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Activatable Fluorescent Molecular Imaging of Peritoneal Metastases following Pretargeting with a Biotinylated Monoclonal Antibody

¹ Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland and ² Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan

Requests for reprints: Hisataka Kobayashi, Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH, Building 10, Room 1B40, MSC 1088, Bethesda, MD 20892-1088. Phone: 301-435-4086; Fax: 301-402-3191; E-mail: Kobayash{at}mail.nih.gov.

	Abstract

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Optical probes that yield high target-to-background ratios are necessary to detect microfoci of cancer that would otherwise escape detection with white light imaging. Target-specific activation of the optical signal at tumor foci is one mechanism by which high target and low background signal can be achieved. Here, we describe a two-step activation process in which the tumors are first pretargeted with a nonfluorescent biotinylated monoclonal antibody [cetuximab (Erbitux) targeting human epidermal growth factor receptor type 1 (HER1)]. Following this, a second agent, neutravidin-BODIPY-FL fluorescent conjugate, is given and binds to the previously targeted antibody, resulting in an 10-fold amplification of the optical fluorescence signal, leading to high tumor-to-background ratios. Spectral fluorescence imaging was done in a mouse model of peritoneal metastasis using a HER1-overexpressing cell line (A431) after pretargeting with biotinylated cetuximab and 3 h after administration of neutravidin-conjugated BODIPY-FL. Both aggregated tumors as well as small cancer implants were clearly visualized in vivo. For lesions 0.8 mm or greater in diameter, the spectral fluorescence imaging had a sensitivity of 96% (178 of 185) and a specificity of 98% (188 of 191). This two-step activation paradigm (pretargeting followed by neutravidin-biotin binding with an attached activatable fluorophore) could be useful in tumor-specific molecular imaging of various targets to guide surgical resections. [Cancer Res 2007;67(8):3809–17]

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Optical imaging has been proposed to improve the detectability of cancer lesions intraoperatively but requires high target-to-background ratios to identify small clusters of cancer cells. Activatable or "smart" optical probes are appealing because they only fluoresce when bound to their target site (1–6). Several strategies have emerged to create such smart optical probes. One approach, developed by Weissleder et al., is based on protease activation (ProSense 680 and 750, VisEn Medical, Inc., Woburn, MA) wherein fluorophores in the quenched state are released from their protein backbone by enzymes found in specific microenvironments, such as those overexpressed in neoplasms and atherosclerotic plaques (2–6). However, maximal fluorescence using extrinsic proteases often requires 24 h and uptake can be seen nonspecifically in organs that normally express the critical enzyme. Moreover, the protease is often found outside the cell and may not accurately reflect the location or margins of the tumor.

Here, we propose an alternative mechanism of target-specific optical activation for in vivo molecular imaging. This strategy is based on a modification of a known observation that the fluorescence of BODIPY conjugated to avidin or streptavidin is greatly (10-fold) increased after binding to biotin. This likely occurs because the photon-induced electron transfer from aromatic amino acids, such as tryptophan or tyrosine, to the excited state of BODIPY is inhibited by biotin-avidin binding, leading to a dequenching of the fluorophore (Fig. 1A ). An in vitro assay based on this phenomenon is in use for measuring endosome fusion in live cells and studying membrane-interacting molecules (7–9). We have taken advantage of this BODIPY-biotin activation for in vivo imaging whereby a biotinylated monoclonal antibody is first given to pretarget peritoneal metastases and is then followed by a neutravidin-conjugated BODIPY-FL (nAv-BDPfl), which activates on binding. This method offers a highly specific and generally applicable paradigm for optical molecular imaging of tiny tumor clusters of peritoneal metastases.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 1. nAv-BDPfl was activated by biotinylated antibody Erbitux. A, schema for the proposed mechanism of fluorescence activation by biotin binding to avidin. B, the fluorescence signal intensities and the emission spectra of 5 µg nAv-BDPfl with and without 5 µg biotin were measured under the presence of 5 µg b-Erb or 5 µg non-b-Erb on the spectral unmixed phantom image. All solutions with nAv-BDPfl have the same emission peak at 540 nm. The signal intensity of nAv-BDPfl with Erbitux was 19.43 ± 1.5 a.u. (mean ± SD) and that of nAv-BDPfl with b-Erb was 173.1 ± 5.1 a.u. (mean ± SD). When 5 µg biotin was added to both solutions, the signal intensity of 5 µg nAv-BDPfl with 5 µg Erbitux was 220.9 ± 16.3 a.u. (mean ± SD) and the signal intensity of 5 µg nAv-BDPfl with 5 µg b-Erb was 223.8 ± 12.0 a.u. (mean ± SD). Without nAv-BDPfl, the signal intensities of 5 µg Erbitux and 5 µg b-Erb were 3.7 ± 0.8 a.u. and 4.2 ± 0.6 a.u. (mean ± SD), respectively.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

At room temperature, 12 µL of 10 mmol/L NHS-LC-Biotin dissolved in DMSO were added to 1 mg (6.7 nmol) of Erbitux or Zenapax in 388 µL of 0.1 mol/L Na₂HPO₄ and incubated for 15 min. Unreacted biotin was separated from the antibody by gel filtration using a Sephadex G50 (PD-10; GE Healthcare, Milwaukee, WI). Biotinylated Erbitux (b-Erb) and biotinylated Zenapax (b-Zen) samples were kept at 4°C.

