoma 9L cells stably expressing ETR (ETR+) were used, with nontransfected (ETR-) cells serving as controls. A conjugate of transferrin and monocrystalline iron oxide (Tf-MION) nanoparticles was synthesized to probe for the activity of ETR. Accumulation of Tf-MION was examined by using cell internalization in culture and MR (n = 6) and nuclear (n = 4) imaging in a mouse model with ETR+ and ETR- tumors implanted in the opposite flanks. Autoradiographic and histopathologic results were correlated with MR findings.

RESULTS: Tf-MION was internalized by ETR+ cells at 37°C but not at 4°C. Rhodamine-labeled Tf-MION and fluorescein-labeled antibody to ETR colocalized in small vesicle-like structures in the cytoplasm. Both findings were consistent with accumulation by the receptor-mediated endocytosis mechanism of ETR. Compared with ETR- tumors, ETR+ tumors accumulated more Tf-MION and had higher signal intensity on T1-weighted MR images and lower signal intensity on T2-weighted images. Autoradiographic findings showed a spatial correlation between MR signal intensity and TF-MION accumulation.

CONCLUSION: ETR+ tumors internalize the MR imaging probe through the action of transferrin receptor in amounts that can be detected with MR imaging.

Index terms: Experimental study • Neoplasms, experimental studies • Neoplasms, radionuclide studies • Genes and genetics


   INTRODUCTION
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 INTRODUCTION
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RESULTS
DISCUSSION
 STATISTICAL CONSULTANT...
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The ability to image transgene expression in vivo and in real time could alter the way gene transfer studies are performed. This technology would enable temporal monitoring of gene expression in single living animals during the administration of transgenes for gene therapy and eliminate the need for animal- and labor-intensive studies (1–3).

Several imaging modalities have been used to image the end point of gene transfer. A nuclear imaging modality utilizes the thymidine kinase gene that is used for gene therapy of different cancers (4–6). The product of this gene transforms radiolabeled nucleoside to toxic nucleotide analogues accumulating in the cell and resulting in its death (7). The enzyme activity of retained nucleotide metabolites can then be imaged by using positron emission tomography (8,9) or scintigraphy (10,11). Another nuclear imaging technique has exploited the sodium/iodide symporter (12) used for both tumor therapy and imaging (13–15). Another strategy to develop a marker transgene is aimed at imaging cell surface receptors, such as the bombesin-binding gastrin-releasing peptide receptor (16) and the somatostatin-binding receptor (17,18). Optical imaging methods include near-infrared fluorescent probes that are activatable by the proteolytic enzyme cathepsin D (19). Another optical imaging modality being developed is optical imaging of luciferase-dependent bioluminescence (20,21).

Two drawbacks to both nuclear and optical imaging techniques are low spatial resolution and lack of anatomic information depicted on the images. Magnetic resonance (MR) imaging would be ideal for imaging transgene expression, because it can provide substantially better spatial resolution and good anatomic and physiologic information, as compared with these features at either nuclear or optical imaging (22,23).

Our efforts to develop a system for visualizing gene expression by using MR imaging were influenced by preliminary studies, the results of which demonstrated the feasibility of MR imaging to depict the activity of endocytotic receptors like the asialoglycoprotein receptor (24,25). In the present study, we used human transferrin receptor, an endocytotic receptor that functions to internalize transferrin. This research was aimed to develop MR imaging for imaging transgene expression by using transferrin receptor as a marker gene and conjugates of transferrin and monocrystalline iron oxide (Tf-MION) as an MR imaging probe for this receptor.

We stably transfected a rat 9L gliosarcoma cell line with a mutant form of human transferrin receptor; this resulted in a cell line that constitutively overexpresses the engineered human transferrin receptor (ETR). These cells do not down regulate the receptor in response to iron uptake, and the increase in receptor expression can be imaged by using superparamagnetic iron oxide–transferrin conjugates both in vitro and in vivo (22,26). Although the results of prior studies have shown the feasibility of using this system to image transgene expression in vivo (22), several questions have remained regarding the mechanism of uptake, including those related to the demonstration of true transferrin receptor–mediated effects and to the correlation between receptor expression and changes in MR signal intensity. Therefore, the purpose of this study was to quantitate and characterize the expression of ETR as a marker gene by using MR imaging.


