2.1.

Synthesis and Characterization of NGR-Cy 5.5

Peptides were purchased as trifluoroacetate (TFA) salts in $>90\%$ purity from Bachem Distribution Services GmbH, Weil am Rhein, Germany. Cy 5.5 monoreactiv NHS-ester was purchased from Amersham Biosciences Europe GmbH, Freiburg, Germany. All chemicals were used as purchased without further purification. Peptides were dissolved in aqueous bicarbonate buffer ( $0.1\mathrm{M}$ , pH 8.4; $1.3\mathrm{mg}∕500\mu \mathrm{L}$ ) at room temperature. To this, a solution of the dye in DMSO $(1.9\mathrm{mg}∕200\mu \mathrm{L})$ was added and stirred for $60\min$ at room temperature in the dark. Purification was accomplished by gradient HPLC using a Knauer system with two K-1800 pumps, an S-2500 UV-detector and a RP-HPLC Nucleosil 100-5 C18 column (eluent A: water, 0.1% TFA; eluent B: acetonitrile, 0.1% TFA). Elution times were $40\text{to}60\min$ , dependent on solution quantity, at a flow rate of $1\mathrm{mL}∕\min$ ). The product fractions were pooled, lyophilized, redissolved in phosphate-buffered saline (PBS) at $1.0\mathrm{mg}∕500\mu \mathrm{L}$ , and frozen at $-20°\mathrm{C}$ . The identities of the labeled products were confirmed by high-resolution electrospray mass spectrometry (HR-ES-MS) on a QUATTRO LCZ (Waters Micromass, Manchester, United Kingdom) spectrometer with a nanospray capillary inlet, $\mathrm{m}∕\mathrm{z}=557.5{\left[\mathrm{M}\right]}^{3-}$ , $837.0{[\mathrm{M}+\mathrm{H}]}^{2-}$ . The fully assembled tracer was assessed by fluorometry and photometry for spectral characteristics (Hitachi, F-4500 fluorescence spectrometer and U-3310 UV/VIS spectrophotometer, Tokyo, Japan).

2.2.

Cell Lines and Reagents

Human fibrosarcoma HT-1080 (ATCC: CCL-1321, Manassas, Virginia) and human adenocarcinoma MCF-7 (ATCC: HTB-22, Manassas, Virginia) cells were cultured in RPMI-1640 or MEM (Invitrogen Corporation, San Diego, California) supplemented with 10% fetal calf serum, penicillin and streptomycin (Biochrom AG, Berlin, Germany). Cells were grown routinely in a monolayer culture at $37°\mathrm{C}$ in a 5% $\mathrm{C}{\mathrm{O}}_2$ humidified air atmosphere. All antibodies were specific for CD13/APN: clone WM15 antibodies (phycoerythrin- and unconjugated) were from Pharmingen (BD Biosciences, San Jose, California), and antibody H-300 for Western blotting and immunohistochemistry was from Santa Cruz Biotechnology (Santa Cruz, California).

2.3.

FACS Analysis by Flow Cytometry

Cells were washed with PBS and subsequently harvested in $5\mathrm{mL}$ Versen-buffer ( $13.7\mathrm{mM}$ NaCl, $10\mathrm{mM}$ EDTA, $2.6\mathrm{mM}$ KCl, $8.1\mathrm{mM}$ $\mathrm{Na}{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_4$ , and $1.4\mathrm{mM}$ $\mathrm{K}{\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_4$ , pH 7.2). Aliquots of $2.5\times {10}^5$ cells were blocked with mouse IgG in PBS/BSA (0.1%) for $15\min$ at $4°\mathrm{C}$ , washed twice with PBS with ${\mathrm{Ca}}^{2+}$ and ${\mathrm{Mg}}^{2+}$ , and resuspended in $150\mu \mathrm{L}$ binding buffer ( $150\mathrm{mM}$ NaCl, $10\mathrm{mM}$ $\mathrm{Mg}{\mathrm{Cl}}_2$ , $10\mathrm{mM}$ $\mathrm{Ca}{\mathrm{Cl}}_2$ , $0.5\mathrm{mM}$ $\mathrm{Mn}{\mathrm{Cl}}_2$ , pH 7.2, diluted 1:20 in PBS/BSA 0.1%). Antibodies $(3\mu \mathrm{g}∕\mathrm{mL})$ were added, and cells were incubated for $45\min$ at $4°\mathrm{C}$ . Cells were washed, resuspended in PBS with 0.1% BSA, and analyzed on a Becton Dickinson FACSCalibur (BD Biosciences, San Diego, California).

