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Near-Infrared Optical Imaging of Protease Activity for Tumor Detection¹

¹ From the Department of Radiology, Center for Molecular Imaging Research, CNY149-5403, Massachusetts General Hospital, 13th St, Boston, MA 02125. From the 1998 RSNA scientific assembly. Received January 8, 1999; revision requested March 5; revision received March 26; accepted July 1. Supported in part by a faculty award grant from the Center for Innovative Minimally Invasive Therapy. Address reprint requests to R.W. (e-mail: weissleder@helix.mgh.harvard.edu).

	Abstract

TOP Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
PURPOSE: To build and test an optical imaging system that is sensitive to near-infrared fluorescent molecular probes activated by specific enzymes in tumor tissues in mice.

MATERIALS AND METHODS: The imaging system consisted of a source that delivered 610–650-nm excitation light within a lighttight chamber, a 700-nm longpass filter for selecting near-infrared fluorescence emission photons from tissues, and a charge-coupled device (CCD) for recording images. The molecular probe was a biocompatible autoquenched near-infrared fluorescent compound that was activated by tumor-associated proteases for cathepsins B and H. Imaging experiments were performed 0–72 hours after intravenous injection of the probe in nude mice that bore human breast carcinoma (BT-20).

RESULTS: The imaging system had a maximal spatial resolution of 60 µm, with a field of view of 14 cm². The detection threshold of the nonquenched near-infrared fluorescent dye was subpicomolar in the imaging phantom experiments. In tissue, 250 pmol of fluorochrome was easily detected during the 10-second image acquisition. After intravenous injection of the probe into the tumor-bearing animals, tumors as small as 1 mm became detectable because of tumor-associated enzymatic activation of the quenched compound.

CONCLUSION: Tumor proteases can be used as molecular targets, allowing visualization of millimeter-sized tumors. The development of this technology, probe design, and optical imaging systems hold promise for molecular imaging, cancer detection, and evaluation of treatment.

Index terms: Animals • Breast neoplasms, experimental studies, 00.32 • Contrast media, experimental studies • Enzymes • Neoplasms, diagnosis, 00.32 • Neoplasms, experimental studies, 00.32

	Introduction

TOP Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
There are many biologic processes that cannot be easily or directly monitored by using magnetic resonance imaging, computed tomography (CT), or nuclear imaging because key molecules in these processes are not distinguishable from each other with these imaging techniques. In the quest for a more fundamental evaluation of the disease process, other parts of the electromagnetic spectrum are being explored for diagnostic imaging (1–8).

The near-infrared region (700–1,000 nm) offers unique advantages for the imaging of pathophysiologic states. Water and most naturally occurring fluorochromes do not absorb substantial amounts of energy in this region. Thus, near-infrared radiation penetrates tissues more efficiently than does visible light or photons in the infrared region.

Exogenously added contrast agents would aid in the specificity and sensitivity of disease detection. Unfortunately, simple near-infrared fluorescent imaging probes are subject to the same limitations as most traditional "contrast agents" that accentuate nonspecific differences such as differences in permeability or perfusion; this results in relatively low target-to-background ratios (usually <4:1). Even the use of "targeted" fluorochromes (eg, labeled antifibronectin antibodies for the imaging of tumor angiogenesis [9]) often yields low target-to-background ratios.

We previously developed a class of contrast agents that fluoresce only after interaction with specific enzymes, for example, those overexpressed in neoplasms (10–14). On intravenous injection, these agents are not fluorescent (Fig 1), but they become fluorescent after enzymatic conversion due to fluorochrome release and abatement of intramolecular optical fluorescence quenching. The feasibility and specificity of these probes has been demonstrated previously by using microscopy, subcellular fractionation experiments, and experiments with selective inhibition of enzymatic activity (15).

View larger version (17K): [in this window] [in a new window]   Figure 1. Diagram depicts the enzyme-sensitive near-infrared fluorescent imaging probe. Biocompatible enzyme-sensitive (Cy5.5)11-PL-methoxypolyethyleneglycol92 (CPGC) probes consist of a cleavable (green line) poly-L-lysine (Lys) backbone to which fluorochromes (dark red circles) are attached. The fluorochromes quench each other (wavy lines) and thus are not detectable. Methoxypolyethyleneglycol (MPEG) side chains have been incorporated to improve biocompatibility and tumor targeting (one of multiple repeating units is shown). Upon enzymatic cleavage, fluorochromes are spatially separated and start to fluoresce (bright red circles); they are detectable at near-infrared fluorescence imaging. H = hydrogen, N = nitrogen.