The protein concentrations of b-Erb and b-Zen samples were determined by measuring the absorption at 280 nm and validated with Coomassie Plus protein assay kit (Pierce Chemical) by measuring the absorption at 595 nm with a UV-Vis system (8453 Value UV-Bis system, Agilent Technologies, Palo Alto, CA) using standard solutions of known concentrations of Erbitux and Zenapax (100, 200, and 400 µg/mL).

The biotin labeling ratio was determined by the HABA assay (Pierce Chemical). HABA/avidin solution (900 µL) was put into a 1 mL cuvette, and the absorbance of this solution at 500 nm was measured and recorded as A₅₀₀ HABA/avidin (11). This measurement procedure was repeated twice with 250 and 500 µg/mL solutions, and the average number of biotin molecules per Erbitux or Zenapax molecule was determined. The number of biotin molecules conjugated to Erbitux and Zenapax was 10 (b-Erb) and 9 (b-Zen), respectively. By changing the concentration of NHS-LC-Biotin, the number of biotin molecules per Erbitux antibody was adjusted to either 2, 12, or 23.

To validate that biotinylation did not compromise the binding ability of antibody, unconjugated Erbitux, 12b-Erb, and 23b-Erb were radiolabeled with ¹²⁵I with modified chloramine-T method. The binding assay to A431 cells was done. In brief, 40 µg of antibodies were dissolved in PBS (pH 7.4) and 400 µCi of Na¹²⁵I were added (GE Healthcare). Then, 6 µg of chloramine-T hydride were added in the reaction mixture and incubated for 5 min. The solution was applied to the Sephadex G50 column (PD-10), and the antibody fractions were collected. The labeling yields were 81%, 72%, and 76% for Erbitux, 12b-Erb, and 23b-Erb, respectively. For the binding assay, 4 ng of each antibody were incubated with 2 x 10⁶ A431 cells in 300 µL of PBS/0.025% bovine serum albumin for 2 h on the ice. To determine the nonspecific binding, two other reaction mixtures containing 40 µg Erbitux were added to block the specific binding. The cells were washed twice and counted with ¹²⁵I to determine the binding fraction.

Synthesis of BODIPY-FL–conjugated Erbitux. Amido-reactive BODIPY-FL was purchased from Molecular Probes, Inc. (Eugene, OR). At room temperature, 500 µg (3.3 nmol) of Erbitux in Na₂HPO₄ were incubated with 10 to 100 nmol (1–10 µL/10 mmol/L) of BODIPY-FL-succinimidyl ester in DMSO at pH 8.5 for 15 min. The mixture was purified with a Sephadex G50 column (PD-10). Erbitux-conjugated BODIPY-FL (Erb-BDPfl) was kept at 4°C as stock solutions.

The protein concentration of Erb-BDPfl samples was determined with Coomassie Plus protein assay kit by measuring the absorption at 595 nm with a UV-Vis system (8453 Value UV-Bis system) using standard solutions of known concentrations of Erbitux (100, 200, and 400 µg/mL). Then, the concentration of BODIPY-FL was measured by the absorption at 508 nm, respectively, with the UV-Vis system to confirm the number of fluorophore molecules conjugated with each Erbitux molecule. By changing the concentration of the Erbitux solution, the number of BODIPY-FL molecules per Erbitux was adjusted to 2.2.

Synthesis of nAv-BDPfl. Neutravidin was purchased from Pierce Biochemical, Inc. (Milwaukee, WI). At room temperature, 400 µg (5.9 nmol) of neutravidin in 198 µL of Na₂HPO₄ were incubated with 12 nmol (2 µL/6 mmol/L) of BODIPY-FL-succinimidyl ester in DMSO for 15 min. The mixture was purified with Sephadex G50 (PD-10). nAv-BDPfl was kept at 4°C.

The protein concentration was determined with Coomassie Plus protein assay kit by measuring the absorption at 595 nm with a UV-Vis system (8453 Value UV-Bis system). Then, the BODIPY-FL concentration was measured by the absorption at 504 nm with a UV-Vis system to confirm the number of BODIPY-FL molecules conjugated with each neutravidin molecule. The number of BODIPY-FL molecules per avidin was 1.3.

