Targeted Signal-Amplifying Enzymes Enhance MRI of EGFR Expression in an Orthotopic Model of Human Glioma

Epidermal growth factor receptor (EGFR) is a member of ErbB receptor kinase family, which is overexpressed predominantly in non–small cell lung cancer, colorectal and squamous carcinomas as well as in glioma cells (1–3). EGFR plays an important role in cancer pathogenesis by readily undergoing ligand-dependent dimerization followed by autophosphorylation of the EGFR dimer resulting in downstream proliferative and antiapoptotic signaling in cancer cells (4). Wild-type EGFRwt overexpression closely correlates with receptor gene amplification and has been previously established as a significant and independent unfavorable predictor of overall survival of glioblastoma patients (5). The truncated EGFRvIII variant of the receptor is constitutively activated and is a hallmark of aggressive gliomas (6, 7). Since EGFR level is low or even undetectable in normal brain cells, this receptor is an appealing molecular target not only for molecular therapies but also as a potential marker molecule for visualization and characterization of gliomas during their response to therapy. Several recombinant monoclonal antibodies (mAb) have been developed during the past decade for achieving specific targeting of the N-terminal extracellular domain III of EGFR with the resultant inhibition of EGF binding and/or receptor dimerization (8–10). Anti-EGFR mAb (11, 12) and EGF ligand (13, 14) have recently been suggested as agents for targeted imaging of EGFR expression using near-IR fluorescence. Unlike in vivo imaging of fluorescence, MRI does not suffer from the drawback of limited depth penetration and scattering of light. However, the sensitivity of MRI to the local molar concentration of contrast agent (CA) is orders of magnitude lower than fluorescence or radionuclide imaging, which limits applicability of MRI for receptor imaging (15–17). Proton MR receptor imaging relies on the ability of CAs associated with the receptor site to shorten relaxation times of nearby water molecules. The number of CA molecules (e.g., the number of chelated paramagnetic cations that can be used for direct labeling of mAbs while still maintaining the appropriate binding affinity of mAbs to the target site) is usually not sufficient for generating adequate MR contrast. Other studies circumvented the problem of insufficient sensitivity by coating iron oxides with mAbs (18–22), or by using gadolinium (Gd)-based targeted micelles (23) and dendrimers (24). MRI sensitivity is thus increased due to either clustering of many Gd cations or, alternatively, due to high superparamagnetism of iron oxide. However, linking of nanosized CAs to antibodies can result in a decrease in tissue penetration after extravasation in tumors and in an increase of nonspecific MR signal (25, 26).

Several studies have looked into alternative uses of mAbs for imaging tumor-associated receptors using contrast-enhanced MRI (27–29). For example, a pretargeting technique has been suggested for enhancing mammary adenocarcinomas by injecting Gd-labeled avidin (25) or dendrimers (29) as a way of achieving specific association with HER-2/neu-bound biotinylated mAb (trastuzumab; ref. 28). We have previously developed a novel pretargeting approach on the basis of an enzyme-mediated amplification of MR signal as a result of accumulation of small molecular weight Gd-labeled substrates at the target site (30). The advantage of this imaging strategy is that small CA molecules accumulate in brain tumors at a faster rate and with less heterogeneity than macromolecular agents (26). The resultant target-to-background contrast ratio can potentially be achieved earlier than in the case of nanoparticle or dendrimer pretargeting due to much faster elimination of the nonreacted CA substrate from the circulation as compared to nonbound Gd-labeled macromolecules or nanoparticles. The other potential advantage is in achieving higher MR signal due to the Gd relaxivity increase that results from the enzyme-activated substrate binding to proteins (31, 32). The increase in relaxivity depends on magnetic field strength and strongly contributes to the MR signal increase at 1.0 to 2.0 Tesla.

The main goals of the current work were (i) to test in vitro the tumor-pretargeted enzyme-mediated amplification system using cells expressing either EGFRwt, or both EGFRwt and EGFRvIII; (ii) to compare in vivo kinetics of MR signal decay after the administration of Gd-labeled peroxidase substrate (diTyr-GdDTPA) with or without pretargeting of the EGF receptor with mAb conjugated to a signal amplification pair of enzymes.

Horseradish peroxidase (HRP)-reducing substrate di(tyramido)-DTPA(Gd) and conjugates (Fig. 1) were synthesized as described in the work of Querol and colleagues (32); mAb conjugates were synthesized using bisaromatic hydrazone method, purified on Superdex200 HPLC columns (GE Life Sciences), and analyzed as described in the work of Bogdanov and colleagues (30).