	MATERIALS AND METHODS

TOP Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Imaging System
The whole-body experimental imaging system for mice (Fig 2) was composed of four parts: (a) an excitation photon source, (b) an imaging chamber with a restraint for the animal, (c) a device for recording emission photons, and (d) a computer to control image acquisition and storage. The excitation source consisted of a fiberoptic illuminator (Fiber Lite PL-800; Dolan-Jenner Industries, Lawrence, Mass) that emitted a broadband white light from a 150-W halogen bulb. A fiberoptic filter holder (Edmund Scientific, Barrington, NJ) attached between the illuminator and the light guide was used to mount the excitation source filter. A bandpass filter with a sharp cut-off was used to supply photons from 610 to 650 nm, with an attenuation of 5 OD at 700 nm (630RDF30 OD 5; Omega Optical, Brattleboro, Vt). Dual gooseneck fiberoptic light guides (EEG 2823; Dolan-Jenner Industries) were positioned to provide a relatively homogeneous excitation source. The output of the excitation light was measured to be 10–100 µW/cm²; the output depended on excitation beam geometry.

View larger version (19K): [in this window] [in a new window]   Figure 2. Diagram depicts the experimental setup for the diffusive-light near-infrared fluorescence imaging system, which has a design analogous to that of fluorescence microscopy systems. Filtered light homogeneously illuminates the animal with 610-650-nm excitation photons. Fluorescent photons are selected with a 700-nm longpass filter, which is optimized for the fluorochrome used in this study. The emission signal is focused with a zoom lens and is recorded with a cooled CCD camera.

Two different charge-coupled devices (CCDs) with analogous setups were used to acquire images. The first system for whole-mouse imaging had an f/1.2, 12.5–75-mm zoom lens that was mounted on a 12-bit, cooled CCD with 600 x 800-pixel resolution (Eastman Kodak). The second system had a cooled CCD with a larger matrix size (1,536 x 1,024 SenSys; Photometrics, Tucson, Ariz).

Software was used for image acquisition and processing and included (a) IPLab software (Signal Analytics; Vienna, Va), which was run on a PowerPC (Apple Computer; Cupertino, Calif); (b) Kodak Digital Science 1D software, which was part of the Kodak Image Station (Eastman Kodak) and which was run on a Pentium (Intel, Santa Clara, Calif)-based personal computer (Dell Computer, Round Rock, Tex); and (c) PhotoShop (Adobe Systems; San Jose, Calif). All images were stored in uncompressed tagged image file format, or tiff, for further analysis.

Image acquisition times were 0.25–300 seconds in the phantom experiments. Whole-body fluorescent images were obtained with acquisition times of 10–60 seconds by using the filters described previously. White-light images were obtained with the shutter open for 0.075 second (minimum acquisition time) and with the voltage decreased across the halogen light so as to not saturate the CCD. The cost of the experimental imaging system was approximately $25,000.

Near-Infrared Probes
The design and synthesis of the enzyme-activatable near-infrared probes is described in more detail elsewhere (15). Briefly, the probes were a modification of a synthetic graft copolymer consisting of poly-L-lysine that is sterically protected by multiple MPEG side chains (16). Each poly-L-lysine backbone contained an average of 92 MPEG molecules and 11 molecules of Cy5.5 to yield CPGC, a molecule with 44 unmodified lysine molecules on the backbone; these molecules are sites for cleavage with enzymes, such as certain cathepsins, that have lysine-lysine specificity.

The optical properties of free Cy5.5 and CPGC were initially assayed by using a fluorescence spectrophotometer (U-4500; Hitachi, Tokyo, Japan). The excitation and emission wavelengths of Cy5.5 were 673 nm and 688 nm, respectively, whereas those of the polymer-bound Cy5.5 were 673 nm and 689 nm, respectively. In the bound state, the near-infrared fluorescence of CPGC was 15-fold lower than that of Cy5.5. Up to 95% of the quenched fluorescence was recovered by means of enzymatic cleavage of the backbone (15).

Evaluation
The field of view and spatial resolution were evaluated at the following incremental focal lengths for the zoom lens: 12.5, 20, 30, 50, and 75 mm. A ruler was imaged to determine image size. The measured field of view reflected the area that was captured with the CCD. Linear pixel density per millimeter was measured at each focal length.

Phantom experiments were performed to determine the detectability threshold with the near-infrared fluorescent probes. Aliquots of serial dilutions of Cy5.5 dye were pipetted into clear plastic wells; each contained a 100-µL aliquot. Images were obtained at serial time points and concentrations. Each sample was imaged adjacent to a 100-µL water sample. IPLab Spectrum software (Signal Analytics) was used for the analysis. After a standard software filter was used to remove CCD–dependent "bad" pixels with a high level of intrinsic electronic noise, region-of-interest measurements were obtained in the sample, water, and background. The signal-to-noise ratio (SNR) was determined as follows: (mean signal - mean noise)/SD for noise.