Measurement of fluorescence enhancement of nAv-BDPfl. To investigate the fluorescence enhancement of nAv-BDPfl on binding to biotinylated antibody, fluorescence intensity and emission spectra of nAv-BDPfl were measured in the presence of Erbitux or b-Erb by the Maestro In-Vivo Imaging System (CRi, Inc., Woburn, MA). Erbitux (5 µg) or b-Erb (5 µg) in 390 µL PBS was placed in a nonfluorescent 96-well plate (Costar, Corning, Inc., Corning, NY) and 5 µg nAv-BDPfl was added to each well. To investigate the activation potential of nAv-BDPfl, high-dose biotin (5 µg) was also added to each of the mixed solutions consisting of nAv-BDPfl and Erbitux and nAv-BDPfl and b-Erb. For the target-specific activation, 5 µg nAv-BDPfl and 5 µg b-Erb in 390 µL PBS and 5 µg nAv-BDPfl and 5 µg b-Zen in 390 µL PBS were placed in a nonfluorescent 96-well plate. A band pass filter from 445 to 490 nm and a long pass filter over 515 nm were used for emission and excitation light, respectively. The tunable filter was automatically stepped in 10-nm increments from 500 to 800 nm, while the camera captured images at each wavelength interval with constant exposure. A region of interest (ROI) as large as each well was drawn to determine the emission spectra using commercial software (Maestro software, CRi). The mean fluorescence intensity in arbitrary unit (a.u.) as well as the SD of each well were measured using ImageJ software.³

Cell culture. EGFR (HER1)-overexpressing A431 human epidermoid carcinoma cells were purchased from the American Type Culture Collection (Manassas, VA). A431 cells were grown in DMEM (Life Technologies, Gaithersburg, MD) containing 10% fetal bovine serum (Life Technologies), 0.03% L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin in 5% CO₂ at 37°C.

Transfection of green fluorescent protein to the A431 cell. Green fluorescent protein (GFP)-expressing plasmid was purchased from Clontech Laboratories, Inc. (Mountain View, CA). The plasmid was transfected into the A431 cells to validate the results with targeted fluorophores (see below). The transfection of GFP was done with an electroporation method using Gene Plus II (Bio-Rad Laboratories, Hercules, CA). Briefly, 3 µg of GFP-expressing plasmid were mixed with 2 x 10⁶ A431 cells in 400 µL of the cell culture medium (DMEM with 10% FCS). The cell suspension was then placed in a pulse cuvette (Bio-Rad Laboratories), and 250 V pulses were delivered after 950 cycles.

Flow cytometry. One-color flow cytometry was done to assess the antibody pretargeting by b-Erb and the fluorescence enhancement of nAv-BDPfl on its binding to biotin on b-Erb. A431 cells (5 x 10⁵) were plated on a 12-chamber culture well and incubated for 16 h. Erbitux (10 µg/mL) or b-Erb (10 µg/mL) was added to the medium, and the cells were incubated for 2 h. For the investigation of target-specific activation, 10 µg/mL b-Erb or 10 µg/mL b-Zen was added to the medium and the cells were incubated for 2 h. For signal amplification study, 10 µg/mL 2b-Erb or 10 µg/mL 12b-Erb was added to the medium and the cells were incubated for 2 h. Then, cells were washed twice with PBS and 10 µg/mL nAv-BDPfl was added to each well and incubated for another 1 h. Cells were washed twice with PBS, and flow cytometry was done using a FACScan cytometer (Becton Dickinson, Franklin Lakes, NJ). The argon ion 488-nm laser was used for excitation, and signals from cells were collected using a 530/30-nm band pass filter. All data were analyzed using CellQuest software (Becton Dickinson). The fluorescing capability of cells in each condition was referred to as the mean fluorescence index (MFI).

Fluorescence microscopy. A431 cells (2 x 10⁴) were plated on a cover glass bottom culture well and incubated for 16 h. Erbitux (10 µg/mL) or b-Erb (10 µg/mL) was added to the medium, and the cells were incubated for 2 h. Cells were washed twice with PBS and 10 µg/mL nAv-BDPfl was added to each well and incubated for another 1 h. Cells were washed twice with PBS, and fluorescence microscopy was done using an Olympus BX51 microscope (Olympus America, Inc., Melville, NY) equipped with the following filters: excitation wavelength of 470 to 490 nm and emission wavelength of 515 nm long pass. Transmitted light differential interference contrast (DIC) images were also acquired.

Tumor model. All procedures were carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), National Research Council, and approved by the local Animal Care and Use Committee. The i.p. tumor implants were established by i.p. injection of 2 x 10⁶ A431 cells or GFP-positive A431 cancer cells (GFP-A431) suspended in 200 µL PBS in female athymic mice (NCI Animal Production Facility, Frederick, MD). Experiments with tumor-bearing mice were done at 10 days after injection of the A431 or GFP-A431 cells.

