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In Vivo Near-infrared Fluorescence Imaging of Carcinoembryonic Antigen–expressing Tumor Cells in Mice¹

¹ From the Institute for Diagnostic and Interventional Radiology, Friedrich-Schiller-University Jena, FZL Erlanger Allee 101, D-07747 Jena, Germany (M.R.L., A.G., C.T., W.A.K., I.H.); and Federal Institute for Materials Research and Testing (BAM), Working Group Optical Spectroscopy, Berlin, Germany (J.P., U.R.). Received January 18, 2007; revision requested March 22; revision received July 17; accepted August 17; final version accepted October 29. Supported by Doktor-Robert-Pfleger Foundation. Address correspondence to I.H. (e-mail: Ingrid.Hilger{at}med.uni-jena.de).

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCE IN KNOWLEDGE References

 
Purpose: To prospectively depict carcinoembryonic antigen (CEA)-expressing tumors in mice with a high-affinity probe consisting of a near-infrared (NIR) fluorochrome and the clinically used anti-CEA antibody fragment arcitumomab.

Materials and Methods: This study was approved by the regional animal committee. By coupling a NIR fluorescent (NIRF) cyanine dye (DY-676) to a specific antibody fragment directed against CEA (arcitumomab) and a nonspecific IgG Fab fragment, a bio-optical high-affinity fluorescent probe (anti-CEA–DY-676) and a low-affinity fluorescent probe (FabIgG–DY-676) were designed. The dye-to-protein ratios were determined, and both probes were tested for NIRF imaging in vitro on CEA-expressing LS-174T human colonic adenocarcinoma cells and CEA-nonexpressing A-375 human melanoma cells by using a bio-optical NIR small-animal imager. In vivo data of xenografted LS-174T and A-375 tumors in mice (n = 10) were recorded and statistically analyzed (Student t test).

Results: The dye-to-protein ratios were determined as 3.0–3.5 for both probes. In vitro experiments revealed the specific binding of the anti-CEA–DY-676 probe on CEA-expressing cells as compared with CEA-nonexpressing cells; the FabIgG–DY-676 probe showed a markedly lower binding affinity to cells. In vivo LS-174T tumors xenografted in all mice could be significantly distinguished from A-375 tumors with application of the anti-CEA–DY-676 but not with that of the FabIgG–DY-676 at different times (2–24 hours, P < .005) after intravenous injection of the probes. Semiquantitative analysis revealed maximal fluorescence signals of anti-CEA–DY-676 to CEA-expressing tumors about 8 hours after injection.

Conclusion: Findings of this study indicate the potential use of the high-affinity probe anti-CEA–DY-676 for specific NIRF imaging in in vivo tumor diagnosis.

© RSNA, 2008

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCE IN KNOWLEDGE References

 
Carcinoembryonic antigen (CEA) is a 180-kDa glycosylated protein and a well-established tumor marker for many different types of cancer, such as colorectal, lung, breast, liver, pancreatic, stomach, and ovarian cancer (1–3). A current method of detection of metastatic CEA-expressing tumors is the determination of serum levels of CEA, for example, to follow up patients after surgical removal of colorectal tumors (3). However, this method does not allow the distinct localization of metastases as required for accurate diagnosis. Basically, the localization of tumors and metastases can be realized only with imaging methods like computed tomography (CT), magnetic resonance (MR) imaging, ultrasonography, or scintigraphic modalities such as positron emission tomography and single photon emission CT (4).

Conventional CT and MR imaging allow tumor staging that is based on anatomic and physiologic information and are not as sensitive as the scintigraphic methods that enable both early and precise diagnosis of developing diseases (5). For radioimmunodetection of colorectal carcinoma, the monoclonal antibody Fab fragment termed arcitumomab (CEA-Scan; Immunomedics, Darmstadt, Germany) labeled with technetium 99m (^99mTc) is already being used in nuclear oncology (6,7). In contrast to this use, optical methods like near-infrared (NIR) fluorescence (NIRF) imaging are nonradioactive and highly sensitive, and are, therefore, potential alternatives to scintigraphic methods (8–13). The use of an indocyanine green–coupled antibody against CEA for visualization of human gastric cancer already has been described but only in an in vitro situation (14). Promising in vivo experiments with trastuzumab (Herceptin; ANS, Plano, Tex), an antibody against the HER-2/neu protein expressed in 30% (three of 10) of breast cancers (15), as well as experiments with the staining of apoptotic tumor cells with annexin V (16), both of which are covalently bound to the NIRF dye Cy5.5, have been described. Thus, the purpose of our study was to prospectively depict CEA-expressing tumors in mice with a high-affinity probe consisting of a NIR fluorochrome and the clinically used anti-CEA antibody fragment arcitumomab.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCE IN KNOWLEDGE References

 
All the procedures used were in accordance with international guidelines on the ethical use of animals and were approved by the animal welfare commission of the Thuringian state government for animal research. The fluorescent dye DY-676 NHS-ester was provided by Dyomics, Jena, Germany. The authors had control of the data and the information submitted for publication.