   MATERIALS AND METHODS
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 STATISTICAL CONSULTANT...
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Cell Lines
Nontransfected gliosarcoma 9L cells (ETR-; Brain Tumor Research Center, University of California, San Francisco) (27) and the stably transfected ETR cell line (ETR+) (26) were grown in Dulbecco modified Eagle medium (Cellgro DMEM; Mediatech, Washington, DC) with 10% fetal bovine serum (Cellgro FBS; Mediatech) and 1% penicillin plus streptomycin (Cellgro; Mediatech) at 37°C in 5.5% carbon dioxide, with media changes every 3–5 days. The medium for the ETR+ cell line was supplemented with 1 mg/mL of geneticin G-418 (Gibco, Grand Island, NY).

Tumor Model
Two million cells of ETR+ were injected into one flank, and two million cells of ETR- were injected into the opposite flank of 10 nu/nu mice anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) (Parke-Davis, Morris Plains, NJ) plus xylazine (12 mg/kg) (Miles, Shawnee Mission, Kan). Each mouse received injections of both ETR+ and ETR- cells (9L), so each mouse served as its own control (10 experimental tumors and 10 control tumors). Seventeen days later, the tumors were 8–10 mm in diameter and were used for subsequent experiments. This model was used in all experiments described in this article. All animal protocols were approved by the Institutional Review Committee on Animal Care at our institution and were conducted in accordance with National Animal Welfare guidelines.

Synthesis and Characterization of Iron Oxide–Transferrin Conjugates
ETR expression was examined by using an iron oxide conjugate with human holotransferrin (Sigma, St Louis, Mo) made from dextran-coated monocrystalline iron oxide, which was synthesized as described elsewhere (28,29). Conjugation of transferrin to monocrystalline iron oxide also was performed as previously described (26). Briefly, the dextran coat of the particles was partially oxidized with sodium periodate (4:1 iron ratio, weight per weight), incubated with radiolabeled transferrin, and then incubated overnight with sodium borohydride (final concentration 1 mg/mL). The conjugate was purified on a 1.5A column (BioGel 1.5A; Bio-Rad, Richmond, Calif).

The iron concentration of the conjugate was determined spectrophotometrically. Ten microliters of iron oxide–transferrin conjugate was added to 1 mL of 6 N of hydrochloric acid solution containing 0.3% hydrogen peroxide and incubated for 1 hour. The iron concentration was obtained from the optical density at 410 nm by using the appropriate standards. The protein concentration was determined by performing a bicinchoninic acid assay (Pierce, Rockford, Ill), with subtraction of a blank due to iron oxide absorption. The number of transferrin molecules per particle was calculated by assuming a molecular weight of 80,000 d for transferrin and that an iron oxide particle contained 2,064 iron atoms (29). T1 and T2 relaxation times were determined by using a nuclear MR spectrophotometer (NMS-120 Minispec; Bruker Instruments, Milton, Ontario, Canada) operating at 0.47 T (20 MHz) and 37°C. Relaxivity was calculated as the slopes of the curves of 1/T1 and 1/T2 versus iron concentration. Particle size was measured by using a submicron particle size analyzer (model N-4; Coulter, Hialeah, Fla).

Synthesis of Radioactive Tf-MION Conjugates
Transferrin was radiolabeled and then coupled to monocrystalline iron oxide, as described earlier. For internalization studies, transferrin was iodinated with sodium iodide 125 (NEN Life Science Products, Boston, Mass) in 0.1 mol/L of sodium carbonate (pH 9.0) in the presence of 1,3,4,6-tetrachloro-3{alpha}-6{alpha}-diphenylglycoluril (IodoGen; Pierce). Iodide was removed by means of gel filtration with a column (model PD-10; Amersham Pharmacia Biotech, Uppsala, Sweden) saturated with bovine serum albumin. For nuclear imaging experiments, the conjugate of monocrystalline iron oxide with indium-labeled apotransferrin (ie, transferrin devoid of iron) (Sigma) was prepared. Two milligrams of apotransferrin was incubated with 2 mCi (7.4 x 107 Bq) of indium chloride 111 (NEN Life Science Products) in 10 mmol/L of citrate buffered saline (pH 6.0) overnight at room temperature. Purification was performed by means of gel filtration, as described earlier. It was calculated that indium 111 (111In)–labeled transferrin contained two indium molecules per molecule of transferrin.

