2.1.

Synthesis of the Probes

The uPA-sensitive probe was synthesized as previously described.¹⁵ The probe, with the excitation and emission maxima of 675 and $694\mathrm{nm}$ , respectively, has a low fluorescence signal in its initial state, but becomes strongly fluorescent after uPA proteolysis. The uPA probe has the peptide substrate sequence of GGSGRSANAKC- $\mathrm{N}{\mathrm{H}}_2$ , which can be recognized by uPA and cleaved between Arg and Ser residues.

2.2.

Cell Culture

HT-1080 fibrosarcoma and HT-29 colon adenocarcinoma (ATCC, Rockville, Maryland) were selected from literatures for their distinct uPA expression.¹⁶ Secreted uPA was reported in the culture medium of HT-1080, but not in HT-29. HT-1080 cells were cultured in Dulbecco’s modified eagle medium (DMEM) (Cellgro Herndon, Virginia), supplemented with 10% heat-inactivated fetal bovine serum, penicillin $(50\mathrm{U}∕\mathrm{ml})$ , and streptomycin $(0.05\mathrm{mg}∕\mathrm{ml})$ , at $37°\mathrm{C}$ in 5% $\mathrm{C}{\mathrm{O}}_2$ . HT-29 cells were cultured similarly, except McCoy’s 5A (Cellgro Herndon) was used as the medium.

2.3.

Fluorescence Intensity Measurements

For specificity of the uPA-sensitive probe, fluorescence intensity measurements with time were recorded on a fluorescence plate reader (GENios Pro, Tecan, Durham, North Carolina). Activation of the probes $\left(1\mu \mathrm{M}\right)$ with MMP-2, MMP-7, MMP-9, cathepsin B, cathepsin D, and uPA ( $5\mu \mathrm{l}$ , $40\mathrm{ng}$ ) was assayed in a 96 well clear bottom plate (Costar, Corning, New York) in phosphate buffered saline (PBS) buffer $\left(200\mu \mathrm{l}\right)$ at $25°\mathrm{C}$ . The sample was excited at $670\mathrm{nm}$ and the emission was monitored at $720\mathrm{nm}$ . The fluorescent signals were recorded every $5\min$ for $135\min$ .

2.4.

Optical Zynography and Plasminogen-Casein Zymography

To determine the specificity of an uPA-sensitive probe, optical zymography was performed as described.¹⁷ Briefly, 10% SDS-PAGE gel was freshly prepared with a uPA-sensitive probe (200 pmol). Different biological samples including uPA (Sigma, Saint Louis, Missouri), cultured cell medium, and tumor tissue extracts were loaded onto the gel. Electrophoresis was then carried out at $100\mathrm{V}$ in Laemmli buffer (BioRad, Hercules, Calfornia). The running buffer was Tris buffer ( $25\mathrm{mM}$ , pH 8.3), containing $0.25\mathrm{M}$ of glycine and 0.1% (w∕v) SDS. After electrophoresis, the enzymes were renatured by incubating the gel in 2.5% (v∕v) Triton X-100 buffer for $2\times 30\min$ at room temperature. It was then washed twice $\left(10\min \right)$ with Tris buffer ( $50\mathrm{mM}$ , pH 7.4), and immediately imaged by using the near-infrared fluorescence (NIRF) reflectance imaging system.¹⁸

Plasminogen-casein zymography was performed as described.¹⁷ In short, 10% (v∕v) SDS-PAGE gels were casted with Glu-Plg $(22\mathrm{mg}∕\mathrm{ml})$ (Calbiochem, La Jolla, California) and ß-casein $\left(30\mathrm{mg}\right)$ (Sigma, Saint Louis, Missouri). After electrophoresis, the separated proteins were renatured by two $30\text{‐}\min$ incubation periods in 2.5% (v∕v) Triton X-100, and the gels were incubated overnight at $37°\mathrm{C}$ in glycine ( $100\mathrm{mM}$ , pH 8.0). Then it was stained for $2\mathrm{h}$ with 2% (w∕v) comassie blue, destained with 30% (v∕v) methanol, 10% (v∕v) acetic acid, dried, and analyzed.