In vivo spectral fluorescence imaging. Erbitux and b-Erb (100 µg each) were diluted in 300 µL PBS and injected into the peritoneal cavities of mice with peritoneally disseminated A431 cancer implants. For the investigation of target-specific activation of nAv-BDPfl, 100 µg each of b-Erb and b-Zen in 300 µL PBS were injected into the peritoneal cavities of the tumor-bearing mice. At 21 h after injection of each antibody, i.p. injection of 100 µg nAv-BDPfl in 300 µL PBS was done (Fig. 2 ). For comparison study between Erbitux and b-Erb, mice were sacrificed with carbon dioxide immediately and 3 h after nAv-BDPfl injection, whereas for comparison study between b-Zen and b-Erb, the mice were sacrificed only 3 h after nAv-BDPfl injection. For the in vivo control study using Erb-BDPfl, the mice were sacrificed 1 and 3 h after i.p. injection of 100 µg Erb-BDPfl.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 2. A schematic strategy for the activatable fluorescence molecular imaging using pretargeting biotinylated antibody followed by quenched neutravidin-BODIPY-FL conjugate.

Semiquantitative comparison of fluorescence intensities of tumors. Aggregated A431 tumors were removed from each of the mice and placed on a nonfluorescent board side by side in each experiment, and spectral fluorescence imaging was done. Semiquantitative side-by-side comparison of fluorescence intensity was done between two tumors pretargeted with Erbitux and b-Erb or b-Zen and b-Erb. A ROI as large as each tumor was drawn to determine the fluorescence intensity as well as the histogram using ImageJ software. The dynamic range of the fluorescent intensity in a.u. was split into equal-sized 256 bins (1–256). All experiments were done in triplicate.

Assessment of the sensitivity and specificity for the detection of peritoneal cancer foci. Side-by-side spectral fluorescence imaging of A431 cancer-bearing mice and normal athymic mice without tumors was done 3 h after i.p. injection of 100 µg nAv-BDPfl. Pretargeting with 100 µg b-Erb was done in both mice 21 h before nAv-BDPfl administration. The abdominal cavity was surgically exposed, and spectral fluorescence images of the peritoneal cavities as well as close-ups of the peritoneal membranes were obtained. A band pass filter from 445 to 490 nm and a long pass filter over 515 nm were used for emission and excitation light, respectively. The tunable filter was automatically stepped in 10-nm increments from 500 to 800 nm, while the camera captured images at each wavelength interval with constant exposure. All experiments were done in triplicate. Sixty fluorescent bumps on the peritoneal membranes of three tumor-bearing mice were randomly selected, and histologic examination (H&E stain) was done to validate the accuracy of b-Erb pretargeting image.

The sensitivity and specificity of spectral imaging for the detection of peritoneal disseminated cancer foci were studied using four tumor-bearing mice. The i.p. tumor xenografts were established 14 days after i.p. injection of 2 x 10⁶ GFP-A431 cancer cells suspended in 200 µL PBS in female athymic mice (NCI Animal Production Facility). Twenty-one hours after i.p. injection of 100 µg b-Erb in 300 µL PBS, 100 µg nAv-BDPfl in 300 µL PBS was injected into the peritoneal cavity of GFP-A431 tumor-bearing mice. Spectral fluorescence images of the peritoneal membranes were obtained. A band pass filter from 445 to 490 nm and a long pass filter over 515 nm were used for emission and excitation light, respectively. The tunable filter was automatically stepped up in 5-nm increments from 500 to 800 nm, while the camera captured images at each wavelength interval with a constant exposure. Standard emission spectra were obtained from the GSA-A431 cell pellets and the nAv-BDPfl solution under the same conditions as this sensitivity and specificity study. The unmixed images of GFP, nAv-BDPfl, and autofluorescence were created using the standard emission spectra. For each mouse, two to three different parts of the peritoneal membranes were randomly selected and spread out on a nonfluorescent plate before close-up spectral fluorescence imaging was done. ROIs were drawn within the nodules depicted by unmixed GFP images, unmixed nAv-BDPfl images, or both. Additional ROIs were drawn in the surrounding nontumorous areas on the unmixed GFP images (standard reference for noncancerous foci). The average fluorescence intensity of each ROI was calculated on the GFP and the nAv-BDPfl spectral unmixed images using commercial software (Maestro software version 2). The minimum possible diameter for the ROI was 0.8 mm; thus, all visible nodules with short axis diameters of 0.8 mm on either image were included for analysis. nAv-BDPfl–positive nodules were defined as having an average fluorescence intensity of 10 a.u. on the unmixed nAv-BDPfl images, whereas nAv-BDPfl–negative nodules were defined as having an average fluorescence intensity of <10 a.u.. The number of foci positive for both nAv-BDPfl and GFP and the number positive only for nAv-BDPfl or GFP were counted. Sensitivity was defined as the number of peritoneal foci positive for both nAv-BDPfl and GFP divided by the number of peritoneal foci positive for GFP. Specificity of nAv-BDPfl was defined as the number of peritoneal foci negative for both GFP and nAv-BDPfl divided by the number of peritoneal foci negative for GFP.