To ensure that the cells overexpressing ETR interpreted the internalization of 111In-labeled transferrin as being similar to that of iodine 125 (125I)–labeled transferrin, cell uptake experiments were performed as previously described (26). Briefly, 100,000 cells were incubated with different amounts of the probe for 1 hour, washed, and detached from the plate surface by using trypsin, and the radioactivity associated with the cells was counted in a gamma counter (1282 Compugamma; Wallac, Finland). The internalization of the 111In-labeled compound was similar to that of the 125I-labeled compound. Seventy-five micrograms of added 111In- or 125I-labeled transferrin resulted in the uptake of 53.5 or 55.7 ng of transferrin, respectively.

Tf-MION was labeled with rhodamine according to the manufacturer’s protocol (Molecular Probes, Eugene, Ore). Fluorescein-labeled antibodies to the human transferrin receptor (ie, fluorescein–antihuman transferrin receptor) (Santa Cruz Biotechnology; Santa Cruz, Calif) were obtained. All experiments were performed in duplicate.

Internalization of 125I–Tf-MION by ETR+ Cells
For internalization experiments, an acid-stripping method was used with slight modifications (30); the cells were washed with acid so that only internalized radioactivity was counted. Briefly, ETR+ cells were first incubated with 125I–Tf-MION at 4°C for 30 minutes. After the incubation, the plate with the cells was transferred to a 37°C environment and incubated for another 15, 30, 45, and 60 minutes. A separate plate with cells remained at 4°C. At each time point, the cells at both 37°C and 4°C were washed with a salt solution (Cellgro Hanks Balanced Salt Solution; Mediatech) at 4°C, incubated for 5 minutes with 0.5 mol/L of sodium chloride plus 0.2 mol/L of acetic acid solution at 4°C, and washed again with the salt solution. Cell-associated radioactivity was counted in the gamma counter.

Subcellular Localization of Tf-MION
ETR- 9L gliosarcoma and ETR+ cells were plated on an eight-chamber polystyrene vessel (Becton Dickinson, Franklin lakes, NJ) at 40% confluency in phenol red–free Dulbecco modified Eagle medium. The cells were incubated with rhodamine-labeled Tf-MION and fluorescein-labeled antihuman transferrin receptor antibodies at 4°C for 30 minutes and then transferred to a 37°C environment for 45 minutes. A separate panel of cells remained at 4°C for the duration of the experiment. After incubation, the cells were washed with the salt solution without phenol red and covered with cover slips (Fisher Scientific, Pittsburgh, Pa) by using fluorescence mounting medium (Fluoromaunt-G; Southern Biotechnology Associates, Birmingham, Ala). The cells were then examined with an inverted fluorescence microscope (Axiovert 100TV; Zeiss, Wetzlar, Germany) equipped with an optical filter set (Omega Optical, Brattleboro, Vt) and an intensified charged coupled device video camera (Photometrics, Tuscon, Ariz) connected to a computer (Power Macintosh 7600/120; Apple Computer, Cupertino, Calif). Neither control experiments with 9L wild-type gliosarcoma lacking ETR nor incubation at 4°C revealed any colocalization of the probes.

Ex Vivo Imaging
Three tumor-bearing mice were injected intravenously with 125I–Tf-MION (3 mg of iron). Six relatively small tumors were then excised, fixed in 4% formaldehyde, and embedded in 1% agarose gel for MR imaging. This experimental setup was chosen as opposed to in vivo imaging, because it allowed direct correlation between MR imaging, autoradiographic, and histopathologic findings. Similar attempts in vivo have been generally unsuccessful because of changes in tumor morphologic structures during histopathologic processing. The ex vivo samples were constructed from 1% agarose gel to prevent drying of tissues and susceptibility artifacts. Tumors from three mice that were not injected with 125I–Tf-MION served as precontrast control lesions.