Gli36ΔEGFR (33) and wild-type Gli36wt cells (34) were propagated on 10% FCS (fetal calf serum) 90% RPMI 1640 in the presence of penicillin/streptomycin and 0.5 μg/mL puromycin (Gli36ΔEGFR). mAb conjugates were cross-titered on 96-well plated live cells using sequential dilutions in the range of 1,000 to 7.5 ng total conjugate (i.e., a mixture of mAb-HRP and mAb-GOX) per well and peroxidase activity associated with the cells was determined as in the work of Bogdanov and colleagues (30).

Flow cytometry of Gli36ΔEGFR and Gli36wt cells was performed by using 1 μg/mL Alexa Fluor 488–labeled EMD72000 or cetuximab (humanized mAb C225; ref. 35).

Cell internalization was studied by using mAb-HRP:mAb-GOX conjugate mixture at 1:2 (w/w ratio). Adherent cells in 6-well plates (4 million cells/well) were incubated with conjugate mixtures either at 4^○C or at 37^○C. The surface-bound conjugates were eluted with 0.5 mL cold 0.2 mol/L glycine, pH 2.5, for 15 minutes. The eluate was immediately neutralized with 1 mol/L Tris, pH 8.0. To extract internalized conjugates, 0.5 mL of 1.0% Igepal CA-630 in the presence of protease inhibitors was added to each well and plates incubated for 15 minutes. The amount of bound and internalized conjugates was determined by measuring the initial rates of HRP/GOX-coupled ABTS (2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) oxidation by adding of 5 mmol/L ABTS, 5 mmol/L glucose (final concentrations) in sodium citrate, pH 5.5, to the sample aliquots and measuring absorbance at 405 nm over time. The serially diluted conjugate mixture at a constant mAb-HRP:mAb-GOX ratio and known concentrations were used for calibration.

Membrane proteins were extracted using CNM compartmental protein extraction procedure (BioChain Institute Inc.) following manufacturer's recommendations with subsequent immunoblotting of protein-normalized lysates on PVDF (polyvinylidene difluoride) membranes using either anti-EGFR mAb (ab3103; AbCam) as primary antibody or EMD72000-HRP conjugates with subsequent digital imaging.

All procedures were performed as approved by the UMMS Institute Animal Care and Use Committee. An orthotopic human glioma xenograft model was obtained by stereotaxically implanting 1 × 10⁵ Gli36ΔEGFR or Gli36wt cells in 3 μL of 10% Matrigel in serum-free RPMI under aseptic conditions at 37°C (2.5 mm posterior to bregma, 2 mm lateral to midline, and depth of 3.5 mm) in the brain of athymic rats (Harlan, 150–180 g, n = 16) 14 days before the first imaging session. Throughout the imaging procedure, the animals were maintained at 37°C and anesthetized using 1.5% isoflurane in a 30% oxygen/70% nitrogen mixture. A 45-mm diameter, 30-mm long birdcage RF (radio frequency) coil was used. A 26-gauge catheter capped with a needle port was placed in the tail vein for CA administration.

MRI measurements were performed using a Philips Achieva 3.0 T/60-cm bore MRI scanner equipped with 80 mT/m actively shielded gradients. To monitor temporal evolution of signal enhancement following CA delivery, multislice, T₁-WT MR images (TR/TE = 700/8.2 ms) were acquired at various time points following CA infusion. Other imaging parameters were: slice thickness = 1.5 mm; slice separation = 0.15 mm; field-of-view = 25.6 mm × 25.6 mm; data acquisition matrix = 256 × 128 (zero padded to 256 × 256), 4 NEX. Two weeks after tumor cell implantation, each animal was imaged on 2 occasions: on day 1 (2 weeks after tumor cell implantation), a precontrast image was acquired followed by i.v. injection of 0.1 mmol/kg diTyr-GdDTPA. Twenty T₁-WT images were then acquired continuously over a 2-hours period. On day 2 (the day after day 1), the animals were first preinjected with 150 μg of mAb-HRP/mAb-GOX mixture at 2:1 (w/w ratio, 0.3 mL) in the tail vein. A separate group of Gli36ΔEGFR tumor animals were preinjected with nonspecific antibody conjugates. Five hours later, a precontrast image was acquired followed by i.v. injection of 0.1 mmol/kg diTyr-GdDTPA. Thirty T₁-WT images were then acquired continuously over a 3-hours period. Precontrast T₂-WT images were acquired on both days to corroborate the presence of tumor observed in the T₁-WT slices. The temporal evolution of the signal decay in the tumor following infusion of diTyr-GdDTPA was evaluated separately for each of the animal groups. Depending on the size of the tumor, one to four slices were selected. Image analysis was performed using region of interest (ROI) for tumor interface and core regions with an aid of ImageJ software (36).

