Enhanced Tumor Detection Using a Folate Receptor-Targeted Near

Lark, R. H., Coney, L. R., Fort, D. W., Frasca, V., and Zurawski, V. R., Jr., et al. ...... IEEE Engineering in Medicine and Biology Magazine 2004...
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Bioconjugate Chem. 2003, 14, 539−545

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Enhanced Tumor Detection Using a Folate Receptor-Targeted Near-Infrared Fluorochrome Conjugate Woo Kyung Moon,† Yuhui Lin, Terence O’Loughlin, Yi Tang, Dong-Eog Kim, Ralph Weissleder, and Ching-Hsuan Tung* Center for Molecular Imaging Research,Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129. Received January 22, 2003; Revised Manuscript Received March 3, 2003

Fluorescence optical imaging technologies are currently being developed to image specific molecular targets in vivo. Detection technologies range from those providing microscopic detail to whole body imaging systems with potential clinical use. A number of target-specific near-infrared imaging probes have recently been developed to image receptors, antigens, and enzymes. The goal of the current study was to evaluate a new near-infrared (NIR) folate receptor (FR)-targeted imaging probe for its ability to improve detection of FR-positive cancers. We hypothesized that modification of folate would retain receptor affinity in vivo, despite the bulkier NIR fluorochrome, NIR2 (em ) 682 nm). Cellular uptake of the NIR conjugates was significantly higher in FR-positive nasopharyngeal epidermoid carcinoma, KB cells, compared to FR-negative human fibrosarcoma, HT1080 cells. When tumors were implanted in vivo, equal-sized KB tumors showed a 2.4-fold higher signal intensity compared to HT1080 tumors (24 h). The maximum signal-to-background ratio (3-fold) was observed at 24 h in KB tumor. Injection of the unmodified NIR2 fluorochrome did not result in persistent contrast increases under similar conditions. Furthermore, tumor enhancement with the NIR2-folate probe persisted over 48 h and was inhibitable in vivo by administration of unlabeled folate. These results indicate that folate-modified NIR fluorochrome conjugate can be used for improved detection of FR-positive tumors.

INTRODUCTION

Light-based imaging technologies are routinely employed clinically (e.g., laparoscopy, colonoscopy, angioscopy, etc.), yet most applications rely on visual inspection of anatomic abnormities. More recently the use of fluorescent imaging probes has been shown to significantly enhance tumor detection (1-4), facilitate identification of small preneoplastic lesions (5), enable in vivo characterization (6), improve the detection of metastatic spread (7), and allow objective assessment of new therapeutic paradigms (8) in animal studies. Potentially those in vivo optical imaging technologies can be translated into clinical applications. It has become clear that imaging in the near-infrared spectrum (700-900 nm) allows most efficient photon migration through the tissue, while autofluorescence is also minimized in this region (9). Despite these obvious opportunities, there is a paucity of near-infrared imaging agents with biocompatibility, stability, and conjugatability. We have previously shown that asymmetric cyanine dyes have ideal properties (10), and currently we are under the process of testing their preclinical utility by conjugating them to affinity ligands. The goal of this study was to determine whether a fairly abundant receptor on tumor cells could serve as a target for near-infrared fluorescence (NIRF)-enhanced optical imaging. While other studies have successfully investigated the somatostatin receptor as a target for NIRF imaging (3, 4), we chose the folate receptor (FR), a * Corresponding author: Ching H. Tung, Ph.D., Center for Molecular Imaging Research, Massachusetts General Hospital, 149 13th St., Rm. 5406, Charlestown, MA 02129. Tel: (617) 7265779, Fax: (617) 726-5708, e-mail: [email protected]. † Current address: Department of Radiology and Clinical Research Institute, Seoul National University Hospital, Korea.

glycosylphosphatidylinositol-anchored protein. The FR is ideally suited for this studies because its was overexpressed in several cancers (including breast, lung, cervical, ovarian, colorectal, renal, and nasopharyngeal cancers) (11-13), low expression in normal tissues (14, 15), proven utility for MR and nuclear imaging (16-21), and cost-effectiveness for possible later clinical trials. We have recently described the synthesis of a folate-nearinfrared fluorochrome conjugate (22). In the current study, we evaluate a FR-targeted conjugate for in vivo NIRF imaging, specifically on tumor enhancement characteristics as a function of time and comparison of the folate and NIRF conjugate to the NIR fluorochrome alone. Our results revealed that a folate-modified NIRF conjugate has potential applications in improved detection of FR positive tumors. MATERIALS AND METHODS