2.4.

Western Blot

Confluent (80 to 100%) HT-1080 and MCF-7 cells were treated with $0.5\mathrm{mL}$ trypsin and harvested by scraping in cold PBS. After two centrifugation steps ( $1500\mathrm{rpm}$ , $5\min$ , $4°\mathrm{C}$ ), cells were resuspended in lysis buffer [0.5% Tween 20, $50\mathrm{mM}$ Tris-HCl pH 8, $250\mathrm{mM}$ NaCl, $5\mathrm{mM}$ EDTA pH 8, $50\mathrm{mM}$ NaF, $0.5\mathrm{mM}$ ${\mathrm{Na}}_3\mathrm{V}{\mathrm{O}}_4$ , $0.9\mathrm{mg}∕\mathrm{ml}$ protease inhibitor cocktail (Sigma, St. Louis, Missouri)], and the protein concentration of each sample was determined. To concentrate the protein, a two-fold volume of cold acetone was added to each sample and placed in $-20°\mathrm{C}$ for $16\mathrm{h}$ , followed by centrifugation at $10,000\mathrm{g}$ , $4°\mathrm{C}$ for $30\min$ . Protein pellets were dissolved in $100\mu \mathrm{L}$ of $6\times$ sodium dodecyl sulfate (SDS) sample buffer (New England Biolabs, Beverly, Massachusetts). Following precipitation, proteins were electrophoretically separated in nonreducing 10% SDS-PAGE and transferred to PVDF membranes (Millipore Corporation, Bedford, Massachusetts). After blocking, immunoblots were incubated with CD13/APN polyclonal antibody H-300 and with peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Sigma, St. Louis, Missouri). Peroxidase activity was revealed using ECL chemiluminescence (Amersham, Buckinghamshire, United Kingdom) and x-ray film, as described by the manufacturers. THP-1 cell lysat (BD Biosciences, San Diego, California) was used as a positive control.

Tumor tissue was frozen in liquid nitrogen, and $0.1\text{to}0.5\mathrm{g}$ of each tissue was homogenized by ultrasound using an Ultra-Turrax T8 (IKA-Labortechnik, Staufen, Germany). The homogenate was resuspended in $300\mu \mathrm{L}$ lysis buffer, additionally sonicated twice at $20\mathrm{kHz}$ for $5\mathrm{s}$ , and centrifuged for $30\text{to}60\min$ at $10,000\mathrm{g}$ to remove cell debris. The supernatant was taken for determining protein concentration following SDS-PAGE and Western blotting.

2.5.

Aminopeptidase N Activity Assay

Surface aminopeptidase activity of intact cells was measured by plating 5000 cells/well in a 96-well plate (Lumox-Multiwell, Greiner Bio-One GmbH, Frickenhausen, Germany). Cells were allowed to recover for $24\mathrm{h}$ , followed by washing with PBS and incubating with $100\mu \mathrm{M}$ L-alanine-4-methyl-7-coumarinylamide-(MCA)-trifluoroacetate (Fluka) in PBS with 0.5% BSA and $20\mathrm{mM}$ HEPES (pH 7.2).²¹ The release of fluorescent product 7-amido-4-methylcoumarin was monitored on a Kodak Gel Logic 200 Imaging System (Kodak, New Haven, Connecticut), with ${\lambda }_{\mathrm{exc}}$ of $360\mathrm{nm}$ and ${\lambda }_{\mathrm{em}}$ of $460\mathrm{nm}$ after $90\min$ . To assay for inhibition of enzymatic activity due to binding of CD13-specific antibody WM15 or the NGR-peptide, cells were pretreated in PBS containing a excess of antibody $(5\mu \mathrm{g}∕\mathrm{mL})$ or peptide $(25\mu \mathrm{g}∕\mathrm{mL})$ at $37°\mathrm{C}$ for $1\mathrm{h}$ .