	Results

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Quality control study. With the same chemical reaction, a single Erbitux molecule could be conjugated with up to 23 biotins. 12b-Erb and 23b-Erb showed no loss of binding ability (72% and 68%) compared with unconjugated Erbitux (70%). In addition, the biotinylation did not increase the nonspecific binding of 12b-Erb (2.9%) and 23b-Erb (2.3%) compared with unconjugated Erbitux (3.1%). Therefore, the conjugation of 12 or 23 biotins did not compromise the binding of antibody to the specific antigen.

nAv-BDPfl activation after binding to a biotinylated antibody. To investigate the fluorescence enhancement of nAv-BDPfl on binding to a biotinylated antibody, a solution of 5 µg nAv-BDPfl was combined with 5 µg b-Erb in 390 µL PBS. As a control, 5 µg nAv-BDPfl was combined with 5 µg Erbitux in 390 µL PBS. All the solutions containing nAv-BDPfl have the same emission peak at a wavelength of 540 nm (Fig. 1B). The fluorescence of nAv-BDPfl increases strongly (10-fold) on binding to the biotinylated antibody b-Erb. The spectral peak of nAv-BDPfl bound to b-Erb was substantially higher than that of nAv-BDPfl in the presence of non-b-Erb. When 5 µg biotin was added to both solutions (nAv-BDPfl + Erbitux and nAv-BDPfl + b-Erb), the spectral peaks became almost identical. The signal intensity of nAv-BDPfl + Erbitux and nAv-BDPfl + b-Erb was 19.43 ± 1.5 a.u. and 173.1 ± 5.1 a.u. (mean ± SD), respectively. However, the coadministration of 5 µg biotin to both nAv-BDPfl + Erbitux and nAv-BDPfl + b-Erb resulted in signal intensities of 220.9 ± 16.3 a.u. and 223.8 ± 12.0 a.u. (mean ± SD), respectively.

Flow cytometry shows significant optical activation of nAv-BDPfl after pretargeting with biotinylated antibody. To investigate whether nAv-BDPfl is activated on cancer cells pretargeted with a biotinylated monoclonal antibody, single-color flow cytometry of A431 cancer cells was done after pretargeting with either b-Erb or Erbitux followed by nAv-BDPfl instillation. The percentage of fluorescence-gated A431 cells that corresponds to activated nAv-BDPfl is shown in Fig. 3A . The percentages of positive cells were 0.5% for A431 cells alone and 1.6% for A431 cells incubated with nAv-BDPfl without prior pretreatment with antibody. The MFI values were 4.8 a.u. for A431 cells alone and 5.1 a.u. for A431 cells incubated with nAv-BDPfl. The percentages of positive cells were 1.1% for cells pretargeted with Erbitux followed by nAv-BDPfl instillation but 99.9% for cells pretargeted with b-Erb followed by nAv-BDPfl instillation. The MFI values of cells pretargeted with Erbitux and b-Erb followed by nAv-BDPfl instillation were 6.8 and 680.0 a.u., respectively. These results indicate that, once a biotinylated antibody pretargets a cell, nAv-BDPfl will be activated on binding to the biotinylated antibody, but in the absence of a biotinylated antibody, nAv-BDPfl will not be activated.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 3. In vitro flow cytometry analysis and fluorescence microscopy show that nAv-BDPfl is activated on the surface of the pretargeted A431 cells. A, the percentages of positive cells (M1) of b-Erb pretargeting followed by nAv-BDPfl instillation (b-Erb + nAv-BDPfl), Erbitux pretargeting followed by nAv-BDPfl instillation (Erb + nAv-BDPfl), and non-pretargeting followed by nAv-BDPfl instillation (nAv-BDPfl) were 99.9%, 1.1%, and 1.6%, respectively. The MFI values of b-Erb + nAv-BDPfl, Erbitux + nAv-BDPfl, and nAv-BDPfl were 680.0, 6.8, and 5.1 a.u., respectively. Only b-Erb + nAv-BDPfl showed the significant rightward shift (>1 order shift). The percentages of positive cells and the MFI value of untreated A431 cells were 0.5% and 4.8 a.u., respectively. B, the MFI value of A431 cells pretargeted with 2b-Erb followed by nAv-BDPfl instillation was 97.6 a.u., whereas the MFI value of A431 cells pretargeted with 12b-Erb followed by nAv-BDPfl instillation was 1,110.6 a.u. The numbers of biotin molecule per antibody were 2 for 2b-Erb and 12 for 12b-Erb. C, fluorescence microscopy of A431 cells after 2-h incubation with 10 µg/mL b-Erb followed by 1-h incubation with 10 µg/mL nAv-BDPfl (b-Erb + nAv-BDPfl) showed strong fluorescence on the surface of the cells, whereas fluorescence microscopy of A431 cells after 2-h incubation with 10 µg/mL b-Erb, 1-h incubation with nAv-BDPfl, and 2-h incubation with Erbitux followed by 1-h incubation with 10 µg/mL nAv-BDPfl showed minimal fluorescence. Original magnification, x200. Photographic exposure time: 1 s for b-Erb, nAv-BDPfl, and Erbitux + nAv-BDPfl; 500 µs for b-Erb + nAv-BDPfl.