Subcutaneous and intramuscular (deep thigh) injections of Cy5.5 allowed the estimation of appropriate amounts of quenched CPGC that were needed to visualize subsurface or deeper tumors. The amounts of probes that were injected are detailed in Results.

BT-20 mammary adenocarcinoma human tumor cells were used for in vivo implantation. Cells were cultured in Dulbecco modified Eagle medium that was supplemented with 10% fetal calf serum (Cellgro Mediatech, Washington, DC). Tumor cells (n = 10⁴–10⁵) were injected into the mammary fat pad or into the thigh in four nude mice (nu/nu; Taconic, Germantown, NY).

Animals were imaged when tumors were less than 3 mm in diameter, except for two mice in which 12–14-mm diameter tumors were evaluated for probe heterogeneity, as detailed in Results. Baseline images in the animals were obtained immediately prior to injection of CPGC to allow for the evaluation of the spatial distribution of native fluorochromes (eg, near-infrared fluorescence in the gut). Animals were then injected via the tail vein with 10 nmol of CPGC in 100 µL of normal saline. Immediate, 24-hour, 48-hour, and 72-hour images were obtained.

The animals were cared for according to the guidelines of the animal facility at our institution. Ketamine hydrochloride (9 mg per 100 g of body weight, subcutaneous administration) and xylazine hydrochloride (0.9 mg/100 g, subcutaneous administration) were used as an anesthetic, and pentobarbital sodium (10 mg/100 g, intraperitoneal administration) was used for sacrifice. For histologic evaluation, animals were euthanized, and tumors were removed and snap frozen in liquid nitrogen. Sections were stained with hematoxylin-eosin or were left unstained for near-infrared fluorescence microscopic evaluation.

The significance of the differences between the near-infrared fluorescence signal mean values of tissues was determined by using two-tailed t tests and SEMs.

	RESULTS

TOP Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Performance
The field of view and spatial resolution of the imaging system ranged from 3.3 x 4.5 to 18.5 x 24.5 and from 60 to 300 µm, respectively. Spatial resolution for deep structures is a complex function of scatter and absorption and is lower than the intrinsic resolution of the system. Figure 3a shows the relationship between the SNR and the length of time during which the image was acquired for a fixed amount (200 pmol) of unquenched dye. Figure 3b demonstrates the relationship between the SNR and the amount of unquenched dye during 30-second acquisitions. Detection of subpicomolar amounts of Cy5.5 was possible at imaging times of 5 minutes (SNR = 4.6 at 0.78 pmol). A 200-pmol phantom was detectable in 250 µsec, with an SNR of 1.9.

View larger version (7K): [in this window] [in a new window]   Figure 3a. (a) Log-log plot depicts the SNR as a function of acquisition time in a 200-pmol unquenched Cy5.5-dye phantom. For a 30-second acquisition, the SNR was 173. (b) Log-log plot depicts the SNR as a function of the amount of unquenched Cy5.5 dye in a phantom, with 30-second acquisitions. A SNR greater than 3 was achieved with 3 pmol of unquenched dye.

View larger version (9K): [in this window] [in a new window]   Figure 3b. (a) Log-log plot depicts the SNR as a function of acquisition time in a 200-pmol unquenched Cy5.5-dye phantom. For a 30-second acquisition, the SNR was 173. (b) Log-log plot depicts the SNR as a function of the amount of unquenched Cy5.5 dye in a phantom, with 30-second acquisitions. A SNR greater than 3 was achieved with 3 pmol of unquenched dye.

In Vivo Imaging of Tumor
Nonenhanced images demonstrated weak endogenous near-infrared fluorescence signal that arose from the small bowel of the animals, presumably due to fluorochromes in the diet. Immediately after the intravenous injection of CPGC, there was little appreciable increase in near-infrared fluorescence signal in any of the organs. Within 12–48 hours after injection, tumors became highly fluorescent in the near-infrared spectrum, with a SNR of 21 at 48 hours (Fig 4) in tumors with a mean diameter of less than 2 mm. The smallest detectable tumor was less than 1 mm in diameter.

View larger version (54K): [in this window] [in a new window]   Figure 4. Light image (left) and near-infrared fluorescence image (right) obtained in a mouse 24 hours after an injection of CPGC shows an implanted breast tumor that measures approximately 2 mm in diameter (acquisition time, 30 seconds). Signal that arises from the tumor (bright central focus) is due to activation of the imaging probe within the tumor. Near-infrared fluorescence signal is scaled linearly and shows the high target-to-background ratio that is achievable with this strategy.

To determine the near-infrared fluorescence signal in individual tissues, animals were sacrificed at the end of the in vivo imaging study, and tissues were excised. Figure 5 shows near-infrared fluorescence signal was highest in tumor and was significantly higher (841 arbitrary units) than blood (74; P < .001), muscle (6.9; P < .001), fat (1313; P < .001), and glandular tissue (1919; P < .001). When tumor sections were sliced, intratumoral near-infrared fluorescence signal was heterogeneous and was predominantly from the tumor periphery, presumably because of the proteolytic enzymatic concentrations in these locations.