MR imaging was performed with a 1.5-T superconducting magnet (Signa 5.0; GE Medical Systems, Milwaukee, Wis) by using a 5-inch surface coil. T1-weighted (300/11 [repetition time msec/echo time msec]) and T2-weighted (2,000/102) spin-echo images were obtained. The section thickness was 3 mm, with a field of view of 10 cm2, a 256 x 192 matrix, and four signals acquired. Two investigators (A.M., R.M.B.) independently calculated signal-to-noise ratios by manually drawing a region of interest around the whole tumor tissue and comparing the signal intensity in the region of interest with that in the background by using computer software (IPLab Spectrum; Scanalytics, Fairfax, Va).

Autoradiography and Histopathologic Analysis
Autoradiography of tumor sections excised from the three mice injected with 125I–Tf-MION was performed immediately following MR imaging. The sections were exposed to phosphor screens overnight and read by using a digital phosphor image analyzer (Molecular Dynamics, Sunnyvale, Calif). After exposure to the phosphor screens, the same sections were subjected to hematoxylin-eosin staining.

Nuclear Imaging of ETR+ and 9L (ETR-) Tumors
To show the difference in tumoral accumulation of the probe—that is, ETR+ versus ETR- tumors—nuclear MR imaging was performed on the excised tumors 24 hours after intravenous injection of 30 µCi (1.11 x 106 Bq) of 111In–Tf-MION per mouse in four mice (0.2 mg of iron). The images were obtained by using a gamma camera (Radioisotope Camera Series 100; Ohio-Nuclear, Cleveland, Ohio), with a pinhole acquisition and a 30-minute acquisition time. The two independent investigators (A.M., R.M.B.) determined the signal intensities by manually drawing a region of interest around the whole tumor tissue and correcting for weight.

Statistical Analyses
The unpaired Student t test and statistical software program (InStat for Macintosh; GraphPad Software, San Diego, Calif) were used to compare MR imaging and nuclear MR imaging data for ETR+ and ETR- tumors. A P value of less than .05 was considered to indicate a significant difference.


   RESULTS
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 STATISTICAL CONSULTANT...
 REFERENCES
 
Physical Properties of Iron Oxide–Transferrin Conjugate
On average, two transferrin molecules were conjugated per iron oxide particle. Iron and protein concentrations of the synthesized conjugate were 1.1 mg/mL (19.6 mmol/L) and 1.51 g/L, respectively. The mean size of the TF-MION particle was 61 nm, as measured according to laser light scattering. The R1 relaxation rate was 31.1 mM-1 sec-1, and the R2 relaxation rate was 75.6 mM-1 sec-1.

Internalization of Tf-MION
The uptake of Tf-MION by ETR+ cells as a function of time and temperature is shown in Figure 1. At 4°C, there was little internalization of ligand Tf-MION, while at 37°C, the ligand continuously accumulated during the 60-minute incubation period. The progressive accumulation of the ligand at 37°C was consistent with the energy-dependent receptor-mediated internalization of bound ligand. This type of temperature dependence for binding and uptake is typical of the other receptor-mediated endocytotic receptors (31–33).



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  		Figure 1. Graph illustrates 125I-Tf-MION internalization by ETR+ cells at 37°C (A) and 4°C (B). At 37°C, cells progressively accumulate the probe, whereas at 4°C, no internalization is seen; these findings are consistent with receptor-mediated endocytotic mechanism. The cells were acid stripped to ensure the removal of cell surface-bound ligands.

 
Cellular Localization of Tf-MION
The fluorescein-labeled antibody used in this study was specific for the introduced human receptor—that is, it did not bind the endogenous rat transferrin receptor. As shown in Figure 2, both probes showed punctuate staining in the cytoplasm, consistent with endosomal localization. The staining intensity and distribution were similar with both probes and consistent with receptor-mediated internalization. Control experiments with the 9L wild-type cells lacking ETR did not reveal substantial staining with either probe (data not shown).



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  		Figure 2. Dual-channel fluorescence microscopy probing for ETR+ by using rhodamine-labeled Tf-MION (left) and fluorescein-labeled antibodies against human transferrin receptor (right) in a mouse model. (Original magnification, x630.) Note the intracellular colocalization of both probes in the endosomes of the ETR+ cells. The arrows point to punctuate endosomal compartments.