Synthesis and Characterization of Fluorescent Probes
The fluorescent cyanine dye DY-676 (excitation wavelength = 676 nm, emission wavelength = 699 nm) was coupled to arcitumomab to produce a high-affinity probe (anti-CEA–DY-676) against CEA (A.G., M.R.L.). Nonspecific Fab antibody fragments were generated from mouse IgG (Dianova, Hamburg, Germany) by using a preparation kit (ImmunoPure Fab Preparation Kit; Pierce Biotechnology, Rockford, Ill) and were subsequently coupled to DY-676, resulting in a low-affinity control probe FabIgG–DY-676 (C.T., M.R.L.). In accordance with the manufacturer's protocol, DY-676 N-hydroxysuccinimide esters were incubated with the respective protein in phosphate-buffered saline (PBS), 100 mmol/L, pH 9.3, to yield a dye-to-protein ratio of 3.0, and the dye-labeled protein was separated from unbound fluorophore by gel filtration (Sephadex; Amersham Biosciences, Uppsala, Sweden). The dye-to-protein ratio was determined for both probes from measurements of the absorbances at 280 and 660 nm according to Mujumdar et al (17), with assumption of a molar absorptivity of 180 000 mol · L^–1 · cm^–1 for DY-676, as determined by the manufacturer. Dye labeling of proteins was performed by three authors (A.G. and C.T., with 1 year of experience each, and M.R.L., with 3 years of experience). The optical spectra of the high-affinity probe anti-CEA–DY-676 and the low-affinity probe FabIgG–DY-676 in a solution of bovine serum albumin (BSA) in PBS (5% wt/vol), and the fluorescence standards rhodamine 101 (Sigma-Aldrich, Steinheim, Germany) dissolved in ethanol and styryl 9M (Lambda Physik, Göttingen, Germany) dissolved in methanol, were recorded with a spectrophotometer (Cary 5000; Varian, Palo Alto, Calif) at 25°C ± 2 (standard deviation). All the spectroscopic measurements and the data evaluation were performed by one author (J.P., with 2 years of experience) under supervision of another author (U.R., with 10 years of experience).

The fluorescence quantum yield, _f, is defined as the ratio of the number of photons emitted to the number of photons absorbed. The fluorescence quantum yields of anti-CEA–DY-676 and FabIgG–DY-676 were calculated from integrated, blank and spectrally corrected emission spectra (wavelength scale; prior to integration, multiplication with ) relative to styryl 9M dissolved in methanol. The fluorescence quantum yield of styryl 9M in methanol was previously determined to be 0.07 by using rhodamine 101 in ethanol (_f = 1.0) as the standard (18). For each compound-solvent pair, the fluorescence quantum yield was determined twice. The photometric and fluorometric probe characterization, the choice of the standards, and the data evaluation were performed by one author (J.P., with 2 years of experience) with the aid and supervision of another author (U.R., with 10 years of experience).

Cell Culture
CEA-expressing LS-174T human colonic adenocarcinoma cells and CEA-nonexpressing A-375 human melanoma cells from the same manufacturer (Cell Lines Service, Heidelberg, Germany) were cultured in Dulbecco's modified Eagle's medium and minimal essential medium (Gibco, Paisely, Scotland), respectively, with 10% (1/10) vol/vol fetal calf serum (Gibco) at 37°C in a 5% (5/100) CO₂ atmosphere and 95% (95/100) humidity. For increased expression of CEA, the LS-174T cells were supplemented with 2 mmol/L sodium butyrate (Sigma-Aldrich, Steinheim, Germany) for 14 days. All cell cultures were performed by two authors (A.G. and C.T., with 1 year of experience each) and two nonauthors (each with 6 years of experience).

Polymerase Chain Reaction
LS-174T and A-375 cells (1 x 10⁷ cells) were harvested by scraping and were washed with PBS (100 mmol/L, pH 7.4). RNA was isolated with a kit (RotiQuick-Kit; Roth, Karlsruhe, Germany) according to the manufacturer's protocol, and RNA concentration was determined with a spectrophotometer (Nanodrop ND1000; Peqlab, Erlangen, Germany). Reverse transcription was performed with a kit (iScript cDNA Synthesis Kit; Biorad, Hercules, Calif). Polymerase chain reaction (PCR) was performed in a 25-µL total volume containing 12.5 µL of PCR MasterMix (Geneo BioProducts, Hamburg, Germany) and 500 pmol/mL of primers. The following primer sequences were used: CEA1 sense 5'-CGC ATA CAG TGG TCG AGA GA-3' and CEA2 antisense 5'-ATT GCT GGA AAG TCC CAT TG-3' to amplify a 560–base pair fragment of CEA (25 cycles, 30 seconds at 94°C, 30 seconds at 61°C, and 30 seconds at 72°C). For the detection of the internal standard glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the following primers were employed: GAPDH1 sense 5'-TCG GAG TCA ACG GAT TTG GTC GTA-3' and GAPDH2 antisense 5'-ATG GAC TGT GGT CAT GAG TCC TTC-3' to amplify a 520–base pair fragment (22 cycles, 30 seconds at 94°C, 30 seconds at 66°C, and 30 seconds at 72°C). DNA fragments were documented after electrophoresis on agarose gel with 2% (2/100) wt/vol agarose (Sigma-Aldrich, Steinheim, Germany) with a gel documentation system (ImageMaster VDS; Amersham Pharmacia Biotech, Freiburg, Germany). All PCR experiments were performed by one author (A.G., with 1 year of experience) with the aid and supervision of another author (M.R.L., with 4 years of experience).