View larger version (82K): [in this window] [in a new window]   Figure 5. Near-infrared fluorescence images show signal from different tissues and tumor sections in one animal. Top row shows homogenized tissues. Bottom row represents 1-mm sections through a tumor and shows the spatial distribution of near-infrared fluorescence signal within it.

	DISCUSSION

TOP Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

Tumor detection is enhanced by two distinct properties of the molecular probe. First, the MPEG component shields the compound and prevents rapid clearance from the plasma (17). This allows the agent to cross the relatively leaky neovasculature of tumors, with integrated accumulation over hours to days (18). Uptake within tumors in vivo is comparable to the uptake of tumor target–directed, cell-internalizing monoclonal antibodies at 24 hours (18). Second, cleavage with specific enzymes allows the spatial separation of fluorochromes (19). In the nonactivated probe, the fluorochromes are near one another; the energy states are coupled such that the characteristic emission photons are not generated. The cleaved probe demonstrates a signal that is 15-fold higher than that of the nonactivated form. A new generation of probes is currently under development; this will result in even higher activation-enhancement ratios.

The currently used CPGC probe is activated by tumor proteases and lysosomal proteases, which include cathepsins B and H (15). The specific peptide spacers that link the fluorochrome and backbone may be easily modified. A wide variety of enzyme specificities are thus theoretically possible. For example, prostate-specific antigen has a proteolytic activity with a specificity for the HSSKLQ peptide sequence (20). Other examples of targetable enzymes include cathepsin D (which is involved in metastagenesis [21] and angiogenesis [22]), caspases (23), and metalloproteinases (24) among others. Since many of the proteolytic enzymes in tumors also represent targets for novel enzymatic inhibitors (25), optical near-infrared fluorescence imaging also could be used to monitor therapy.

The current first-generation imaging system has an acceptable resolution and detection threshold but leaves room for improvement, which would allow deeper or multichannel imaging. Each of the components of the imaging system may be improved for a further increase in sensitivity of over 1,000-fold compared with that of the current system. The excitation source (halogen light with bandpass filter) may be replaced with a dye laser to increase the excitation photon flux from 100 µW/cm² to over 100 mW/cm² without substantial tissue heating.

The narrower spectral range also improves the output signal by allowing excitation that is closer to the maximum absorption peak. Emission filters may be serially combined to decrease the transmission of excitation photons. An additional notch filter with absorption at the laser excitation frequency would further aid in the elimination of this source of noise. A liquid nitrogen-cooled CCD would allow the integration of signal for longer periods by decreasing the electronic noise. Longer imaging times would further improve the detection threshold. Furthermore, near-infrared fluorescence tomographic imaging systems that could be adapted to allow cross-sectional tomographic imaging of the developed probes are under development (26,27).Practical applications: Near-infrared fluorescence imaging is a relatively new imaging modality. There are numerous potential advantages that could take this modality from being a current research tool to being a routine clinical diagnostic technique in the future. Specifically, molecular events may be imaged, which will allow the detection and localization of disease states before anatomic changes become apparent. Two near-term applications are the detection of primary breast cancer and lymph node metastases and the detection and localization of prostate cancer; imaging would reflect the increases in neoplastic and metastasis-related enzymatic activity.

Advancements in optical imaging technology and probe development are coupled; improvements in one area result in advancement in the other. We have shown the feasibility of obtaining images on which the optical signal intensity is related to specific enzyme activity. We hypothesized that the intrinsic molecular contrast used in fluorescence microscopy could thus be extended to optical microscopy. The activatable probes we have described may be useful for the entire range of imaging between the two scales of spatial resolution and image size in these modalities. Furthermore, the developed probes have potential utility in near-infrared epifluorescence microscopy, confocal fluorescence microscopy, two-photon microscopy, optical coherence tomography, diffuse optical tomography (26), laser CT (27,28), and surface imaging.

	Footnotes

 
Abbreviations: CCD = charge-coupled device CPGC = (Cy5.5)11-PL-methoxypolyethyleneglycol92 MPEG = methoxypolyethyleneglycol SNR = signal-to-noise ratio

Author contributions: Guarantors of integrity of entire study, U.M., R.W.; study concepts, all authors; study design, R.W., U.M.; definition of intellectual content, all authors; literature research, U.M., R.W.; experimental studies, U.M., R.W., C.H.T.; data acquisition and analysis, U.M., R.W.; statistical analysis, U.M., R.W.; manuscript preparation and editing, U.M., R.W.; manuscript review, all authors.

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TOP Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 

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