2.6.

In Vitro CD13/APN Ligand Binding Studies

Cells were seeded in 24-well plates and incubated in culture medium overnight. For binding studies, cells were washed twice with PBS and resuspended in $150\mu \mathrm{L}$ cation-buffer diluted 1:20 in PBS/BSA (0.1%). Cy 5.5 (up to $30\mu \mathrm{M}$ ) or NGR-Cy 5.5 $\left(12.5\mu \mathrm{M}\right)$ was added into each well. After an incubation period of $1\mathrm{h}$ at $37°\mathrm{C}$ , cells were washed twice with PBS and resuspended in binding buffer. For blocking studies, free NGR-peptide $\left(125\mu \mathrm{M}\right)$ was added to each well for $45\min$ at $37°\mathrm{C}$ . Cells could be directly visualized by fluorescence microscopy ( $40\times$ objective magnification, Nikon TE 2000-S, Tokyo, Japan). The microscope was equipped with a mercury vapor lamp, $620∕775\mathrm{nm}$ and $545∕675\mathrm{nm}$ (excitation/emission) filters (AHF Analysentechnik, Tuebingen, Germany), a Nikon DXM1200F camera, and ACT1/DXM1200F software (Nikon, Tokyo, Japan).

2.7.

Immunohistochemistry

Tumors were removed and embedded in buffered formalin (4%, pH 7.0). Immunohistochemistry has been carried out commercially by Habedank-LID (Berlin, Germany) using the specific anti–CD13mAb H-300. For the in vivo visualization of NGR-Cy 5.5 uptake in tumor tissue, tumors were removed $24\mathrm{h}$ p.i., embedded in Tissue-Tek O.C.T. (Sakura, Zoeterwoude, Netherlands), snap-frozen, and serial-sectioned $\left(10\mu \mathrm{m}\right)$ . After drying, tissue fluorescence was evaluated by fluorescence microscopy.

2.8.

Tumor Xenografts

All animal studies were approved by the institutional review boards (# G76/2005). Female athymic nude mice $(nu∕nu)$ were obtained from Charles River Laboratories (Sulzfeld, Germany). At $4\text{to}6\text{weeks}$ of age, $3\times {10}^6$ HT-1080 or MCF-7 cells suspended in $100\mu \mathrm{L}$ PBS and Matrigel (BD Biosciences, San Diego, California) were injected subcutaneously in the flank and thoracic wall, respectively. When tumors reached $4\text{to}6\mathrm{mm}$ in diameter, the tumor-bearing mice were anesthetized by an i.p. injection of ketamine ( $125\mathrm{mg}∕\mathrm{kg}$ bw) and xylazine ( $12.5\mathrm{mg}∕\mathrm{kg}$ bw) and subjected to in vivo imaging studies.

2.9.

In Vivo Optical Imaging Systems – Fluorescence Reflectance Imaging

The combination of x-ray and optical fluorescence reflectance imaging (FRI) was performed using the Image Station In Vivo FX Imaging System (Kodak Molecular Imaging Systems, New Haven, Connecticut), which is equipped to provide multiwavelength fluorescence, luminescence, x-ray, and radioisotopic imaging. Fluorescence is provided by a $150\text{-}\mathrm{W}$ halogen illuminator with selectable bandpass excitation and emission filters. For Cy 5.5 excitation, $625\pm 18\mathrm{nm}$ and emission $700\pm 17.5\mathrm{nm}$ were employed. X-ray energy is provided by a $35\text{-}\mathrm{Kvp}$ , $150\text{-}\mu \mathrm{amp}$ , microfocus source in an enclosed chamber above the animal. A specialized radiographic phosphor screen is moved into the imaging path under the animal, which transduces the x-ray energy to light. Light from the fluorescence and/or the radiographic screen is captured with the $4\text{-}\text{million}\text{-}\text{pixel}$ cooled CCD camera equipped with a $10\times$ zoom lens. Images were captured 1, 3, 5, and $24\mathrm{h}$ after probe injection. Image acquisition times were $30\mathrm{s}$ per animal at the emission wavelength. Optical images were co-registered with x-ray background images, and regions of interest were selected and analyzed with the Kodak MI 4.0 software.