In Vitro NIRF Imaging
Cultured LS-174T and A-375 cells were harvested by scraping and were washed in PBS (100 mmol/L, pH 7.4) to remove medium remnants by two authors (A.G., C.T.). In total, 1 x 10⁶ cells (one group per cell line, four independent experiments) were incubated with 1 µg anti-CEA–DY-676 or FabIgG–DY-676, respectively, for 1 hour at 4°C in 100 µL PBS with 1% (1/100) wt/vol BSA and 100 mmol/L ethylenediaminetetraacetic acid. Unbound probes were removed by washing them with PBS (A.G., C.T.). For bio-optical NIRF imaging, the cells were sedimented in 1.5-mL reaction tubes. Measurements were performed with the whole-body small-animal bio-optical NIRF imaging system (bonSAI; Siemens, Erlangen, Germany) by using the 660- and 735-nm filter combination for excitation and emission with an acquisition time of 1.5 seconds and a binning factor of two (512 x 696-pixel matrix size, 9.3 x 9.3 µm/pixel). Images were analyzed semiquantitatively with a region of interest (ROI)-based calculation by using software (Syngo; Siemens). ROIs of approximately 100 pixels were placed manually on visible cell pellets. In vitro imaging was performed by three authors (M.R.L., with 3 years of experience, and A.G. and C.T., with 1 year of experience each).

For further information about probe binding on LS-174T and A-375 cells treated with either anti-CEA–DY-676 or FabIgG–DY-676, approximately 50 000 cells (two independent experiments) were sedimented on object slides (Histobond; Marienfeld, Bad Mergentheim, Germany), air-dried, stained with 10 µg/mL 4'-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Steinheim, Germany) by two authors (A.G. and C.T., with 1 year of experience each), and embedded with a mounting medium (Permafluor; Immunotech, Marseille, France). Confocal laser scanning microscopy was performed by one author (M.R.L., with 3 years of experience) by using a system with image examiner software (LSM510; Zeiss, Jena, Germany).

In Vivo NIRF Imaging of Tumors in a Mouse Model
Two-month-old female BALB/c/SCID mice (n = 10) supplied by the local Institute of Animal Research weighing approximately 23 g were housed with standard conditions; food and water were available ad libitum. To analyze the specific accumulation of the DY-676–labeled probes, CEA-expressing LS-174T cells were implanted in the ventral sites of the right shoulders and CEA-nonexpressing A-375 cells were implanted in the left shoulders of each mouse, with two tumors per animal and 1 x 10⁷ cells in 300 µL of a gelatinous protein mixture for cell culture (Matrigel; BD Biosciences, Heidelberg, Germany). Because tumors were implanted subcutaneously, variations in injection depths were negligible. Between 7 and 10 days (mean, 8 days) prior to imaging experiments, all mice received a low pheophorbide diet (Altromin, Lage, Germany) to reduce undesired autofluorescence. When the tumors had grown to approximately 8 mm in diameter, the ventral thorax and throat regions in all animals were shaved by two authors (A.G., C.T.) and two nonauthors. Animals were classified in two groups with five animals per group. One author (M.R.L., with 3 years of experience) and two nonauthors (with 6 years of experience each) injected into the tail vein either 40 µg of anti-CEA–DY-676 per mouse or 40 µg of FabIgG–DY-676 per mouse in each group. In all animals, fluorescence emission was recorded before and at 0, 2, 4, 8, 16, and 24 hours after injection of the probes with the whole-body small-animal NIRF imaging system, as described previously. The acquisition time was 1.5 seconds, with a binning factor of one (1024 x 1392-pixel matrix size, 4.6 x 4.6 µm/pixel). Relative fluorescence intensity at the LS-174T and the A-375 tumor region were analyzed semiquantitatively by using manually selected circular ROIs around the tumors by two authors (A.G. and C.T.) by using corresponding whole-body light macroscopic images as reference (ROI, approximately 3100 pixels), as described previously in relation to muscle. In vivo imaging was performed by three authors (A.G. and C.T., with 1 year of experience each, and M.R.L., with 3 years of experience).

Ex Vivo NIRF Imaging of Tumors
To provide additional information to the in vivo data, 24 hours after probe injection, three randomly selected mice per group of five were sacrificed, and NIRF imaging of mice with an opened abdominal wall was performed as described previously. Afterward, the tumors, lungs, livers, hearts, spleens, and kidneys were harvested, and the fluorescence emission of the organs was recorded. The acquisition time was 0.5 second together with a binning factor of one. Ex vivo imaging was performed by two authors (A.G. and C.T., with 1 year of experience each) with the supervision of another author (M.R.L., with 3 years of experience).