Following the MRI studies, animals were euthanized and their brains were removed. Under a dissecting microscope, histologic slices were obtained ±2 mm anterior/posterior of the needle track and then embedded in OCT medium. Frozen sections (6 μm) were fixed in acetone, treated by TBS/EDTA, pH 8, at 65°C for inhibiting endogenous phosphatase activity and blocked using 2% serum/TBS. The sections were incubated with anti-EGFR mouse mAb (Abcam) or anti-HRP mAb (Abcam) followed by anti-mouse alkaline phosphatase (AP)-linked antibodies and BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium; Roche). Staining for residual peroxidase activity was achieved by using a DAB kit (Vector Labs). Immunofluorescent staining was performed on frozen sections blocked with 2% serum, 0.05% Tween-20, PBS, pH 7.4. Alexa Fluor 488–labeled EMD72000, Cy3-labeled mouse anti-rat CD31 mAb (clone TLD-3A12; Abcam) were used for visualizing EGFR and endothelial CD31 expression, respectively. For detecting HRP in the tissue sections, digoxigenin-labeled mouse anti-HRP mAb (clone 2H11; Abcam) was used. Digoxigenin-labeled antibody binding was visualized using custom Cy5.5-labeled anti-digoxigenin F(ab′)₂ (Roche; ref. 37).

Detailed methods are included in Supplementary Materials.

The synthesis of peroxidase (HRP) and glucose oxidase (GOX) conjugates of humanized chimeric antibody (EMD72000; Merck) that catalyze oligomerization of substrate di(tyramido)-DTPA(Gd) (Fig. 1) was performed using conditions optimized to facilitate enzyme conjugation with minimal antibody binding affinity loss (30). Gel electrophoresis of purified conjugates showed the formation of mAb-GOX (290 and 460 kD bands) and mAb-HRP (220 and 400 kD bands; Fig. 2A). Since GOX is a 2-subunit enzyme, the band corresponding to the 69-kD deglycosylated GOX subunit was present in the SDS-treated mAb-GOX conjugates (Fig. 2A, lane 4). The conjugation reaction conditions prevented the formation of cross-linked products as evidenced by the lack of binding specificity to EGFR-positive cells. Immunoblotting experiments showed that optimized conjugates reacted with the same set of receptor variants (i.e., EGFRwt in Gli36wt and EGFRwt/EGFRvIII in Gli36ΔEGFR cell lysates) as did the control anti-EGFR antibody (Fig. 2B-a2). Overall, Gli36ΔEGFR cells contained 2.8 times more mAb-reactive EGFR than Gl36wt cells and this result correlated with the results of flow cytometry performed using fluorescent-labeled cetuximab (see Supplementary Fig. 1SA). However, EMD72000 showed stronger preference for Gli36ΔEGFR cells, that is, 7.2 times higher binding levels than Gli36wt cells (Supplementary Fig.1SB). Using ¹¹¹InDTPA-labeled EMD72000 mAb, we determined that Gli36ΔEGFR had 4.6 ± 0.3 × 10⁵ binding sites per cell as opposed to a control nonbinding anti-EpCAM antibody, which showed no detectable binding to the surface of Gli36ΔEGFR cells. Optimization of the mAb-HRP:mAb-GOX conjugate ratios was performed to determine the conditions that provide sustainable release of hydrogen peroxide as a result of d-GOX, without inhibiting the binding of mAb-HRP to cells (see Supplementary Fig. 1SB and C). These experiments showed no detectable loss of cell viability observed below 0.25 μg mAb-GOX per well. Cross-titration demonstrated that the highest HRP activity was detectable at the weight ratios of mAb-HRP:mAb-GOX of 1:2 in the range of 0.03 (0.06) to 0.25 (0.5) μg of mAb-GOX (mAb-HRP) conjugate per well. In contrast to Gli36ΔEGFR, the same dilutions of conjugates added to Gli36wt cells resulted in lower levels of cell-associated enzymatic activity. The titration of mAb-HRP:mAb-GOX conjugate mixture (1:2 w/w) allowed determination of the effective EC₅₀ equal to 0.21 μg (Fig. 2C). Finally, we studied binding and internalization of optimized conjugate mixtures (Fig. 2D) in both cell lines by measuring the complementing enzymatic activities, which are essential for MR signal amplification. The differences observed during flow cytometry were further confirmed in these experiments showing that Gli36ΔEGFR cells were binding 10 times more of mAb conjugate-mediated enzymatic activity than Gli36wt, of which about 25% remained externalized on the surface of the cells.