2.J.

Fluorescence Mediated Tomography

All tomographic optical imaging studies were performed with a small-animal fluorescence mediated tomography (FMT) system from VisEn Medical, Inc. (Woburn, Massachusetts) that has been described in detail elsewhere.⁶ The central piece of the system is the animal-imaging chamber configured in a parallel plate geometry. The chamber is scanned by a movable optical fiber that can excite a user-defined matrix of typically 50,000 to 100,000 source-detector projection pairs. The light source is software-selectable from two high-power laser diodes at $670\mathrm{nm}$ and $745\mathrm{nm}$ , operating in the range of $5\mathrm{mW}\text{to}150\mathrm{mW}$ . Detection is performed with a thermoelectrically cooled CCD camera by means of an imaging lens and appropriate bandpass filters (three-cavity interference at $675\mathrm{nm}$ or $755\mathrm{nm}$ for excitation measurements, and $705\mathrm{nm}$ or $770\mathrm{nm}$ for emission measurements). Tomographic reconstruction is based on the normalized Born approximation, a diffraction optical tomographic technique that uses a diffusion-type theoretical photon propagation model in biological tissue. The underlying assumption of the mathematical model has been propagation in a homogeneous diffuse medium with average optical absorption and scattering properties those of small rodents at the relevant wavelengths. However, it has been demonstrated that because of the appropriate data normalization scheme applied, the algorithm is very accurate even in highly heterogeneous media and in vivo.⁵ The instrument is calibrated on each channel using euthanized mice with surgical implants of known fluorochrome concentration and is able to recover fluorescence depth, size, and concentration in three dimensions from tissue at any depth. The system exhibits strict linearity over a broad range of biologically relevant concentration and detection sensitivities in the order of femtomoles.²² Animal scan times are in the range of $2\text{to}5\min$ ; tomographic reconstruction times on a standard desktop PC are about $1\text{to}3\min$ .

For in vivo experiments, $2\mathrm{nmol}$ NGR-Cy 5.5 were injected into the tail vein of each mouse ( $n=5$ for MCF-7, $n=6$ for HT-1080 xenografts). FRI and FMT were performed 1, 2, 5, and $24\mathrm{h}$ (FRI additional 5, 10, and $30\min$ ) post injection. Blocking studies were accomplished by injecting $10\mathrm{mg}∕\mathrm{kg}$ NGR-peptide in $100\mu \mathrm{L}$ PBS $\left(200\mathrm{nmol}\right)$ $10\min$ before injection of the labeled peptide ( $n=6$ for HT-1080 xenografts). For biodistribution studies, FRI images of isolated organs (tumor, muscle, heart, spleen, kidney, liver, and lung) were carried out $24\mathrm{h}$ after injection of the tracer.

2.K.

Statistical Analysis

Data are presented as $\text{mean}\pm \mathrm{SEM}$ (standard error of the mean). Statistical analysis of in vivo tumor fluorescence was conducted using a two-tailed, unpaired student t-test. A p-value $⩽0.05$ was considered to be significant.

3.1.

Synthesis and Characterization of NGR-Cy 5.5

The NHS ester of the near infrared fluorophore Cy 5.5 is reacted with an equimolar amount of the cyclo-[Cys-Asn-Gly-Arg-Cys]-Gly-Lys (NGR) peptide and purified by reversed phase HPLC (purity $⩾95\%$ ). The retention time was $28.5\min$ . Yields of the NGR-Cy 5.5 conjugate were typically 75 to 90%, as calculated with ${\epsilon }_{675}=\mathrm{250,000}{(\mathrm{mol}∕\mathrm{L})}^{-1}{\mathrm{cm}}^{-1}$ from the absorption spectra measured with the PBS solution. The excitation and emission spectrum of the targeted probe remained unchanged as compared to the nonmodified fluorophore showing excitation and emission peaks at $675\mathrm{nm}$ or $694\mathrm{nm}$ , respectively.