Histologic Analysis of Tumors
To control CEA expression levels of xenografts, the harvested tumors were cut into 3-µm-thick paraffin sections and placed on polylysine-coated object slides (Menzel, Braunschweig, Germany). Subsequently, tissue sections were immunostained with polyclonal rabbit antihuman CEA or IgG isotype control (control for staining specificity) and peroxidase with a staining kit (LSAB+SystemHRP; Dakocytomation, Glostrup, Denmark). Cytoplasmic structures were stained with a solution of 0.1% (1/1000) eosin (Sigma-Aldrich, Munich, Germany) in water. Experiments were performed by an individual with 1 year of experience with the supervision of an author (I.H., with 10 years of experience).

Statistical Analysis
Fluorescence intensities (arbitrary units, in vitro) and fluorescence intensity values (in vivo) were presented as means ± standard deviations or standard error, as indicated, and statistical analysis was performed by using an unpaired Student t test for in vitro experiments. For in vivo results, an unpaired Student t test was used to show significant differences (P < .005) in the binding of both probes to LS-174T tumors, and a paired Student t test was used to show significant differences (P < .005) in the binding of anti-CEA–DY-676 to LS-174T or A-375 tumors, as they were implanted in the same animal. Statistical analysis was performed by three authors (M.R.L., with 3 years of experience, and A.G. and C.T., with 1 year of experience each).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCE IN KNOWLEDGE References

 
Spectroscopic Properties of Anti-CEA–DY-676 and FabIgG–DY-676
The spectroscopic studies with anti-CEA–DY-676 and FabIgG–DY-676 revealed dye-to-protein ratios of 3.0–3.5 in PBS and very similar absorption and emission spectra for both probes. The fluorescence quantum yield values of anti-CEA–DY-676 and FabIgG–DY-676 were determined to be 0.048 and 0.070, respectively (Table).

View larger version (57K): [in this window] [in a new window] [Download PPT slide]   Figure 1: Analysis of CEA expression in cultured cells by using reverse-transcription PCR. Agarose gel electrophoresis of GAPDH (lanes 1 and 3, 520 base pairs) and CEA (lanes 2 and 4, 560 base pairs) transcripts of isolated messenger RNA from A-375 and LS-174T human carcinoma cell lines.

View larger version (77K): [in this window] [in a new window] [Download PPT slide]   Figure 2a: Bio-optical NIRF recordings of in vitro labeled LS-174T and A-375 cells. (a) Whole-body macroscopic NIRF image of 1.5-mL reaction cups containing pellets of LS-174T and A-375 cells after incubation with anti-CEA–DY-676 or FabIgG–DY-676 for 1 hour (representative data from four independent experiments). (b) Semiquantitative ROI-based evaluation of NIRF images of cell pellets (106 cells) after incubation with medium only, FabIgG–DY-676, or anti-CEA–DY-676 (four different experiments). Data are mean ± standard deviation and show significant differences between LS-174T and A-375 cells after incubation with anti-CEA–DY-676 (P < .005). Different fluorescence intensity values of LS-174T cells incubated with anti-CEA–DY-676 or FabIgG–DY-676 are significant (P < .005).

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 2b: Bio-optical NIRF recordings of in vitro labeled LS-174T and A-375 cells. (a) Whole-body macroscopic NIRF image of 1.5-mL reaction cups containing pellets of LS-174T and A-375 cells after incubation with anti-CEA–DY-676 or FabIgG–DY-676 for 1 hour (representative data from four independent experiments). (b) Semiquantitative ROI-based evaluation of NIRF images of cell pellets (106 cells) after incubation with medium only, FabIgG–DY-676, or anti-CEA–DY-676 (four different experiments). Data are mean ± standard deviation and show significant differences between LS-174T and A-375 cells after incubation with anti-CEA–DY-676 (P < .005). Different fluorescence intensity values of LS-174T cells incubated with anti-CEA–DY-676 or FabIgG–DY-676 are significant (P < .005).

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 3a: Confocal laser scanning microscopic analysis of in vitro labeled cells. Microscopic images of (a) LS-174T and (c) A-375 cells labeled with anti-CEA–DY-676 and (b) LS-174T and (d) A-375 cells labeled with FabIgG–DY-676. NIRF signals (>650-nm line pairs) are red, phase-contrast images are gray, and 4'-6-diamidino-2-phenylindole nucleus staining is blue (scale bar = 20 µm).

View larger version (73K): [in this window] [in a new window] [Download PPT slide]   Figure 3b: Confocal laser scanning microscopic analysis of in vitro labeled cells. Microscopic images of (a) LS-174T and (c) A-375 cells labeled with anti-CEA–DY-676 and (b) LS-174T and (d) A-375 cells labeled with FabIgG–DY-676. NIRF signals (>650-nm line pairs) are red, phase-contrast images are gray, and 4'-6-diamidino-2-phenylindole nucleus staining is blue (scale bar = 20 µm).