 
Comparison of Induced Signal Intensity Changes with Tf-MION Distribution
Figures 3a and 3b show representative T1- and T2-weighted MR images of an ex vivo specimen from an animal injected with 125I–Tf-MION. The T1-weighted images showed significantly different signal-to-noise ratios between the ETR+ (41.3 ± 1.6 [mean ± SD]) and ETR- (32.1 ± 0.8) tumors (P < .001). Comparison of the ETR+ tumors from animals not injected with 125I–Tf-MION with those from the animals after injection of the probe revealed significant differences in signal-to-noise ratio on the T1-weighted images (35.4 ± 2.5 versus 41.3 ± 1.6, respectively; P < .05). Compared with the ETR- tumors from the injected animals, the ETR- tumors from the noninjected animals did not show significant differences in signal-to-noise ratio on the T1-weighted images (30.3 ± 3.5 versus 32.1 ± 0.8, respectively; P > .05).

The T2-weighted images showed significant differences (P < .001) in signal-to-noise ratio between the tumors (12.8 ± 0.7 for ETR+ versus 20.1 ± 0.9 for ETR-) with and those without injected contrast agent. There was a significant difference in signal-to-noise ratio on the T2-weighted images of the ETR+ tumors from the injected animals compared with those from the noninjected control animals (12.8 ± 0.7 versus 31.2 ± 4.1; P < .05). The ETR- tumors from the animals also showed a significant difference in signal-to-noise ratio on the T2-weighted images (20.1 ± 0.9 versus 26.4 ± 2.7, respectively; P < .05), indicating that there was an accumulation of the probe in the ETR- tumors; this finding was consistent with previously reported biodistribution data (22).

Autoradiographic findings (Fig 3c) showed a general correlation with MR imaging results. The portions of the ETR+ tumors with the highest signal intensity on T1-weighted images and the lowest signal intensity on T2-weighted images showed the highest TF-MION accumulation on the autoradiographs. Conversely, much lower accumulation was seen in the ETR- tumors, except for occasional linear areas consistent with large vessels (Fig 3c). Histopathologic evaluation was performed to examine the tumors for necrosis and tissue homogeneity. Findings in the specimens shown in Figure 3d indicated that the tumors consisted of a solid mass of viable cells and that there were no necrotic lesions in the tissues.



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  		Figure 3. T1-weighted spin-echo (300/11) (A) and T2-weighted spin-echo (2,000/102) (B) ex vivo MR images of ETR+ (left) and ETR- (right) tumors excised from mice. On the T1-weighted images, there is increased signal intensity in the ETR+ tumor due to Tf-MION accumulation. On the T2-weighted images, substantially decreased signal intensity is seen within the ETR+ tumor, as compared with the signal intensity seen within the ETR- tumor. This is secondary to increased accumulation of Tf-MION within the ETR+ tumor. (C) Autoradiographs of ETR+ (left) and ETR- (right) tumors. Note the increased accumulation of Tf-MION within the ETR+ tumor but not within the ETR- tumor. (D) Corresponding hematoxylin-eosin-stained tumor sections (magnification bar, 0.5 cm) show the viable solid tumor mass with no regions of necrosis.

 
To further demonstrate that the signal intensity differences between ETR+ and ETR- tumors were due to probe accumulation, we also imaged the excised tumors from animals injected with 111In–Tf-MION. Figure 4 shows an example of ex vivo nuclear MR images of ETR+ and ETR- tumors from four animals, along with the tumor weights. For each animal, higher amounts of radioactivity were seen in the ETR+ tumor. The tumor weights were essentially the same for the ETR+ and ETR- cell lines. The weight-adjusted counts were significantly higher for the ETR+ tumors than for the ETR- tumors (320.4 ± 45.1 versus 154.1 ± 30.2, respectively; P < .05).



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  		Figure 4. Ex vivo nuclear MR images of ETR+ (left) and ETR- (right) tumors excised from four mice. For each animal, higher amounts of radioactivity (ie, brighter signal) were observed in the ETR+ tumor. Note that the tumor weights are essentially the same for the ETR+ and ETR- cell lines.