3.2.

CD13 Protein Expression in HT-1080 and MCF-7 Cells

To determine the cellular origin of the CD13/APN receptor and its activity in HT-1080 and MCF-7 cells, cell lines were analyzed for CD13 protein expression by FACS, Western blot, and immunohistochemistry as well as by measurement of the specific aminopeptidase activity. For flow cytometry, cells were incubated with PE-labeled WM15 antibody directed against the human APN, and binding was analyzed (Fig. 1 ). Approximately 87% of the HT-1080 cells expressed the APN-receptor. In contrast, the receptor was hardly detected on MCF-7 adenocarcinoma cells [0.24%; see also Fig. 1b]. CD13 Western blotting of HT-1080 and MCF-7 cells and tumor tissues with the H-300 antibody, recommended for detection of CD13 of mouse, rat, and human origin, confirmed the apparent ${\mathrm{M}}_{\mathrm{r}}$ of APN at 150,000. THP-1 cell lysate was used as a positive control. The APN enzyme was expressed only on HT-1080 cells and tumor tissue, while MCF-7 cells and tumors are negative for CD13 expression (not shown). Proper catalytic function of the CD13 protein was verified using a specific neutral aminopeptidase activity assay [Ref. 23; Fig. 1c]. CD13 activity in HT-1080 and MCF-7 cells is correlated with the expression measured by flow cytometry and Western blot analysis. The enzyme activity in HT-1080 cells could be specifically blocked by the WM15 antibody²⁴ and the NGR-peptide.

Fig. 1

Expression of CD13/APN in vitro. Human HT-1080 [(a), black curve] and MCF-7 [(a), red curve, which is enlarged in (b)] carcinoma cells were analyzed by flow cytometry using monoclonal antibody WM15-PE (CD13/APN specific) or isotype control (mouse-IgG1-PE). The results are presented in histograms (i.e., distribution of cells according to their fluorescence intensity). Numbers indicate the percentage of cells that were CD13/APN positive. (c) APN activity assay using L-alanine-4-methy-coumarinylamide-(MCA)-trifluoroacetate as substrate. The release of fluorescent product 7-amido-4-methylcoumarin was monitored on positive HT-1080 fibrosarcoma and negative MCF-7 adenocarcinoma cells. To assay for inhibition of enzymatic activity, binding of CD13-specific antibody WM15 and the NGR-peptide was measured on HT-1080 cells. (Color online only.)

3.3.

In Vitro NGR Binding Assays

Based on the CD13 protein expression data, the binding properties of cyclo-[Cys-Asn-Gly-Arg-Cys]-Gly-Lys-Cy 5.5 (NGR-Cy 5.5) to the cell lines were evaluated by fluorescence microscopy (Fig. 2 ). Negligible signals were detected when cells were incubated with the nonmodified Cy 5.5 dye alone (up to $30\mu \mathrm{M}$ ; data not shown), and also for MCF-7 cells following incubation with up to $12.5\mu \mathrm{M}$ of the labeled tracer [Fig. 2c]. The labeled peptide bound distinctly to HT-1080 cells [Fig. 2a]. The fluorescence was distributed over the cell surface and membrane-associated. Blocking of the signal was possible with a tenfold excess of unlabeled NGR-peptide [ $125\mu \mathrm{M}$ ; Fig. 2b].

Fig. 2

Binding affinity of NGR-Cy 5.5 to tumor cell lines with different CD13/APN expression level. HT-1080 fibrosarcoma cells [(a) and (b)] and MCF-7 adenocarcinoma cells (c) were incubated with $12.5\mu \mathrm{M}$ NGR-Cy 5.5 to assess the target affinity of the probe. In accordance with FACS analysis and Western blot data, HT-1080 cells producing high CD13/APN levels revealed strong cellular fluorescence [(a): fluorescence microscopy; left: phase contrast], which could be selectively blocked by coincubation with the unlabeled NGR-peptide (b). MCF-7 cells (c), which lack CD13 expression, did not bind the targeted fluorochrome (objective magnification: $40\times$ ).