nAv-BDPfl is activated on the surface of pretargeted A431 cells. To investigate the location where nAv-BDPfl is activated, fluorescence microscopy and DIC images were obtained. Fluorescence microscopy of A431 cells pretargeted with b-Erb followed by nAv-BDPfl administration showed strong fluorescence on the surface of the cells (Fig. 3C). Intracellular fluorescence was not observed after 2 h of pretargeting with b-Erb and 1 h after incubation with nAv-BDPfl, whereas fluorescence microscopy of A431 cells incubated with b-Erb alone or nAv-BDPfl alone or A431 cells pretargeted with non-b-Erb followed by nAv-BDPfl instillation showed minimal fluorescence (Fig. 3C). These results indicate that nAv-BDPfl is activated on the surface of A431 cancer cells on binding to the pretargeting biotinylated antibody b-Erb; however, internalization of the b-Erb-nAv-BDPfl complex is minimal within this incubation period.

Peritoneal cancer foci are clearly visualized in vivo using the b-Erb pretargeting strategy. To show that nAv-BDPfl can be activated at target cancer foci in an i.p. cancer model using A431 cancer cells, the spectral fluorescence imaging was done of a surgically exposed mouse after pretargeting with Erbitux or b-Erb. Twenty-one hours after i.p. injection with 100 µg Erbitux or 100 µg b-Erb in tumor-bearing mice, 100 µg nAv-BDPfl was injected into the peritoneal cavity of each mouse. Immediately after injection with nAv-BDPfl, the aggregated tumors were clearly visualized in a mouse pretargeted with b-Erb, whereas the tumors were not depicted in a mouse pretargeted with non-b-Erb (Fig. 4A ). Some background signal contamination was due to the unbound b-Erb. At 3 h after injection of nAv-BDPfl, the tumor remained bright but the background signal decreased markedly by the transperitoneal clearance of noncellular b-Erb and nAv-BDPfl complexes (Fig. 4A) resultantly in a high signal-to-background image. Close-up images of the peritoneal membranes showed submillimeter cancer foci with optical enhancement (Fig. 4A). As a control, Erbitux directly conjugated to BODIPY-FL (Erb-BDPfl) was injected into A431 tumor-bearing mice and spectral fluorescence imaging of the peritoneal cavity as well as the peritoneal membrane was done 1 or 3 h after i.p. injection. Unlike the pretargeting with b-Erb followed by nAv-BDPfl instillation, the Erb-BDPfl failed to visualize the peritoneal cancer foci due to the high background signals both 1 and 3 h after injection with Erb-BDPfl (data not shown).

View larger version (48K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Peritoneal cancer foci were clearly visualized by pretargeting with biotinylated antibody and subsequent administration of nAv-BDPfl. A, immediately after i.p. injection of nAv-BDPfl, the mouse pretargeted with b-Erb clearly showed the aggregated tumors (small yellow arrow) as well as solitary tumor nodules (large yellow arrow and small white arrow). However, several background signal contaminations were also seen (yellow arrowheads and white arrowheads). Blue arrow, mouse pretargeted by Erbitux could not depict the tumors. At 3 h after i.p. injection of nAv-BDPfl, the mouse pretargeted with b-Erb more clearly showed the aggregated tumors (yellow arrow) as well as the solitary implant (white arrow). Submillimeter small implants (white arrowheads) were also detected on the close-up image of the peritoneal membrane. Although nonspecific fluorescence from the cancer implants (pink arrow) was detected on the close-up image of the mouse pretargeted with Erbitux, macroscopic aggregated tumors (blue arrow) were not visualized on the spectral fluorescence image of the peritoneal cavity. Red, autofluorescence; green, nAv-BDPfl fluorescence. B, ex vivo spectral fluorescence image of aggregated tumor nodules instilled with nAv-BDPfl after pretargeting with Erbitux or b-Erb. Top, ROI was drawn as large as each tumor by referring both white light image and spectral unmixed blue light image. The signal intensity of tumors pretargeted with b-Erb was higher than that with Erbitux both immediately and at 3 h after injection with nAv-BDPfl. The histograms (bottom) showed that the b-Erb has a larger proportion of counts for pixels (Y axis) in the higher fluorescent signal intensity range (X axis) than Erbitux both immediately and 3 h after injection of nAv-BDPfl, indicating that the fluorescence intensity of the tumor pretargeted with b-Erb is higher than that with Erbitux.