Imaging Probe. The folate-targeting optical probe (Figure 1), NIR2-folate, consisting of a near-infrared fluorochrome (10) and folic acid, was synthesized as previously described (22). Briefly, folic acid was first reacted with 2,2′-(ethylenedioxy)bis(ethylamine) using diisopropylcarbodiimide as the coupling agent in DMSO. The N-hydroxysuccinimide activated ester of nearinfrared fluorochrome (NIR2) (10) was linked to the amino-derivatized folic acid in 0.1 M NaHCO3/DMF. The final conjugate was purified by C-18 reverse-phase HPLC and characterized by mass spectroscopic analysis. The NIR2-folate has an excitation maximum at 665 nm and an emission maximum at 686 nm. Modeling of Target Interaction. Interaction of NIR2-folate and FR was studied using a model structure of riboflavin binding protein (RFBP) with bound riboflavin (23). The crystal structure of RFBP and riboflavin

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Figure 1. (A) Synthetic scheme of folate receptor (FR)-targeting near-infrared fluorescence probe (NIR2-folate). (B) Stereoview of computer modeling of NIR2-folate and its receptor analogue, riboflavin binding protein (RFBP). The RFBP is rendered in white as a vdw surface. Folic acid and NIR2 were colored in red and gold, respectively. The ring of folate is sandwiched between the planar residues tyr 75 and trp 156. The spacer (blue) extends from the binding pocket, allowing free rotation of NIR2.

was used to determine a model active site for simulated docking of NIR2-folate in the ligand fit module of Cerius2 (Accelrys, San Diego, CA) (24). The docking algorithm performed 106 cycles of Monte Carlo simulations. First, a search using ligand torsion angles was used to generate random conformations. Docking scores were then determined by three algorithms, a softened Lennard-Jones 6-9 potential, the evaluation of buried polar surface area for attractive and repulsive interactions. The top 20 conformations placed the folate moiety of NIR2-folate in the binding pocket, in a parallel plane between trp156 and tyr75 of RFBP, which correspond to trp168 and tyr82 of FR. The RFBP van der Waals surface was generated using the program MSMS (25). All models were rendered with Raster3D (26). Characterization of Cell Lines. A human nasopharyngeal epidermoid carcinoma, KB, and a human fibrosarcoma, HT1080, cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). These cell lines were selected because of putative FR overexpression (KB) or lack of detectable FR expression (HT1080) (11). Both cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO2 in folate-

deficient medium RPMI 1640 with 10% fetal calf serum (Gibco BRL/Life Technologies, Rockville, MD). To confirm receptor expression levels, we first determined cellular binding/internalization using 3H-folate. KB or HT1080 (106 cells) grown in 12-well plates were incubated at 37 °C for different times (1, 10, 30, 60, 120 min) with 50 nM 3H-folate (specific activity 34.5 Ci/mmol, American Radiolabeled Chemical Inc, St. Louis, MO). At the end of the incubation, cells were harvested using 0.1% Triton X-100, and the radioactivity (pmol/106 cells) was determined using a scintillation counter. For competitive inhibition studies, KB cells were incubated with different amounts of folic acid or NIR2-folate probe (5, 50, 500, and 5000 nM). Cellular Uptake of the NIR2-Folate Probe. Similar to previous uptake experiments, the NIR2-folate probe was tested in cell culture using KB and HT1080 cells grown at 70% confluency on glass cover slips. The culture medium was replaced with 0.5 mL of fresh medium containing 1 µM NIR2-folate probe and incubated for 1 h at 37 °C. Cells were washed three times and fluorescence microscopy was performed using an