 

   DISCUSSION
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
STATISTICAL CONSULTANT...
 REFERENCES
 
We have explored the properties of an engineered form of the transferrin receptor (ETR+), which has been suggested as a marker of gene expression for MR imaging (22,26). Studies of the cellular accumulation of Tf-MION by ETR+ cells in the present investigation revealed a temperature dependence that was consistent with receptor-mediated endocytosis of the contrast agent. At 37°C, progressive intracellular accumulation of Tf-MION can be measured over time. However, the administration of Tf-MION to the cells at 4°C results in little to no accumulation of the contrast agent. The intracellular accumulation of Tf-MION has been visualized both with an antibody specific for the human transferrin receptor and with a ligand for the receptor. Both probes showed a similar intracellular distribution that consisted of punctuate cytoplasmic staining, similar to previous findings describing the transferrin receptor–mediated internalization of native holotransferrin (34–36). Furthermore, we demonstrated previously that Tf-MION uptake into ETR+ cells could be effectively inhibited by transferrin but not by monocrystalline iron oxide, and that this uptake was mediated by the protein (transferrin) bound to monocrystalline iron oxide (26). Our study results suggest that the accumulation of Tf-MION is by means of receptor endocytosis of the transferrin receptor.

Nuclear MR imaging of explanted tumors showed a higher accumulation of 111In-labeled Tf-MION in ETR+ tumors than in ETR- tumors. We obtained a more detailed view of probe distribution throughout the ETR+ tumor by performing autoradiography. The spatial correlation between MR signal intensity and tracer accumulation at autoradiography supported quantitative differences in accumulation of the probe in vivo. The two techniques showed a similar heterogeneous pattern of uptake throughout the ETR+ tumors that presumably was due to regional differences in tumor growth rates, to vascular density, and/or to differences in vascular permeability of the given tumor. The heterogeneous tumor uptake was not due to necrotic regions of tumor failing to take up probe, since no necrosis was evident at histopathologic examination. In addition, MR imaging of tumors from the noninjected animals did not reveal any differences in signal intensity between the ETR+ and ETR- tumors. We thus conclude that the signal intensity change seen at MR imaging was due to the accumulation of Tf-MION in the ETR+ tumors.

In the present study, Tf-MION decreased the signal intensity on T2-weighted images and increased the signal intensity on T1-weighted images. This behavior may seem paradoxical, since superparamagnetic iron–based contrast agents are sometimes thought of as active with T2-weighted pulse sequences. In fact, monocrystalline iron oxide affects both T1 and T2, and the particular ratio of these effects leads to increases in signal intensity at T1-weighted pulse sequences, with physiologically attainable iron concentrations (ie, between 50 and 500 µmol/L). Increases in signal intensity on T1-weighted images achieved by using monocrystalline iron oxide have been observed in ex vivo studies and are predicted on the basis of the spin-echo signal intensity (37).

Our approach to imaging the transferrin receptor involves several amplification strategies to overcome the limited sensitivity of MR imaging for depicting contrast agent enhancement. Our probe of receptor function consists of conjugates of Tf-MION, with approximately two transferrin molecules attached to the surface of a single crystal of iron. Since each crystal consists of 2,064 iron atoms (29), there are approximately 1,000 iron atoms per mole of transferrin, allowing substantially more iron uptake for each cycle of the transferrin receptor than that achieved with native holotransferrin (two iron atoms per mole of holotransferrin). In addition, superparamagnetic iron is far more detectable, per mole of metal, than are paramagnetic chelates such as holotransferrin. If used with susceptibility sensitive gradient-echo pulse sequences, superparamagnetic iron has about 100 times the susceptibility of paramagnetic chelates (38). If used with spin-echo pulse sequences, the R1 and R2 relaxivities of the described iron oxide–transferrin conjugate (see Results) are substantially higher than those of gadopentetate dimeglumine, which are 4–5 mM-1 sec-1 (39). Finally, we used ETR+ mutants, which do not down regulate transferrin receptor expression with the accumulation of intracellular iron because of a lack of down-regulatory elements (26).