Target-specific activation of nAv-BDPfl was shown by in vitro and in vivo. To show that the optical activation of nAv-BDPfl was target specific, in vitro spectral fluorescence imaging was done using b-Erb and b-Zen as a control. Two cell-free mixed solutions consisting of 5 µg nAv-BDPfl and 5 µg b-Zen (b-Zen + nAv-BDPfl) in 390 µL PBS and 5 µg nAv-BDPfl and 5 µg b-Erb (b-Erb + nAv-BDPfl) in 390 µL PBS were placed on a nonfluorescent 96-well plate, and spectral fluorescence imaging was done. Unmixed images were generated (Fig. 5A ) and a ROI was placed in each well. The signal intensities of b-Zen + nAv-BDPfl and b-Erb + nAv-BDPfl were 155.2 ± 9.4 a.u. and 180.8 ± 16.6 a.u. (mean ± SD), respectively. Based on the phantom study, the activation potential of b-Zen and b-Erb was comparable (<15% difference in fluorescence intensity of the b-Erb + nAv-BDPfl solution). Flow cytometry showed a significant rightward shift when A431 cells were pretargeted with 10 µg/mL b-Erb for 2 h followed by 1-h incubation with 10 µg/mL nAv-BDPfl (Fig. 5B); however, only a minimal shift was observed in A431 cells pretargeted with 10 µg/mL b-Zen for 2 h followed by 1-h incubation with 10 µg/mL nAv-BDPfl (Fig. 5B).

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Target-specific activation of nAv-BDPfl was verified both in vitro and in vivo. A, spectral unmixed image of phantom consisting of two mixed solutions, 5 µg nAv-BDPfl and 5 µg b-Erb (b-Erb + nAv-BDPfl) and 5 µg nAv-BDPfl and 5 µg b-Zen (b-Zen + nAv-BDPfl). The signal intensities of b-Zen + nAv-BDPfl solution and b-Erb + nAv-BDPfl solution were 155.2 ± 9.4 a.u. and 180.8 ± 16.6 a.u., respectively. The signal intensities were almost comparable between the two solutions. B, one-color flow cytometry of A431 cancer cells was done after 2-h incubation with 10 µg/mL b-Zen or 10 µg/mL b-Erb followed by 1-h incubation with 10 µg/mL nAv-BDPfl. The percentages of positive cells (M1) and the MFI values were 0.5% and 6.0 a.u. for untreated A431 control cells, 18.3% and 16.2 a.u. for A431 cells pretargeted with b-Zen followed by nAv-BDPfl instillation, and 97.5% and 577.6 a.u. for A431 cells pretargeted with b-Erb followed by nAv-BDPfl instillation, respectively. Nonspecific increase in the fluorescence intensity was observed in a small portion of cells pretargeted with b-Zen (b-Zen + nAv-BDPfl), whereas most cells pretargeted with b-Erb (b-Erb + nAv-BDPfl) showed substantial increase in the MFI values. C, spectral fluorescence images of the peritoneal cavities as well as the close-up peritoneal membranes of A431 tumor-bearing mice were done 3 h after injection with 100 µg nAv-BDPfl. Pretargeting with 100 µg b-Zen (left) or 100 µg b-Erb was done 21 h before the administration of nAv-BDPfl. Spectrally unmixed images successfully visualized the aggregated tumors (yellow arrows) in a mouse pretargeted with b-Erb, but due to the low signal-to-background ratio, tumors (blue arrow) could not be visualized in a mouse pretargeted with b-Zen. Close-up composite image of the peritoneal membranes showed that macroscopic tumors (large yellow arrow) as well as the submillimeter small implants (yellow arrowhead) were clearly depicted with b-Erb pretargeting, but the signal intensities of tumors (blue arrowheads) as well as the signal-to-background ratio of b-Zen + nAv-BDPfl were lower than those of b-Erb + nAv-BDPfl. Red, autofluorescence; green, nAv-BDPfl fluorescence. D, ex vivo images and histograms of aggregated tumor nodules instilled with 100 µg nAv-BDPfl after pretargeting with 100 µg b-Zen or 100 µg b-Erb. A ROI was drawn as large as each tumor by referring both white light image and spectral unmixed blue light image. The signal intensity of tumors pretargeted with b-Erb was higher than that with b-Zen at 3 h after injection with nAv-BDPfl. The histogram of fluorescent intensity of ROI drawn on each tumor showed that the b-Erb has a larger proportion of counts for pixels (Y axis) in the higher fluorescent signal intensity range (X axis) than b-Zen after injection of nAv-BDPfl, indicating that the fluorescence intensity of the tumor pretargeted with b-Erb is higher than that with b-Zen.