View larger version (84K): [in this window] [in a new window] [Download PPT slide]   Figure 3c: Confocal laser scanning microscopic analysis of in vitro labeled cells. Microscopic images of (a) LS-174T and (c) A-375 cells labeled with anti-CEA–DY-676 and (b) LS-174T and (d) A-375 cells labeled with FabIgG–DY-676. NIRF signals (>650-nm line pairs) are red, phase-contrast images are gray, and 4'-6-diamidino-2-phenylindole nucleus staining is blue (scale bar = 20 µm).

View larger version (73K): [in this window] [in a new window] [Download PPT slide]   Figure 3d: Confocal laser scanning microscopic analysis of in vitro labeled cells. Microscopic images of (a) LS-174T and (c) A-375 cells labeled with anti-CEA–DY-676 and (b) LS-174T and (d) A-375 cells labeled with FabIgG–DY-676. NIRF signals (>650-nm line pairs) are red, phase-contrast images are gray, and 4'-6-diamidino-2-phenylindole nucleus staining is blue (scale bar = 20 µm).

View larger version (101K): [in this window] [in a new window] [Download PPT slide]   Figure 4a: NIRF whole-body in vivo imaging of xenografted CEA-expressing and CEA-nonexpressing tumors in mice. Whole-body light macroscopic and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with xenografted LS-174T and A-375 tumors that have been injected intravenously with (a–e) anti-CEA–DY-676 or (f–j) FabIgG–DY-676 before and after probe injection.

View larger version (75K): [in this window] [in a new window] [Download PPT slide]   Figure 4b: NIRF whole-body in vivo imaging of xenografted CEA-expressing and CEA-nonexpressing tumors in mice. Whole-body light macroscopic and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with xenografted LS-174T and A-375 tumors that have been injected intravenously with (a–e) anti-CEA–DY-676 or (f–j) FabIgG–DY-676 before and after probe injection.

View larger version (92K): [in this window] [in a new window] [Download PPT slide]   Figure 4c: NIRF whole-body in vivo imaging of xenografted CEA-expressing and CEA-nonexpressing tumors in mice. Whole-body light macroscopic and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with xenografted LS-174T and A-375 tumors that have been injected intravenously with (a–e) anti-CEA–DY-676 or (f–j) FabIgG–DY-676 before and after probe injection.

View larger version (98K): [in this window] [in a new window] [Download PPT slide]   Figure 4d: NIRF whole-body in vivo imaging of xenografted CEA-expressing and CEA-nonexpressing tumors in mice. Whole-body light macroscopic and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with xenografted LS-174T and A-375 tumors that have been injected intravenously with (a–e) anti-CEA–DY-676 or (f–j) FabIgG–DY-676 before and after probe injection.

View larger version (91K): [in this window] [in a new window] [Download PPT slide]   Figure 4e: NIRF whole-body in vivo imaging of xenografted CEA-expressing and CEA-nonexpressing tumors in mice. Whole-body light macroscopic and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with xenografted LS-174T and A-375 tumors that have been injected intravenously with (a–e) anti-CEA–DY-676 or (f–j) FabIgG–DY-676 before and after probe injection.

View larger version (113K): [in this window] [in a new window] [Download PPT slide]   Figure 4f: NIRF whole-body in vivo imaging of xenografted CEA-expressing and CEA-nonexpressing tumors in mice. Whole-body light macroscopic and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with xenografted LS-174T and A-375 tumors that have been injected intravenously with (a–e) anti-CEA–DY-676 or (f–j) FabIgG–DY-676 before and after probe injection.

View larger version (83K): [in this window] [in a new window] [Download PPT slide]   Figure 4g: NIRF whole-body in vivo imaging of xenografted CEA-expressing and CEA-nonexpressing tumors in mice. Whole-body light macroscopic and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with xenografted LS-174T and A-375 tumors that have been injected intravenously with (a–e) anti-CEA–DY-676 or (f–j) FabIgG–DY-676 before and after probe injection.

View larger version (92K): [in this window] [in a new window] [Download PPT slide]   Figure 4h: NIRF whole-body in vivo imaging of xenografted CEA-expressing and CEA-nonexpressing tumors in mice. Whole-body light macroscopic and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with xenografted LS-174T and A-375 tumors that have been injected intravenously with (a–e) anti-CEA–DY-676 or (f–j) FabIgG–DY-676 before and after probe injection.

View larger version (96K): [in this window] [in a new window] [Download PPT slide]   Figure 4i: NIRF whole-body in vivo imaging of xenografted CEA-expressing and CEA-nonexpressing tumors in mice. Whole-body light macroscopic and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with xenografted LS-174T and A-375 tumors that have been injected intravenously with (a–e) anti-CEA–DY-676 or (f–j) FabIgG–DY-676 before and after probe injection.