Folate Receptor-Targeted NIR Fluorochrome Conjugate

inverted epifluorescence microscope (Zeiss Axiovert, Thornwood, NY). Animal Preparation. All animal studies were approved by the institutional animal care committee. Ketamine (90 mg per kilogram of body weight) and xylazine (10 mg/kg) given subcutaneously were used as anesthetics, and for euthanasia, pentobarbital (100 mg/kg) was administered intraperitoneally. To induce solid tumors, 106 KB or HT1080 cells were injected subcutaneously into mammary fat pad and the lower abdomen of 36 nude mice (average weight 20 g). Within 7-17 days after implantations, each mouse developed 3-4 tumors of 1-14 mm (mean 4.1 mm) in size. To study tumor heterogeneity, the tumors with different size were included in the experiments. For dualtumor experiments, six mice were injected with 106 of KB and HT1080 cells on the ipsilateral and contralateral side, respectively. Immunohistology. For immunohistochemical staining, tumors were frozen sectioned into 7 µm thick slices and fixed with 4% paraformaldehyde for 5 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in 0.3% normal horse serum in phosphatebuffered saline for 5 min. The mouse immunoglobulin was blocked using M.O.M. immunodetection kit (Vector laboratories, Burlingame, CA), and sections were incubated with FR-recognizing MAb LK26 (15 to 20 µg/mL immunoglobulin; Signet laboratories, Dedham, MA). Sections were washed and then incubated with biotinylated horse anti-mouse immunoglobulin for 30 min at room temperature, followed by avidin-biotin-horseradish peroxidase complex. The final reaction product was visualized with diaminobenzidine. Tissue sections were viewed using a Nikon Eclipse 800 microscope and images were digitally captured using a CCD-SPOT RT digital camera and compiled using Adobe Photoshop software (v5.5). In Vivo Studies. Thirty-six mice bearing KB and/or HT1080 tumors (n ) 60 each) were divided into three groups so that each group had 12 mice with 40 tumors; five mice with 18 KB tumors, five mice with 18 HT1080 tumors, and two mice with both KB and HT1080 tumors. Group 1 was injected with the NIR2-folate probe (2 nmol/mouse), group 2 received free NIR2 fluorochrome (not conjugated to folate, 2 nmol/mouse), and group 3 was injected with the mixture of NIR2-folate probe (2 nmol/ mouse) and free folic acid (600 nmol folate/mouse). NIRF imaging was performed before and 1, 4, 24, 48 h after tail vein injection of the probes. In two animals from each group, NIRF images were also acquired daily up to 7 days (168 h) to study the in vivo kinetics of the probe. Image and Data Analysis. Imaging was performed using NIRF reflectance imaging system, which has been described in detail previously (27). Exposure time was 30 s per image, with maximum input photon flux delivered by the light source. Images were analyzed using commercially available software (Digital Science 1D software; Kodak). Regions of interest (300-320 pixels for each location) were placed manually within the visible tumor margins, the representative adjacent nontarget tissue (adjacent thigh), and a reference standard containing 10 nM free Cy5.5 fluorescent dye (Amersham Pharmacia, Piscataway, NJ). For determination of tumor contrast, mean fluorescence intensities of the tumor area (T) and of the corresponding area at the ipsilateral thigh (N) were calculated for each animal. Dividing T by N yielded the contrast between tumor tissue and normal tissue. Statistical Analysis. Data are presented as means and standard error of the mean. Statistical analysis of

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Figure 2. Cellular uptake of 3H-folate in the KB and HT1080 tumor cell lines. KB and HT1080 tumor cells were incubated with 3H-folate (50 nM) up to 120 min and cellular binding and uptake was quantitated by scintillation counting. The 3H-folate is internalized to significant amounts in the KB cells whereas the HT1080 cells show essentially no uptake of 3H-folate reflecting the receptor positive nature of the KB cells and the absence of receptors in the negative control cell line.

in vivo tumor fluorescence intensity (arbitrary unit) and the contrast between tumor and normal tissue was conducted by using a two-tailed paired Student t test. A P value of 0.05 or less was considered to indicate a statistically significant difference. RESULTS