The HRP/GOX-mediated increase of relaxivity (i.e., strength of MR CA) in the reaction mixture containing the paramagnetic substrate diTyr-GdDTPA was compared between low external magnetic field and the field of the MR imaging unit that was used for animal experiments. Mixtures of conjugates and paramagnetic substrate diTyr-GdDTPA in the presence of d-glucose always resulted in shorter T₁ relaxation times (i.e., higher average longitudinal relaxivity r1, see Supplementary Table S1). The measured difference in molar relaxivity between the substrate alone and reaction mixtures containing HRP and GOX conjugates was clearly greater at lower magnetic field (0.47 T vs. 3 T). The reaction resulted in 2.7 times higher r1 if measured at 0.47 T as opposed to 5% increase of r1 at 3 T. The simple addition of proteins in the solution did not result in large relaxivity gains. However, in the presence of both plasma proteins and mAb conjugates, the relaxivity increase was measurable and higher at 3 T and showed a 20% r1 increase.

The paramagnetic substrate-mediated enhancement of human glioma xenografts (Gli36ΔEGFR and Gli36wt) was studied using MRI in vivo. To account for tumor heterogeneity, the experiments were performed on consecutive days in each animal. On the first day, only the CA substrate was administered (day 1). On the next day–after CA had been completely eliminated–a second experiment was conducted with preinjection of mAb conjugates 5 hours before injecting the CA (day 2). Figure 3A shows sequential T₁-WT rat brain MR images depicting Gli36ΔEGFR xenograft enhancement as a function of time post-i.v. injection of diTyr-GdDTPA. The top row of images shows the temporal washout of diTyr-GdDTPA with no anti-EGFR mAb conjugate pretreatment (day 1). The bottom row of images shows the temporal washout of diTyr-GdDTPA at 5 hours following pretreatment with anti-EGFR mAb conjugates (day 2) in the same slice for the same animal. For both days, the T₁-WT images showed strong initial enhancement of the tumor within minutes after i.v. diTyr-GdDTPA injection. However, following i.v. injection of diTyr-GdDTPA with EGFR-targeted conjugate pretreatment (Fig. 3A, day 2: 8 minutes), the initial enhancement was significantly higher than without EGFR-targeted conjugate pretreatment (Fig. 3A, day 1: 9 minutes), especially in the tumor interface region. Moreover, images obtained after the EGFR-targeted conjugate preinjection (Fig. 3A, bottom row) showed more detailed tumor structure with areas of focal enhancement in both the tumor/normal brain interface and core at all time points postinjection of the paramagnetic MR CA. With or without conjugate pretreatment, the tumor interface consistently showed higher T₁-WT signal enhancement compared with the core.

Animals preinjected with anti-EGFR mAb conjugates displayed a delayed retention of MR CA compared with animals not receiving EGFR-targeted conjugate pretreatment. Without a preinjection of anti-EGFR conjugates (day 1), most of the diTyr-GdDTPA had washed out of both the tumor/normal brain interface and core regions by 117 minutes after i.v. injection (Fig. 3A, top row). However, after allowing the EGFR-targeted conjugates to accumulate in the tumors (day 2), significant T₁-WT hyperintensity persisted in the tumor interface at approximately the same time point (Fig. 3A: day 2 - 116 minutes) post-diTyr-GdDTPA injection. Furthermore, T₁-WT enhancement in the tumor interface remained visible for 170 minutes after CA administration (Fig. 3A: day 2 - 170 minutes). In contrast to EGFR-targeted conjugates preinjection, no retention of MR signal was observed when Gli36ΔEGFR-bearing animals were preinjected with anti-EpCAM–targeted conjugates which do not bind to Gli36ΔEGFR cells (Fig. 3B, compare top and bottom rows).