View larger version (100K): [in this window] [in a new window] [Download PPT slide]   Figure 4j: NIRF whole-body in vivo imaging of xenografted CEA-expressing and CEA-nonexpressing tumors in mice. Whole-body light macroscopic and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with xenografted LS-174T and A-375 tumors that have been injected intravenously with (a–e) anti-CEA–DY-676 or (f–j) FabIgG–DY-676 before and after probe injection.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 5: Graph shows results of semiquantitative evaluation of whole-body NIRF in vivo recordings in mice with xenografted tumors. Analysis of manually selected ROIs on NIRF images for five animals before and after intravenous injection of anti-CEA-DY-676 and for five animals before and after intravenous injection of FabIgG–DY-676. Ratios of the relative mean fluorescence intensity values of LS-174T tumors and A-375 tumors to those of muscle tissue. Data are mean ± standard error.

View larger version (124K): [in this window] [in a new window] [Download PPT slide]   Figure 6a: Ex vivo NIRF recordings in mice with xenografted tumors and removed organs. Whole-body macroscopic light and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with opened abdominal wall with xenografted LS-174T and A-375 tumors 24 hours after injection of (a, b) anti-CEA–DY-676 or (c, d) FabIgG–DY-676. (e–h) NIRF (660-nm excitation wavelength and 730-nm emission wavelength) and whole-body light macroscopic images of removed organs of mice that have been injected intravenously with (e, f) anti-CEA–DY-676 or (g, h) FabIgG–DY-676. Images show representative data from three randomly selected mice per group (n = 5). 1 = LS-174T tumor, 2 = A-375 tumor, 3 = heart, 4 = lung, 5 = liver, 6 = kidney, 7 = spleen.

View larger version (101K): [in this window] [in a new window] [Download PPT slide]   Figure 6b: Ex vivo NIRF recordings in mice with xenografted tumors and removed organs. Whole-body macroscopic light and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with opened abdominal wall with xenografted LS-174T and A-375 tumors 24 hours after injection of (a, b) anti-CEA–DY-676 or (c, d) FabIgG–DY-676. (e–h) NIRF (660-nm excitation wavelength and 730-nm emission wavelength) and whole-body light macroscopic images of removed organs of mice that have been injected intravenously with (e, f) anti-CEA–DY-676 or (g, h) FabIgG–DY-676. Images show representative data from three randomly selected mice per group (n = 5). 1 = LS-174T tumor, 2 = A-375 tumor, 3 = heart, 4 = lung, 5 = liver, 6 = kidney, 7 = spleen.

View larger version (113K): [in this window] [in a new window] [Download PPT slide]   Figure 6c: Ex vivo NIRF recordings in mice with xenografted tumors and removed organs. Whole-body macroscopic light and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with opened abdominal wall with xenografted LS-174T and A-375 tumors 24 hours after injection of (a, b) anti-CEA–DY-676 or (c, d) FabIgG–DY-676. (e–h) NIRF (660-nm excitation wavelength and 730-nm emission wavelength) and whole-body light macroscopic images of removed organs of mice that have been injected intravenously with (e, f) anti-CEA–DY-676 or (g, h) FabIgG–DY-676. Images show representative data from three randomly selected mice per group (n = 5). 1 = LS-174T tumor, 2 = A-375 tumor, 3 = heart, 4 = lung, 5 = liver, 6 = kidney, 7 = spleen.

View larger version (76K): [in this window] [in a new window] [Download PPT slide]   Figure 6d: Ex vivo NIRF recordings in mice with xenografted tumors and removed organs. Whole-body macroscopic light and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with opened abdominal wall with xenografted LS-174T and A-375 tumors 24 hours after injection of (a, b) anti-CEA–DY-676 or (c, d) FabIgG–DY-676. (e–h) NIRF (660-nm excitation wavelength and 730-nm emission wavelength) and whole-body light macroscopic images of removed organs of mice that have been injected intravenously with (e, f) anti-CEA–DY-676 or (g, h) FabIgG–DY-676. Images show representative data from three randomly selected mice per group (n = 5). 1 = LS-174T tumor, 2 = A-375 tumor, 3 = heart, 4 = lung, 5 = liver, 6 = kidney, 7 = spleen.

View larger version (86K): [in this window] [in a new window] [Download PPT slide]   Figure 6e: Ex vivo NIRF recordings in mice with xenografted tumors and removed organs. Whole-body macroscopic light and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with opened abdominal wall with xenografted LS-174T and A-375 tumors 24 hours after injection of (a, b) anti-CEA–DY-676 or (c, d) FabIgG–DY-676. (e–h) NIRF (660-nm excitation wavelength and 730-nm emission wavelength) and whole-body light macroscopic images of removed organs of mice that have been injected intravenously with (e, f) anti-CEA–DY-676 or (g, h) FabIgG–DY-676. Images show representative data from three randomly selected mice per group (n = 5). 1 = LS-174T tumor, 2 = A-375 tumor, 3 = heart, 4 = lung, 5 = liver, 6 = kidney, 7 = spleen.