Modeling of NIR2-Folate and Receptor Interaction. Before synthesizing the NIR2-folate probe, computer modeling was used to study the potential ligand and receptor interaction. The crystal structure of FR has not yet been solved, RFBP, a prototype protein for the riboflavin and folate binding protein family, was used instead (28). FR has approximately 30% sequence identity with RFBP, suggesting a high degree of structural homology. The binding site of RFBP contains a tryptophan, trp156, and a tyrosine, tyr75, oriented in parallel planes, forming a hydrophobic pocket. The isoalloxazine ring of riboflavin binds tightly between these two residues (23). Since FR also contains the same two residues, trp168 and tyr82, in its putative binding site, we used the crystal structure of RFBP to model the binding of a NIR2-folate conjugate. As expected, simulated docking experiments resulted in folate binding the active site of RFBP in an almost identical manner to riboflavin (Figure 1B). The binding pocket is deep and narrow, though it widens sufficiently at the protein surface to accommodate a long spacer for conjugation. On the basis of this, the 2,2′-(ethylenedioxy)bis(ethylamine) was selected as the linking arm. Docking experiments with attachment of NIR2 directly to folate or with a shorter spacer arm resulted in poor binding because of steric hindrance. Additionally, there are 12 tryptophans in proximity to the active site in both RFBP and FR. These residues are likely responsible for the complete quenching of riboflavin fluorescence upon binding. The spacer forces NIR2 to remain at least 10 Å from these residues, minimizing any quenching effects. Validation of Tumor Model. The HT1080 and KB cell lines were first characterized in terms of their putative capability of 3H-folate binding and uptake. Figure 2 summarizes the data showing significant uptake of 3H-folate by KB cells, but essentially no uptake by HT1080 cells. For KB cells, 50% of saturation of available FR by 3H-folate was reached in 20 min and uptake reached a plateau in 60 min. At peak maximum we observed 12 pmol of 3H-folate /106 cells under the chosen experimental conditions. In competition assays, there was

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Figure 3. Cellular uptake of NIR2-folate probe. KB and HT1080 tumor cells were incubated with NIR2-folate probe (0.1 µM) for 30 min at 37 °C, and cells were visualized by NIRF microscopy. Note strong fluorescence signals at the plasma membrane of KB cells suggesting the efficient binding of the NIR2-folate probe to FR present in the cell surface (magnification: 20×).

Figure 4. Immunohistochemical analysis of FR expression (top) and hematoxylin-eosin staining (bottom) of KB and HT1080 tumors. The staining of FR is strong positive in KB tumor cells whereas it is negative in HT1080. Note the localization of the LK26 antibody in the membrane and cytoplasm of the KB cells (original magnification, 40×).

a 60% decrease in bound 3H-folate in the presence of an equimolar amount (50 nM) of the free folic acid (4.97 pmol/106 cells) or NIR2-folate probe (5.01 pmol/106 cells). As the concentration of the free folic acid or NIR2-folate probe was increased to 5000 nM, binding of 3H-folate also decreased to 15% of its initial value, free folic acid at 1.86 pmol/106 cells or NIR2-folate probe at 1.92 pmol/106 cells. Competition by the NIR2-folate probe was similar to that of unconjugated folic acid. These results confirmed that fluorochrome attachment does not interfere with FR binding.

To determine the localization of fluorescent folate within cells, fluorescence microscopy was performed on KB cells incubated with the NIR-2 folate probe. The cells showed extensive, bright fluorescence signal whereas there was essentially no binding or uptake of the NIR2folate probe in the negative control, HT1080 cells (Figure 3). Fluorescence signal was seen primarily in the distribution of the plasma membrane of KB cells and in punctate vesicles in the interstitial compartment. FR Expression in Tumor. Before testing the NIR2folate probe in vivo, tumor expression of FR was further

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Figure 5. In vivo imaging of folate receptor. (a) White light and NIRF images obtained 24 h after intravenous injection of the NIR2-folate probe in a representative animal. FR-positive KB tumors were implanted on the right of the chest and the low abdomen, and FR-negative HT1080 tumor on the left of the chest. (b) Enlarged NIRF images of the chest tumors. Note the strong fluorescence signal in the FR-positive KB tumors compared with the control HT1080 tumors. (The imaging shows 2.7-fold difference in signal). (c) Enlarged NIRF images of the low abdomen KB tumors (1 mm).