The corroborative histology performed after the final MRI session showed areas of EGFR-positive staining and revealed 2 tumor masses (2.7–3 mm in diameter) with multiple microdeposits around the tumor/normal brain interface (Fig. 3D, arrows). These same features were also easily identifiable on MR images after the injection of anti-EGFR conjugate followed by the CA (Fig. 3C). These areas of tumor/brain were also positively stained for HRP activity using diaminobenzidine (Fig. 3D). By performing immunofluorescent visualization of EGFR and CD31 expression, we observed the presence of multiple blood vessels feeding the expanding tumor (Fig. 4A). Visualization of HRP accumulation by using anti-HRP antibodies (Fig. 4B) verified the results obtained using peroxidase enzymatic activity detection in brain sections (Fig. 3D).

Detailed kinetic analysis of in vivo MR signal enhancement as a function of time was performed to assess the efficacy of EGFR-targeted imaging. Figure 5 shows the normalized T₁-WT signal intensities in the Gli36ΔEGFR tumor interface and core regions as a function of time post-CA injection. These plots were derived from the tumor images of the same representative animals shown in Figure 3A and B. The relative percent change in T₁-WT signal intensity of the interface and core regions was significantly higher with EGFR-targeted conjugate administration (day 2, Fig. 5B), as compared with without pretreatment (day 1, Fig. 5A), at the initial time points. With EpCAM-targeted conjugate administration (day 2), there was no significant change in initial T₁-WT signal intensities of the interface and core regions between days 1 and 2; both days showed similar washout behavior of the interface and core signal intensity decay (SID) curves (Fig. 5).

Interestingly, the SID curves for tumor interface and core regions in animals pretreated with EGFR-targeted conjugates (day 2; Fig. 5B) depict 2 separate decay components (long τ₁ and short τ₂), whereas the washout curves for the corresponding regions without EGFR-targeted conjugate injection (day 1; Fig. 5A) show only a single decay component (τ₀). On the basis of the χ² analysis using an F test (Supplementary Material), a biexponential function was found to best model the SID curves for the tumor interface and core regions after injection of EGFR-targeted conjugates (day 2). Without EGFR-targeted conjugate preinjection (day 1), a monoexponential function was found tobestmodel both the regions. For treatment with and without EpCAM-targeted conjugates and Gli36wt tumors (treated with and without EGFR-targeted conjugates), a monoexponential elimination function was the best model for all SID curves [see Table 1 for all decay time constant (DTC) values].

Imaging of receptor expression in cancer with high spatial resolution usually requires MRI assisted with imaging probes (38, 39). Due to the inherent nonspecific uptake of nanosized probes in nontarget cells and slow elimination of nonbound CA from blood, the specific imaging signal is frequently obscured. With a goal of imaging EGF receptor expression in gliomas, we investigated a novel strategy on the basis of specific local retention of paramagnetic products of di(tyramido)-DTPA(Gd) (diTyr-GdDTPA; Fig. 1), that is, a substrate of peroxidase (30, 40). We applied the strategy in models of orthotopic human gliomas that either expressed the wild-type EGFR or both the wild-type receptor and EGFRvIII (33). The anti-EGFR mAb (EMD72000) was covalently linked with HRP and GOX, that is, to the enzymes thatfunction as a self-complementing enzymatic signal amplification system (30). In cell culture experiments, these conjugates showed a remarkable preference for truncated EGFRvIII mutant form expressed in Gli36ΔEGFR cells but not in Gli36wt counterpart. This effect can be explained by the steric constraints in accessibility of epitope (Ser460-Gly461) on EGFR domain III recognized by EMD72000 antibody (41). Since even after prolonged incubation in cell culture 25% of mAb conjugates were resistant to internalization, we anticipated that our strategy enables reliable MR imaging of EGFRvIII with high specificity due to the retention of CA at the sites of mAb conjugate colocalization.

To test this assumption, we designed experiments that directly compared in vivo elimination kinetics of diTyr-GdDTPA in the same tumor-bearing animals. These experiments were performed initially in the absence of mAb conjugates and then after the preinjection of a mixture of conjugates at the optimized HRP:GOX ratio. As a result, we were able to compare kinetics of the measured MR signal decrease due to the washout of diTyr-GdDTPA and its reactive products from the total tumor volume, as well as from volumes corresponding to highly vascularized tumor “interface” (Fig. 4 and Supplementary Fig. 2S) and the remaining “core” volumes. From the quantitative kinetic analysis of the MR signal intensities, we concluded that the initial MR signal intensity enhancement for both interface and core regions was significantly higher for Gli36ΔEGFR tumors that were pretreated with EGFR-targeted mAb conjugates (day 2) as compared with the same tumors before the injection of conjugates (day 1) or Gli36wt tumors. When nontargeted mAb conjugates (control) were preinjected into Gli36ΔEGFR tumors, no significant signal change was observed compared with day 1. Binding of paramagnetic products of enzymatic reaction are more efficient CAs and their binding to the EGFRvIII-positive tumors resulted in a long-term retention/enhancement (Fig. 3A), whereas oligomerization or binding to proteins in the extracellular compartment resulted in a short-term tumor enhancement. The effects caused by paramagnetic products binding were previously observed and characterized in several similar enzyme-mediated reactions (32, 42).