To evaluate the specificity of a uPA-sensitive probe, equal amounts of tumor-associated proteases $\left(40\mathrm{ng}\right)$ , including MMP-2, MMP-7, MMP-9, cathepsin B, cathepsin D (Calbiochem, La Jolla, California), and uPA were analyzed using optical zymography. To acquire secreted uPA proteins, cells were grown to approximately 80% confluent, the culture medium was removed, and the cells were gently rinsed with Hanks' balanced salt solution (HBSS) twice to remove fetal bovine serum. The cells were incubated with serum-free DMEM medium for an additional $24\mathrm{h}$ , then the medium was collected and centrifuged at $1500\mathrm{rpm}$ for $5\min$ to remove cell debris. An equal amount of Laemmli sample buffer was mixed with the cell medium for optical zymography $\left(5\mu \mathrm{l}\right)$ and for plasminogen-casein zymography $\left(2\mu \mathrm{l}\right)$ . For preparation of tumor tissue extracts, tumor tissue was homogenized in Tris buffer ( $50\mathrm{mM}$ , pH 7.5) containing 0.25% (v∕v) of Triton-X 100. Minced tissue was ultrasonicated and centrifuged at $\mathrm{10,000}\mathrm{rpm}$ for $10\min$ . The supernatant was collected for electrophoresis.

2.5.

Western Blot

Following electrophoresis, the separated proteins from either the conditioned culture mediums or the tumor extracts were transferred to a polyvinylidene fluoride membrane (Hybond™-P, Amersham, Piscataway, New Jersey). The membrane was then blocked with 5% (w∕v) nonfat dry milk (BioRad, Hercules, California) in 0.1% (v∕v) Tween 20-TBS buffer for overnight at $4°\mathrm{C}$ . Primary mouse antibody against human urokinase (1:2000 dilution, American Diagnostica, Stamford, Connecticut) was added into the buffer for further incubation for $8\mathrm{h}$ . The membrane was washed with 0.1% (v∕v) Tween 20-Tris-buffered saline (TBS) buffer ( $3\times 15\min$ ) and allowed to incubate in 0.1% (v∕v) Tween 20-TBS buffer containing secondary goat antimouse IgG $(\mathrm{H}+\mathrm{L})$ HRP conjugated (1:1000, ImmunoPure®, Pierce, Rockford, Illinois) for $1.5\mathrm{h}$ at room temperature. After washing, the uPA was detected with ECL™ Western Blotting Detection Reagents (Amersham) and the image was developed on a Kodak BioMax Light Film.

2.6.

Near-Infrared Fluorescence Reflectance Imaging System

Imaging of the optical-zymographic gel as well as the animals was performed using a NIRF reflectance imaging system, which has previously been described.¹⁸ A broadband white light from a $150\text{‐}\mathrm{W}$ halogen bulb was used as an excitation photon source. Bandpass filters (Omega Optical, Brattleboro, Vermont) were employed to adjust the corresponding excitation wavelength for Cy 5.5 $\left(615\text{to}645\mathrm{nm}\right)$ . SDS-PAGE gels or the mice were exposed to the excitation light source. The corresponding bandpass emission filter (Omega Optical, Brattleboro, Vermont) was used to filter the fluorescence emission $\left(680\text{to}720\mathrm{nm}\right)$ . A $12\text{‐}\text{bit}$ monochrome CCD camera (Kodak, Rochester, New York) equipped with a $12.5\text{‐}75\text{‐}\mathrm{mm}$ zoom lens was used as a detector. Focusing was done manually. Exposure time and aperture were specifically $5\min$ ∕f1.2 for optical zymography and $120\mathrm{s}$ ∕f1.2 for animal imaging.

2.7.

In-Vivo Imaging

All animal studies were approved by the institutional animal care committee. Tumor cell suspensions $(2\times {10}^6\text{cells}∕50\mu \mathrm{l})$ were implanted at both sides of mammary fat pads under intraperitoneal anesthesia (Ketamine, $90\mathrm{mg}∕\mathrm{kg}$ , BenVenue, Bedford, Ohio and Xylazine, $10\mathrm{mg}∕\mathrm{kg}$ , Phoenix, Saint Joseph, Missouri). Tumor size was monitored daily until its diameter reached $5\text{to}10\mathrm{mm}$ , $2\text{to}3\text{weeks}$ after implantation. An uPA-sensitive probe ( $10\mathrm{nmol}$ , $100\mu \mathrm{l}$ ) was injected intravenously via tail vein under intraperitoneal anesthesia. All mice were imaged 6 and $24\mathrm{h}$ post probe injection. Arbitrary units, which indicate real tumor signals, were determined by subtracting tumor signal intensities with the background.