characterized by immunohistology with FR recognizing Mab LK26. The staining showed strong immunoprecipitation in KB tumor tissues, indicating that the receptor remains overexpressed following implantation (Figure 4). Antibody staining showed primarily membrane and cytoplasmic staining of the KB cells. In contrast, HT 1080 tumor sections were essentially negative for folate receptor. Hematoxylin-eosin staining revealed multiple mitotic figures present in the rapidly proliferating HT1080 fibrosarcoma, while relatively well differentiated epidermoid cells were seen in the KB tumors. Tumor Detection with the Folate Conjugate. Following intravenous administration of the NIRF-folate probe, KB tumors showed significantly higher fluorescence signal intensity compared to HT1080 tumors (Figure 5). Mice bearing KB tumors, tumoral fluorescence could be detected as early as 1 h after administration of the probe (728 ( 109 AU), which peaked at 4 h (1210 AU ( 127) and then decreased (870 AU ( 98 AU at 24 h, 459 AU ( 48 AU at 48 h, and 255 AU ( 39 at 72 h). In tumors of equal size, there was a 2.4-fold (870 AU ( 98/366 AU ( 41, P < 0.01) higher fluorescence intensity in the FR-positive KB tumors compared with the control HT1080 tumors at 24-h images (Figure 6). In this set of experiments we also compared tumoral enhancement with the free NIR2 dye. At the 24 h time point, NIR-2 fluorochrome did not result in appreciably higher signal than background. Similarly, in competition studies, fluorescence signal of FR-positive KB tumor was reduced to that of FR-negative HT1080 tumors (Figure 6). We next determined tumor to background contrast (an essential parameter for tumor detectability) and plotted these ratios as a function of time after injection for the three experimental groups (Figure 7). At 1 h time point, all agents had similar tumor/background ratios and these ratios were only moderately elevated in KB tumors. At 4 h after injection, a significantly higher tumor/background ratio for the NIR2-folate was observed when compared to the NIR2 compound. Importantly for clinical applications, tumor/background ratios remained elevated

Figure 6. In vivo fluorescence signal of tumors and normal tissues. The fluorescence signal was obtained 24 h after intravenous injection of the NIR2-folate probes (2 nmol), the NIR2 compounds (2 nmol), and the NIR2-folate probes (2 nmol) with folic acid (600 nmol). Fluorescence intensity generated by the NIR2-folate probes in the FR-positive KB tumor is significantly higher than FR-negative HT1080 tumors whereas there is no significant difference between KB and HT1080 tumors after injection of the NIR2 compounds. In competition study performed with coinjection of the NIR2-folate probe and folic acid, fluorescence intensity of FR-positive tumors is reduced to that of FR-negative tumors.

with this probe for at least 24-48 h indicating its potential utility for endoscopic and intraoperative use (Figure 7). The tumoral fluorescence signal was reduced rapidly after 72 h (255 AU ( 39) and returned to the baseline (115 AU ( 17) in 5 days. Organ distribution of the probe was also examined after dissection. Highest fluorescence signal was observed in kidney because of high FR expression (29). Tumor, liver, lung, and intestine were at about the similar level. DISCUSSION

The results show that in vivo receptor imaging is feasible using a NIR fluorochrome-labeled targeting ligand. After intravenous injection, the targeted probe

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In conclusion, in vivo tumor detection by optical imaging was feasible using a FR-targeted NIRF probe. Compared to nontargeted fluorescence dye (NIR2), intravenous injection of the targeting probe (NIR2-folate) was shown to significantly increase tumor contrast 4-48 h in a nude mouse xenograft model. These receptortargeted imaging methods may facilitate both improved cancer diagnosis and staging, and early assessment of the effectiveness of treatment, by providing a more accurate in vivo characterization of tumor receptor status. Supporting Information Available: Computer modeling of NIR2-folate and its receptor analogue, riboflavin binding protein. This material is available free of charge via the Internet at http://pubs.acs.org. Figure 7. Time course of KB tumor with various probes. Tumor-to-normal contrast of FR-positive KB tumors after intravenous injection of the NIR2-folate probe, the NIR2 compounds and the NIR2-folate probe with folic acid was measured up to 48 h. Note the significantly higher tumor-tonormal contrast between 4 and 48 h after injection of the NIR2folate probes compared with the NIR2 compounds injection or coinjection of the NIR2-folate probes and folic acid.