Overall, up to 8 x 106 nanoparticles per cell can be internalized into ETR+ cells within an hour (26), and this amount of superparamagnetic iron per cell was sufficient to enable the detection of a single cell by using microscopic MR imaging (40). Therefore, in vivo, as few as one labeled cell per 50-µm3 voxel (spatial resolution of MR imaging in small living animals) can cause sufficient signal intensity abnormalities to be detectable at MR imaging.

Our use of a cycling endocytotic receptor, the transferrin receptor as a reporter gene, differs from amplification strategies that involve enzymes for imaging gene expression with MR. Beta-galactosidase activity has been imaged by using the synthesis of a gadolinium chelate that has reduced effects on water relaxation due to a galactopyranosyl ring blocking water access to the gadolinium ion (23). Beta-galactosidase cleaves the sugar residue and thus exposes the gadolinium ion to water and increases the effect on water relaxation. The beta-galactosidase activity of specific cells of Xenopus laevis embryos has been imaged after intracellular injections of the substrate. A second enzyme amplification system that has been investigated as a reporter gene for MR imaging is tyrosine. After transfection with the tyrosine gene, 293 cells in culture exhibited increased signal intensity due to the accumulation of melanin (41).

In contrast to these approaches, the expression of the transferrin receptor with tumor cells implanted with ETR+ has been imaged after intravenous injection of Tf-MION in mice (22). This experimental design is substantially closer to the protocol required for the clinical imaging of a reporter gene at MR imaging than is that involving the use of beta-galactosidase or tyrosinase reporter genes in their current status. Further improvements in the chemistry of the Tf-MION probe have been achieved (42), and experiments on MR imaging of in vivo gene transfer by means of viral and nonviral DNA delivery systems are underway.

Practical applications: The proposed study has several potential practical applications. First, the use of a practical MR marker gene of gene expression may permit monitoring of gene therapy in which exogenous genes are introduced into the body to eliminate a genetic defect or to add an additional gene function to tumor cells. Second, because it has become increasingly important to image endogenous gene expression during development and pathogenesis, it may be possible, with advances in the establishment of transgenic animal models, to create transgenic animals that encode imaging marker genes, which are under the control of different promoters. In this way, the promoter activity can be directly imaged during pathogenesis.


   STATISTICAL CONSULTANT COMMENTARY
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 STATISTICAL CONSULTANT...
REFERENCES
 
A reviewer was concerned that the small size of the experimental groups—that is, three or four mice—would not permit the rejection of a null hypothesis, regardless of the P value, when a paired t test was performed to evaluate the treatment effect. It should be noted that the calculation of the P value for the paired t test takes the sample size into account through the degrees of freedom associated with the distribution of the test statistic. As a result, a small P value is associated with an event that has a low probability of occurring, regardless of the sample size. Since the P value is the probability of observing a value of the test statistic that is more extreme than the observed value, given that the hypothesis being tested (the null hypothesis) is true, a small P value leads to the conclusion that the observed data may not be consistent with the null hypothesis.

The data in the Table may help to visualize the relationship between the P value, the number of observations, and the value of the test statistic, t. For example, it can be seen that when the sample size is two, the value of the test statistic cannot be smaller than 12.706 for the P value to be .05 or less. It can be seen also that the test statistic must be at least 63.657 before the associated P value can be less than or equal to .01.


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  		Relationship between P Values, Numbers of Observations, and t Test Statistic Values

 


   FOOTNOTES
 
Abbreviations: ETR = engineered human transferrin receptor, Tf-MION = transferrin and monocrystalline iron oxide

Author contributions: Guarantors of integrity of entire study, A.M., R.W.; study concepts, A.M., J.P.B., R.W.; study design, A.M., R.M.B.; literature research, A.M., L.J.; experimental studies, A.M., R.M.B.; data acquisition, A.M., R.M.B., R.W.; data analysis/interpretation, A.M., L.J., R.W.; statistical analysis, A.M.; manuscript preparation, A.M., L.J., J.P.B., R.W.; manuscript definition of intellectual content, A.M., L.J., R.W.; manuscript editing, L.J., R.W.; manuscript revision/review and final version approval, all authors.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 STATISTICAL CONSULTANT...
 REFERENCES
 

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