2.8.

Histologic Studies

Tumors were excised, frozen, and sliced into $10\text{‐}\mu \mathrm{m}$ sections. Both HT-1080 fibrosarcoma and HT-29 adenocarcinoma were stained with hematoxylin-eosin (Sigma). Immunohistochemistry was performed using a primary goat polyclonal antibody against human uPA (American Diagnostica, Stamford, Connecticut) and biotinylated secondary rabbit antigoat antibody (Vector Laboratories, Burlingame, California). Endogenous peroxidase activity was eliminated using 3% (v∕v) ${\mathrm{H}}_2{\mathrm{O}}_2$ . Peroxidase conjugated avidin (Vector Laboratories) was incubated, allowing avidin-biotin complex formation. The activity of peroxidase was visualized using $3,3^{\prime }$ —diaminobenzidine substrate, and the sections were counterstained with hematoxylin. Control sections were processed identically, except for the lack of the primary antibody.

To determine the location of the fluorescence signals, NIRF fluorescence microscopy was performed. Tumors were snap frozen and cryosectioned into $10\text{‐}\mu \mathrm{m}$ -thick slices. A fluorescence microscope (Nikon, Eclipse 80i, Japan), and a cooled change-coupled device (CCD) camera (Photometrics, Cascade 512B) adapted with a bandpass filter were used for image capture.

2.9.

Statistical Analysis

Data are presented as means with standard deviation. Statistical analysis of in-vivo tumor fluorescence was conducted by using a two-tailed paired Student t test ( $n=16$ tumors for uPA-sensitive probe in HT-1080, and $n=10$ tumors for uPA-sensitive probe in HT-29.). P values smaller than 0.05 were considered of statistically significance.

3.1.

In-Vitro Urokinase-Type Plasminogen Activator-Sensitive Probe Specificity

To verify the selectivity of the uPA-sensitive probe, several tumor associated proteases, including MMP-2, MMP-7, MMP-9, cathepsin B, and cathepsin D were tested using newly developed optical zymography.¹⁷ The gel was prepared as in traditional zymography, except casein was replaced with the uPA-sensitive probe. After electrophoresis, the renatured uPA activated∕hydrolyzed the quenched probe, resulting in fluorescence signal increase. Except uPA, none of the tested proteases was found to activate the uPA-sensitive probe under the testing condition, as seen by the fluorescent band in optical zymography [Fig. 1a ]. The specificity of the uPA-sensitive probe was also confirmed by measurement of fluorescent changes with pure proteases in PBS buffer. As shown in Fig. 1b, only uPA could activate the uPA-sensitive probe, resulting in a ten-fold increase of fluorescence signal. In contrast, the signal intensity of the uPA-sensitive probe incubated with other proteases remained optically silent.

Fig. 1

In-vitro selectivity of the uPA probe to tumor associated proteases. (a) Optical zymography of tumor-associated proteases revealed only uPA cleaved the uPA-sensitive probe, yielding a fluorescent band at $48\mathrm{kD}$ . (b) Fluorescence measurement showed uPA digested uPA-sensitive probe, which generated increased fluorescence signal up to 10 fold. Other proteases did not digest the uPA-sensitive probe.

3.2.

In-Vivo Tumor Imaging

Previously, HT-1080 human fibrosarcoma and HT-29 human colon adenocarcinoma cell lines have been reported as having distinct uPA expression in cell culture.¹⁹ Analyzing their culture medium, a high level of uPA was found in HT-1080, but an extremely low level of uPA was detected in HT-29. Surprisingly, both cell lines expressed a high level of uPAR.^{19, 20} It is known that uPAR and uPA are strongly associated with tumor progression.²¹ For a HT-29 tumor to grow in vivo, it may need to recruit uPA expressed by neighboring stroma cells through its surface uPA receptors. We thus explored this possibility by using the developed uPA-sensitive probe.