The retention of MR signal enhancement in tumors that were pretreated with specific targeted conjugates relative to those that were not, resulted in a slower CA elimination consistent with the binding of the CA to the target cells in the presence of targeted enzyme amplification pair. As expected, signal enhancement of the tumor interface was initially higher than that of the rest of the tumor volume (“core”), independent of whether the animals were preinjected with conjugates or not. This was caused by high vascular density of the tumor interface (Fig. 3), which corresponds to an area of enhanced transvascular permeability and higher functional blood volume (43). The higher local permeability translates into higher concentrations of targeted conjugates as well as the substrate compared with less vascularized core. The results of direct staining for HRP activity or detection of HRP in tumors by using immunofluorescence suggest this difference (Figs. 3 and 4).

The inevitable variations in tumor size and tumor heterogeneity dictated the need in a parameter that would allow comparing MRI results obtained in different animals and tumors. We determined that kinetic analysis of temporal decay of the normalized MR signal intensities can be used for this purpose (Table 1). The calculated DTC values can be correlated to tumor perfusion, vascular permeability, and the volume of the extravascular extracellular space (EES; see Supplementary Material).

By performing SID analysis, we found that in the absence of conjugate preinjection, the washout of diTyr-GdDTPA was monoexponential throughout the tumor volume generating a single DTC (τ₀) in both tumor models (Fig. 5A, Table 1). In each case, τ₀ was attributed to the washout of the free CA since the substrate does not bind to plasma proteins. This assignment is justified because diTyr-GdDTPA is not expected to have any affinity for tumor cells in the absence of receptor-targeted conjugates. The control EpCAM-targeted conjugates in Gli36ΔEGFR tumors (day 2) as well as EGFR-targeted conjugates in Gli36wt cells (day 2) similarly showed monoexponential decay (Table 1) since control anti-EpCAM conjugates did not bind to glioma cells of our study and Gli36wt cells were binding very low amounts of EMD72000 conjugates.

Following the preinjection of Gli36ΔEGFR-bearing animals with EGFR-targeted conjugates on day 2, the diTyr-GdDTPA elimination changed from monoexponential to biexponential for both the tumor interface and core regions (Fig. 5, Table 1). The elimination of free (and/or oligomerized) substrate was responsible for short DTC (τ₂), whereas the long DTC (τ₁) component was attributed to paramagnetic reaction products retained by tumor cells. Importantly, the comparison of τ₁ and τ₂ DTC values suggest that in addition to dynamic acquisition delayed MR imaging can also be useful when imaging receptors: the retention of tumor-bound paramagnetic product resulting from a specific enzymatic reaction can be detectable in the tumor interface for several hours postadministration of the substrate while the free substrate is eliminated within an hour.

In conclusion, we performed quantitative analysis of MR dynamic signal enhancement in human glioma xenografted animals that were (i) imaged first by using “nonspecific” paramagnetic CA and (ii) imaged with the same CA after preinjecting of anti-EGFR antibody conjugates. The enzyme linked to EGFR-targeted antibodies converted the CA into reactive products thereby profoundly changing the tissue elimination kinetics. Therefore, in addition to imaging of the MR signal enhancement associated with tumor tissues in vivo, the amplification strategy results in a kinetic signature. The analysis of images acquired dynamically or by using endpoint imaging can establish the presence of cell surface marker and quickly delineate the areas where this marker molecule can be targeted for therapeutic purposes.

No potential conflicts of interest were disclosed.

The authors thank Dr. Suresh Gupta, Jamie O'Callaghan, and Dr. Hye-Won Kang for assistance with several aspects of this work including cell culture experiments and animal surgery. They also thank Merck KGaA (Darmstadt, Germany) for providing EMD72000 antibody and Dr. Mary Mazzanti for editing the text.

This work was supported by 5R01EB000858 grant to A. Bogdanov.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.