3.4.

Immunohistochemistry

Immunohistochemistry on tumor tissue slides with CD13 antibody H-300 [Figs. 3c and 3d ] exhibited membranous CD13/APN antigen expression on HT-1080 cells and only background staining in MCF-7 xenografts. Specific in vivo binding of NGR-Cy 5.5 in HT-1080 tumor tissue could be visualized in Fig. 3e, exhibiting an intense, membranous fluorescence in tumor tissue $24\mathrm{h}$ after injection of the optical tracer, whereas MCF-7 tissue showed only background staining [Fig. 3f].

Fig. 3

In vivo expression of CD13/APN in tumor xenografts. Immunohistochemical staining of CD13/APN and fluorescence microscopy of tumor slides taken $24\mathrm{h}$ after injection of NGR-Cy 5.5. Paraffin sections from HT-1080 and MCF-7 tumor xenografts were stained with: H&E [(a) and (b)] and CD13 [(c) and (d); H-300; $5\mu \mathrm{g}∕\mathrm{ml}$ ]. Nuclei were counterstained with hematoxylin. HT-1080 tumors exhibited a strong, membranous CD13 expression (c), whereas MCF-7 tumors showed only background CD13 staining (d). Red arrows exemplify CD13 staining. HT-1080 tissue sections (e) exhibited a strong cellular fluorescence indicating a high binding of the optical tracer, while MCF-7 tissue showed only background staining. Objective magnification 20 and $40\times$ .

3.5.

In Vivo Target Assessment and Optical Imaging

Figure 4a shows typical NIR fluorescence reflectance images of mice with HT-1080 $(n=6)$ and MCF-7 $(n=5)$ tumors, respectively, $24\mathrm{h}$ after injection with NGR-Cy 5.5. After clearing of the unbound circulating fluorochromes, the HT-1080 tumor xenografts could be clearly delineated from the surrounding background tissue up to $24\mathrm{h}$ with maximal fluorescence intensities of $2756.2\pm 209.4\mathrm{AU}$ , visible $3\mathrm{h}$ post injection [Fig. 4b]. Compared to the targeted probe, the blocking experiment revealed lower fluorescence intensities (e.g., $1\mathrm{h}$ post injection: $2339.3\pm 269.9\mathrm{AU}$ versus $1999.5\pm 213.5\mathrm{AU}$ ; $n=6$ ; $\mathrm{p}<0.05$ ). Target-to-background ratios (TBR) after tracer injection ranged around $1.61\pm 0.52$ ( $1.54\pm 0.56$ for the blocking experiment). MCF-7 xenografts showed a weaker fluorescence, indicating a lower expression of the CD13/APN receptor [ $1407.5\pm 122.0\mathrm{AU}$ $3\mathrm{h}$ p.i.; Fig. 4b]. In HT-1080 tumor xenografts, the nonmodified Cy 5.5 revealed a fluorescence intensity of $1404.4\pm 243.8\mathrm{AU}$ $(n=7)$ $24\mathrm{h}$ post injection (data not shown).

Fig. 4

In vivo fluorescence reflectance imaging of HT-1080 and MCF-7 xenografts. HT-1080 fibrosarcoma $(n=6)$ and MCF-7 adenocarcinoma $(n=5)$ bearing mice were i.v. injected with the NGR-Cy 5.5 probe and imaged the following $24\mathrm{h}$ using a multichannel fluorescence reflectance imager (FRI). (a) FRI image $24\mathrm{h}$ past NGR-Cy 5.5 injection. Tumors were marked with an arrow. (b) The fluorochrome showed a strong retention over the time into the tumor (solid line), revealing a significantly higher target to background ratio as compared to APN-negative MCF-7 xenografts (dashed line). Tracer uptake could be blocked by preinjection of the unlabeled peptide (not shown).