Sensitivity and specificity of b-Erb pretargeting of peritoneal metastases are 96% and 98%, respectively. To validate that there is no nAv-BDPfl signal from normal tissues, side-by-side spectral fluorescence imaging of A431 cancer-bearing mice and normal mice without tumors was done 3 h after i.p. injection of 100 µg nAv-BDPfl. Pretargeting with 100 µg b-Erb was done 21 h before nAv-BDPfl injection. Spectrally unmixed nAv-BDPfl images showed that there were no nodular foci or bumps on the peritoneal membranes in normal mice, whereas there were several fluorescent foci on the peritoneal membranes of A431 tumor-bearing mice (Fig. 6A ). The histology of 60 fluorescent bumps randomly selected from the tumor-bearing mice (n = 3) confirmed that all 60 bumps were A431 cancer foci. The sensitivity and specificity of spectrally unmixed nAv-BDPfl imaging for the detection of GFP-A431 peritoneal cancer foci were studied using dual-color in vivo spectral fluorescence imaging of GFP and nAv-BDPfl (Fig. 6B). A total of 188 peritoneal tumor foci in four mice was identified by the unmixed nAv-BDPfl images, the unmixed GFP images, or both. Additionally, 188 ROIs were created in the nontumorous areas (i.e., where no tumors were visible on the GFP images). One hundred and seventy-eight foci showed nAv-BDPfl fluorescence intensities of 10 a.u. among the 185 GFP-positive foci (Fig. 6C). One hundred and eighty-eight foci showed nAv-BDPfl fluorescence intensities of <10 a.u. among the 191 GFP-negative foci (i.e., fluorescence intensities of <10 a.u. on unmixed GFP images). Thus, the spectral unmixed nAv-BDPfl imaging had a sensitivity of 96% (178 of 185) and a specificity of 98% (188 of 191).

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Sensitivity and specificity of b-Erb/nAv-BDPfl spectral fluorescence imaging to detect peritoneal cancer foci are 96% and 99%, respectively. A, spectral fluorescence images of the peritoneal cavities as well as the peritoneal membranes of A431 tumor-bearing mice [Tumor (+)] or non–tumor-bearing normal mice [Tumor (–)] were done 3 h after injection with 100 µg nAv-BDPfl. Pretargeting with 100 µg b-Erb was done 21 h before administration of nAv-BDPfl. Spectrally unmixed image and composite image successfully visualized the aggregated tumors (white arrow) in a tumor-bearing mouse, but tumor was not detected in a normal mouse. Close-up image of the peritoneal membranes showed several macroscopic bumps (yellow arrow) in a tumor-bearing mouse, but no fluorescent bumps were detected in a normal mouse. Histologic examination (H&E stain) of 60 randomly selected fluorescent bumps showed that all 60 bumps were A431 cancer foci. B, the spectral fluorescence image was unmixed based on the spectral patterns of GFP and nAv-BDPfl as well as the autofluorescence. The emission peaks of GFP and nAv-BDPfl were 545 and 555 nm, respectively. Then, a composite image consisting of GFP (blue), nAv-BDPfl (yellow), and autofluorescence (black and white) was made. Most foci detected by unmixed nAv-BDPfl image were located in correspondence with the foci visualized by unmixed GFP image. SI, signal intensity. C, two-color in vivo fluorescence intensity plots of cancer foci and noncancerous foci. All visible foci with signal intensities 10 on spectrally unmixed GFP images and diameters 0.8 mm were defined as cancer foci (n = 185). For comparison, 188 ROIs were drawn in the surrounding peritumoral areas. Three foci were found only by the unmixed nAv-BDPfl images. When foci positive for nAv-BDPfl were defined as those whose fluorescence intensities 10, sensitivity and specificity of spectral unmixed nAv-BDPfl images to detect the presence of cancer foci were 96% and 98%, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

This method benefits from a target amplification effect because 10 biotin molecules are conjugated to each antibody. Thus, a single antibody, such as anti-HER1, can bind up to 10 nAv-BDPfl molecules, greatly amplifying the net fluorescence. Taken together with the 10-fold activation of fluorescence by biotin binding, this method has the potential to achieve 100-fold higher signal-to-background ratio compared with the use of a nonbiotinylated antibody-fluorophore conjugate. In addition, this method does not require the enzymatic activation or biological clearance of unbound reagents or internalization of nAv-BDPfl complex (12, 13). Thus, this method has the potential to be highly specific and highly sensitive for the detection of tiny cancer deposits.

In conclusion, a two-step pretargeting and activation technique can be used to visualize submillimeter peritoneal cancer implants. This method can be applied for targeting any cancer-specific receptors or antigens by use of their corresponding specific antibody while using a general activatable fluorophore, which remains the same. The combination of the target amplification due to a 10-fold ratio of biotin to pretargeting antibody, the 10-fold activation of nAv-BDPfl after binding to biotin, and the gain in contrast with the use of spectrally unmixed imaging allow the detection of tiny foci of peritoneal implants. We hope this method may be useful in guiding physicians to detect and treat disease that would otherwise escape detection.

	Acknowledgments

 
Grant support: Intramural Research Program of the NIH, NCI, Center for Cancer Research.

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.

	Footnotes

 
³ http://rsb.info.nih.gov/ij/plugins/mri-analysis.html

Received 10/13/06. Revised 1/20/07. Accepted 2/ 1/07.

	References

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