View larger version (86K): [in this window] [in a new window] [Download PPT slide]   Figure 6f: Ex vivo NIRF recordings in mice with xenografted tumors and removed organs. Whole-body macroscopic light and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with opened abdominal wall with xenografted LS-174T and A-375 tumors 24 hours after injection of (a, b) anti-CEA–DY-676 or (c, d) FabIgG–DY-676. (e–h) NIRF (660-nm excitation wavelength and 730-nm emission wavelength) and whole-body light macroscopic images of removed organs of mice that have been injected intravenously with (e, f) anti-CEA–DY-676 or (g, h) FabIgG–DY-676. Images show representative data from three randomly selected mice per group (n = 5). 1 = LS-174T tumor, 2 = A-375 tumor, 3 = heart, 4 = lung, 5 = liver, 6 = kidney, 7 = spleen.

View larger version (77K): [in this window] [in a new window] [Download PPT slide]   Figure 6g: Ex vivo NIRF recordings in mice with xenografted tumors and removed organs. Whole-body macroscopic light and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with opened abdominal wall with xenografted LS-174T and A-375 tumors 24 hours after injection of (a, b) anti-CEA–DY-676 or (c, d) FabIgG–DY-676. (e–h) NIRF (660-nm excitation wavelength and 730-nm emission wavelength) and whole-body light macroscopic images of removed organs of mice that have been injected intravenously with (e, f) anti-CEA–DY-676 or (g, h) FabIgG–DY-676. Images show representative data from three randomly selected mice per group (n = 5). 1 = LS-174T tumor, 2 = A-375 tumor, 3 = heart, 4 = lung, 5 = liver, 6 = kidney, 7 = spleen.

View larger version (130K): [in this window] [in a new window] [Download PPT slide]   Figure 6h: Ex vivo NIRF recordings in mice with xenografted tumors and removed organs. Whole-body macroscopic light and NIRF (660-nm excitation wavelength and 730-nm emission wavelength) images of mice with opened abdominal wall with xenografted LS-174T and A-375 tumors 24 hours after injection of (a, b) anti-CEA–DY-676 or (c, d) FabIgG–DY-676. (e–h) NIRF (660-nm excitation wavelength and 730-nm emission wavelength) and whole-body light macroscopic images of removed organs of mice that have been injected intravenously with (e, f) anti-CEA–DY-676 or (g, h) FabIgG–DY-676. Images show representative data from three randomly selected mice per group (n = 5). 1 = LS-174T tumor, 2 = A-375 tumor, 3 = heart, 4 = lung, 5 = liver, 6 = kidney, 7 = spleen.

View larger version (136K): [in this window] [in a new window] [Download PPT slide]   Figure 7a: Histologic immunostaining of xenografted LS-174T and A-375 tumors. Microscopic images show sections of (a) CEA-expressing LS-174T tumor and (b) nonexpressing A-375 tumor after eosin and peroxidase staining with rabbit polyclonal antibody against CEA (scale bar = 100 µm). Insets display eosin and peroxidase staining with IgG isotype control for staining specificity.

View larger version (108K): [in this window] [in a new window] [Download PPT slide]   Figure 7b: Histologic immunostaining of xenografted LS-174T and A-375 tumors. Microscopic images show sections of (a) CEA-expressing LS-174T tumor and (b) nonexpressing A-375 tumor after eosin and peroxidase staining with rabbit polyclonal antibody against CEA (scale bar = 100 µm). Insets display eosin and peroxidase staining with IgG isotype control for staining specificity.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCE IN KNOWLEDGE References

CEA-expressing LS-174T cells were selectively detected in our study with a whole-body small-animal NIRF imaging system as compared with CEA-nonexpressing cells. Similar results were obtained when the HER-2/neu receptor (15) or somatostatin (10) were tagged to cells with whole-body NIRF imaging technology.

The results of the in vitro bio-optical NIRF images were confirmed by using confocal laser scanning microscopy and showed that CEA molecules on the surface of LS-174T cells were selectively labeled with anti-CEA–DY-676, whereas no such labeling occurred for A-375 cells. As a control probe, the low-affinity probe FabIgG–DY-676 could not bind to any of the tested cell lines. These data confirm the specific binding of the designed high-affinity probe. Moreover, reverse-transcription PCR results verified that LS-174T cells highly express CEA messenger RNA and that no transcripts were found in A-375 control cells.

In vivo whole-body fluorescence imaging in mice with xenografted LS-174T and A-375 tumors showed that CEA-expressing tumors within mice could be clearly differentiated from CEA-nonexpressing tumors after injection of anti-CEA–DY-676. Particularly, between 8 and 24 hours after application of the high-affinity probe, the most pronounced relative fluorescence signals in the CEA-expressing tumors as compared with the CEA-nonexpressing tumors were observed. Similar results were obtained in other studies (10,15,16) in which the feasibility of imaging of further target molecules was reported. After application of the nonspecific probe FabIgG–DY-676, neither xenografted LS-174T tumors nor A-375 tumors could be discovered on in vivo whole-body NIRF images.