FMT confirmed the FRI results, exhibiting a strong fluorochrome distribution in the target tissue throughout the tumor (Fig. 5 ). Quantitative data analysis revealed a fluorochrome concentration $5\mathrm{h}$ after injection of $306.7\pm 54.3\mathrm{nM}$ NGR-Cy 5.5 (Fig. 5), while blocking with the free peptide resulted in an average fluorochrome concentration of $195.3\pm 21.9\mathrm{nM}$ NGR-Cy 5.5 (Fig. 5). TBR values for this time-point ranged around $7.53\pm 4.04$ and $3.33\pm 2.13$ for the blocking experiment. Tumor fluorescence reached maximum intensities after $5\mathrm{h}$ and showed a slow clearance over the time [Fig. 5g]. Fluorochrome concentrations in nontarget tissue were significantly lower and ranged around $116.0\pm 18.3\mathrm{nM}$ .

Fig. 5

Fluorescence-mediated tomography of HT-1080 and MCF-7 tumor bearing nude mice. FMT experiments were performed to visualize and quantify the three-dimensional (3-D) fluorochrome distribution in deeper tissue sections. The fluorochrome concentration is displayed color encoded (scale: $1\text{to}256\mathrm{nM}$ ). FMT was carried out $24\mathrm{h}$ after injection of $2\mathrm{nmol}$ NGR-Cy 5.5 probe in HT-1080 (a) and MCF-7 (e) tumor–bearing nude mice. In order to assess probe specificity, $200\mathrm{nmol}$ of the unlabeled peptide were i.v. injected $10\min$ before application of the NGR-Cy 5.5 probe (c). FMT revealed intense fluorochrome retention $60\min$ after probe injection in HT-1080 tumors (b), which could be significantly reduced after pretreatment with the nonmodified peptide (d). Compared to the fluorescence measured in HT-1080 tumors, the fluorochrome accumulation in MCF-7 tumors (f) was substantially lower. Tumor fluorescence washout over the time was slower in HT-1080 than in MCF-7 tumor tissue (g), black $\text{bar}=\text{native}$ HT-1080; gray $\text{bar}=\text{blocked}$ HT-1080; white $\text{bar}=\mathrm{MCF}\text{-}7$ .

3.6.

Biodistribution

The distribution of Cy 5.5-labeled NGR-peptide was measured in tumor, muscle, heart, lung, spleen, kidneys, and liver from nude mice bearing HT-1080 fibrosarcoma $(n=6)$ and MCF-7 adenocarcinoma $(n=5)$ tumors. Data were obtained by FRI measuring $24\mathrm{h}$ post injection of the tracer. A representative example of NGR-Cy 5.5 biodistribution in HT-1080 tumor–bearing mice is shown in Fig. 6 . The highest concentration of optical tracer $24\mathrm{h}$ after injection was determined in kidneys ( $792.6\pm 272.6\mathrm{AU}$ for HT-1080 and $725.3\pm 52.6\mathrm{AU}$ for MCF-7), followed by the accumulation in liver $(398.0\pm 86.9\mathrm{AU})$ , tumor $(331.9\pm 151.7\mathrm{AU})$ , and lung $(306.5\pm 38.6\mathrm{AU})$ . Preinjection of the unlabeled peptide resulted in decreased uptake in all dissected tissues (data not shown).

Fig. 6

Representative overlay of white light and color-encoded FRI images of dissected organs of HT-1080 tumor–bearing nude mice. Animals $(n=6)$ were sacrificed $24\mathrm{h}$ after intravenous injection of $2\mathrm{nmol}$ NGR-Cy 5.5. Enhanced fluorochrome accumulation was detected in kidneys, liver, and lung tissue [(1): HT-1080 tumor, (2) heart, (3) spleen, (4) lung, (5) liver, (6) kidneys], indicating a high tracer washout of tumor tissue and a renal excretion. The fluorochrome concentration is displayed color encoded (scale: $1\text{to}256\mathrm{nM}$ ).