(NIR2-folate) was shown to significantly increase tumor fluorescence intensity and contrast to normal tissues in a nude mouse xenograft model compared to nontargeted fluorescence dye (NIR2). The specificity of this FRtargeted probe was confirmed in vitro and in vivo studies. Our data indicate that the NIR2-folate probe has significant advantages over nonspecific fluorochromes for in vivo imaging, the latter often being used for nontargeted image enhancement (30, 31). Although NIR fluorochrome alone is capable of increasing tumor fluorescence, the actual contrast enhancement is typically lower. In KB tumors, the tumor contrast was about 3.0, 4 h after injection of the NIR2-folate probe. This is equivalent to 94% of maximum contrast value (3.2 ( 0.41) at 24 h. Rapid contrast enhancement of the tumor during the first few hours after injection is another major advantage of small molecular targeting agents (4). Two additional experiments confirmed that the fluorescent probe uptake is receptor dependent. Compared to KB tumor, HT1080 tumor expressed significantly less FR, which was reflected by a 2.4-fold lower fluorescence signal. In addition, the in vivo competition assay showed that the availability of free folate was able to compete off the receptor binding to NIR2-folate probe. The results supported the hypothesis that fluorochrome-labeled folic acid can still be recognized by its receptor. Interestingly, the competition experiment also showed that folate probe binding was not completely blocked by 300-fold excess of free folic acid (Figure 6). It is speculated that short blood half-life of folic acid may cause incomplete blockage; however, more studies are needed. Optical imaging is a noninvasive method and does not depend on radiolabeled contrast agents such as those in nuclear medicine; thus, there is no exposure of the patient to ionizing radiations. However, tissue penetration of near-infrared light in living tissue may limit the use of near-infrared contrast agents in tumor diagnostics to certain special applications, such as detection of superficial lesions, especially with endoscopic techniques and optical mammography (5, 32, 33). Recently, new imaging systems for deeper targets are under development to overcome these limitations. A tomographic reconstruction method has shown promise for in vivo imaging of fluorescent signal by surrounding the subject with photon sources and detectors (34).

ACKNOWLEDGMENT

The authors would like to thank Dr. Philip Low for the technique suggestion. This research was supported by NIH P50-CA86355, NO1-CO17014, R33-CA88365, and NSF BES-0119382. W.K.M. was partly supported by the Korean Science & Engineering Foundation. LITERATURE CITED (1) Weissleder, R., Tung, C. H., Mahmood, U., and Bogdanov, A. (1999) In vivo imaging of tumors with protease-activated near-infrared fluorescent probes. Nat. Biotech. 17, 375-8. (2) Tung, C. H., Mahmood, U., Bredow, S., and Weissleder, R. (2000) In vivo imaging of proteolytic enzyme activity using a novel molecular reporter. Cancer Res. 60, 4953-4958. (3) Achilefu, S., Dorshow, R. B., Bugaj, J. E., and Rajagopalan, R. (2000) Novel receptor-targeted fluorescent contrast agents for in vivo tumor imaging. Invest Radiol. 35, 479-85. (4) Becker, A., Hessenius, C., Licha, K., Semmler, W., Wiedenmann, B., and Groetzinger, C. (2001) Receptor targeted optical imaging of tumors with near-infrared flurorescent ligands. Nat. Biotech. 19, 327-31. (5) Marten, K., Bremer, C., Khazaie, K., Sameni, M., Sloane, B., and Tung, C. H. et al. (2002) Detection of dysplastic intestinal adenomas using enzyme-sensing molecular beacons in mice. Gastroenterology 122, 406-14. (6) Bremer, C., Tung, C. H., Bogdanov, A., Jr., and Weissleder, R. (2002) Imaging of differential protease expression in breast cancers for detection of aggressive tumor phenotypes. Radiology 222, 814-8. (7) Bremer, C., Bredow, S., Mahmood, U., Weissleder, R., and Tung, C. H. (2001) Optical imaging of matrix metalloproteinase-2 activity in tumors: feasibility study in a mouse model. Radiology 221, 523-9. (8) Bremer, C., Tung, C. H., and Weissleder, R. (2001) In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat Med. 7, 743-8. (9) Weissleder, R., and Ntziachristos, V. (2003) Shedding light onto live molecular targets. Nat Med, 9, 123-8. (10) Lin, Y., Weissleder, R., and Tung, C. H. (2002) Novel nearinfrared cyanine fluorochromes: synthesis, properties, and bioconjugation. Bioconjugate Chem. 13, 605-10. (11) Ross, J. F., Chaudhuri, P. K., and Ratnam, M. (1994) Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines. Physiologic and clinical implications. Cancer 73, 2432-43. (12) Miotti, S., Bagnoli, M., Ottone, F., Tomassetti, A., Colnaghi, M. I., and Canevari, S. (1997) Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines. J. Cell Biochem. 65, 479-91. (13) Toffoli, G., Cernigoi, C., Russo, A., Gallo, A., Bagnoli, M., and Boiocchi, M. (1997) Overexpression of folate binding protein in ovarian cancers. Int. J. Cancer 74, 193-8. (14) Leamon, C. P., DePrince, R. B., and Hendren, R. W. (1999) Folate-mediated drug delivery: effect of alternative conjugation chemistry. J. Drug Target. 7, 157-69.

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