Prior to the imaging experiments, the difference in uPA expression of these two cell lines was confirmed by using Western blot, traditional plasminogen-casein zymography, and newly developed optical zymography. As previously reported,¹⁹ it was found that only HT-1080 cell line secreted uPA, but not HT-29 cells (Fig. 2 ). The dark band in Western blot indicated the uPA expression, whereas white bands in both zymographies demonstrated the proteolytic activity of uPA. The bands generated by the HT-1080 cell medium in all assays were identified to be $48\text{‐}\mathrm{kD}$ uPA.

Fig. 2

uPA secretion in HT-1080 human fibrosacoma and HT-29 colon adenocarcinoma cell lines. Western blot, plasminogen-casein zymography, and optical zymography revealed uPA-secretion ability in HT-1080 human fibrosarcoma cells. HT-29 colon adenocarcinoma did not secrete uPA in culture.

HT-1080 and HT-29 tumors were implanted in the upper chest of nude mice (HT-1080 $n=16$ , and HT-29 $n=10$ ). Whole body optical imaging showed negligible NIR fluorescence signals ( $75\pm 1$ AU) prior to probe injection. After probe administration, an intense fluorescence signal was observed in the HT-1080 experimental groups, $735\pm 52$ AU at $6\mathrm{h}$ and increased to $1206\pm 107$ AU at $24\mathrm{h}$ (Fig. 3 ). The average tumor to normal tissue ratio was 2.8 and 3.2, respectively. Interestingly, the fluorescence intensity of the supposedly low uPA expressing HT-29 tumor was about the same as those of the HT-1080 tumor, at $959\pm 106$ AU and $1217\pm 87$ AU at 6 and $24\mathrm{h}$ , respectively. The average contrasts of tumor to normal tissue were 3.1 and 3.1 at 6 and $24\mathrm{h}$ , respectively. No statistical significance was found between HT-1080 and HT-29 tumors. The results suggested that the uPA sensitive probe was activated in both types of tumors.

Fig. 3

In-vivo imaging of uPA activity. (a) A representative near-infrared fluorescence image of HT-1080 and HT-29 bearing mice $24\mathrm{h}$ after probe injection. The color map indicated fluorescence intensity (AU). (b) Bar graph shows the NIRF signals in tumors at 6 (white bar) and 24 (black bar) h after probe injection. There were no significant differences in signal intensities in HT-1080 and HT-29 groups at 6 and $24\mathrm{h}$ after probe administration.

3.3.

Urokinase-Type Plasminogen Activator Expression in Tumor Tissue Extract

To correlate the fluorescence signal with uPA expression in vivo, proteins were extracted from tumors and analyzed by Western blot. As expected, uPA was found in both tumors [Fig. 4a ], despite the differences observed in cell culture experiments. Moreover, these results were also confirmed by plasminogen-casein zymography [Fig. 4b].

Fig. 4

uPA expression in tumors. The uPA expression revealed by (a) Western blot analysis and (b) plasminogen-casein zymography of tumor extract demonstrated both HT-1080 fibrosarcoma and HT-29 colon adenocarcinoma secreted UPA.

3.4.

Histological Correlations

Hematoxylin-eosin staining showed increased nuclear cytoplasm ratio as well as the presence of multiple mitotic figures in the both HT-1080 fibrosarcoma and HT-29 colon adenocarcinoma (Fig 5 ). The expression of uPA in HT-1080 and HT-29 tumors was readily detected by immunohistochemistry. The majority of the enzyme was located pericellularly in both tumors. No significant difference in uPA expression was found in both types of tumors. NIRF signal generation was confirmed by fluorescent microscopy of the tumors. Considerable fluorescence was detected in the interstitium and tumor cells of HT-1080 fibrosarcoma and HT-29 adenocarcinoma.

Fig. 5

Histology of tumor sections. Histology of HT-1080 fibrosarcoma (top row) and HT-29 (bottom row). The tumors revealed increased nuclear cytoplasm ratio in H∕E staining (a) and (d). Immunohistochemistry targeting on the expression of uPA revealed that both tumors express uPA in vivo (b) and (e). Fluorescent microscope showed a strong fluorescence signal in both HT-1080 and HT-29 tumors (c) and (f). The signals are distributed more intracellularly in HT-1080, while HT-29 showed fluorescent signal both intracellularly and in the interstitium.