Semiquantitative analysis of NIRF images showed that the fluorescence signals reached their maximum level about 8 hours after injection of the contrast agent, and this finding is supported by findings in earlier reports (10) about radiolabeled contrast agents. Moreover, the increased signal ratios of LS-174T tumor to muscle after anti-CEA–DY-676 application revealed a significantly higher binding of that probe as compared with the binding of FabIgG–DY-676. Considering the specific binding of anti-CEA–DY-676 to LS-174T tumors as compared with that to A-375 tumors, the semiquantitative results are not as clear as expected from the qualitative images and the in vitro experiments. The fact that, in vivo, the signal ratio of LS-174T tumors (approximately 2.70) was increased by a factor of only 1.35 of the signal ratio of A-375 tumors (approximately 2.00) after application of the anti-CEA–DY-676 probe could be attributed to the differing tumors, particularly variations in blood flow, vascularization, and interstitial pressure. This assumption is supported by the presence of relatively higher fluorescence intensity values in A-375 tumors compared with LS-174T tumors after injection of the low-affinity probe. Although differences between both tumors appeared to be significant only at 24 hours after probe injection, qualitative discrimination can be performed much earlier.

For further studies, our results should provide the basis for corresponding diagnostic protocols related to the detection of tumors with NIRF optical imaging. Considering the results from whole-organ fluorescence, the liver seems to be the preferred place for storage and metabolism, according to previous investigations with macromolecular contrast agents (13) that indicate a quite similar distribution and catabolism of fluorochrome- and radiolabeled antibodies. The signals in the liver are attributed to the accumulation of the probe by the reticuloendothelial system, where, in particular, macromolecular substances are preferably accumulated (15). Moreover, comparatively high fluorescence intensity values were observed in the kidneys and lungs of mice treated with anti-CEA–DY-676. These findings are consistent with findings in studies about metabolism with the use of arcitumomab and other low–molecular-weight proteins (21–23), although no data were recorded at different times after probe injection.

Besides the distinct accumulation of the high-affinity probe in the liver, lung, and kidney, serious adverse effects such as anaphylaxis seem to be rare as reported in diagnostic applications of the CEA antibody (24). A low toxicity of the modular fluorescent probe used in our study can be expected, as the fluorescent dye, which is not yet clinically approved, shows similarities to the clinically approved indocyanine green.

The in vivo findings presented in our study are based on simple planar fluorescence reflectance imaging, with penetration depths of less than 7 mm. By using three-dimensional high-resolution (submillimeter-range) tomographic systems, more detailed information about shape and exact location of tumors at depths up to 20 cm would be available (8). Limitations for the application of optical methods in general might be the absorbance and scattering of light within biological tissues, which leads to a lower sensitivity as compared with the sensitivity of radiotracer methods. With new optical scanners that use time-resolved techniques for recording the temporal response of photons (time of flight), these problems can be solved. One limitation for the use of anti-CEA–DY-676 is the detection of tumors that are located very close to the liver because of the reported nonspecific probe accumulation by the reticuloendothelial system.

In conclusion, our study findings show that CEA-expressing tumor cells can be successfully labeled and imaged in their original size in mice by using a high-affinity fluorescent contrast agent as an alternative to radioactive ^99mTc-labeled arcitumomab and that, accordingly, selective tumor labeling and whole-body imaging could also be performed in the in vivo situation.

	ADVANCE IN KNOWLEDGE

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCE IN KNOWLEDGE References

 

• A high-affinity probe consisting of the cyanine dye DY-676 and the clinically used antibody fragment arcitumomab directed against carcinoembryonic antigen (CEA) was shown to be suitable for in vitro and in vivo fluorescence imaging of CEA-expressing tumors in mice.
  

	ACKNOWLEDGMENTS

 
The authors thank Harald Schubert, PD (Institute for Animal Research, Jena, Germany), Drs Peter Czerney, Frank Lehmann, and Matthias Wenzel (Dyomics, Jena, Germany), and Yvonne Heyne, Brigitte Maron, and Nancy Richter (Institute for Diagnostic and Interventional Radiology, Jena, Germany), and Monika Spieles (Federal Institute for Materials Research and Testing, Berlin, Germany) for excellent technical assistance. We express our gratitude to Drs Dietmar Pfeifer and Knut Rurack (Federal Institute for Materials Research and Testing, Berlin, Germany) for help with the measurements and data evaluation.

	FOOTNOTES

 

Abbreviations: BSA = bovine serum albumin • CEA = carcinoembryonic antigen • GAPDH = glyceraldehyde-3-phosphate dehydrogenase • NIR = near infrared • NIRF = near-infrared fluorescence • PBS = phosphate-buffered saline • PCR = polymerase chain reaction • ROI = region of interest

See Materials and Methods for pertinent disclosures.

Author contributions: Guarantors of integrity of entire study, M.R.L., W.A.K., I.H.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, M.R.L., J.P., U.R.; experimental studies, M.R.L., A.G., C.T., J.P., U.R., I.H.; statistical analysis, M.R.L., A.G., C.T., J.P., U.R.; and manuscript editing, M.R.L., J.P., U.R., I.H.

	References

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