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Tumor Biology |
Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We hypothesized that specific biocompatible reporter probes can be
synthesized to image single target proteases in vivo. We
attached NIRF fluorochromes through specific peptide substrates
directly to a delivery vehicle (Fig. 1)
. Although it is conceivable to design alternative low molecular weight
probes in which a fluorescence donor and a quencher are directly
attached to a substrate peptide (2
, 5)
, such compounds are
typically subject to fast excretion in vivo. For this
reason, and to improve tumoral delivery of the NIRF probes, we used a
novel, long-circulating synthetic PGC that has recently been
tested in clinical trials (6)
. The copolymer accumulates
in tumors by slow extravasation through permeable neovasculature,
reaching up to 2–6% injected dose/g tissue in mice within 24–48 h
after injection in some tumor models (7)
. Uptake of the
polymer into tumor cells occurs by pinocytosis and is comparable in
magnitude to that of tumor-specific internalizing monoclonal
antibodies. The intracellular release of NIRF probes results in a
fluorescent signal that can be detected in vivo at
subnanomolar quantities and at depths sufficient for
experimental or clinical imaging, depending on the NIRF image
acquisition technique (4)
.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The structure, physicochemical
characteristics, and biological behavior of the graft copolymer have
been described previously, and a similar compound has been tested in
clinical trials (6)
. The preparation of the entire NIRF
probe has been described in more detail elsewhere (12)
.
Briefly, PGC (Mr 450,000) was reacted
with a large excess of iodoacetyl anhydride (Aldrich, Milwaukee, WI) to
convert all available amino groups of the
poly-L-lysine backbone into iodol groups. The
CaD-specific peptide
Gly-Pro-Ile-Cys(Et)-Phe-Phe-Arg-Leu-Gly-Lys(FITC)-Cys-NH2
[the italicized amino acids correspond to CaD substrate
(5)
] was synthesized using an automatic synthesizer (PS3;
Rainin, Woburn, MA). N-(9-Fluorenyl)methoxycarbonyl
chemistry with
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate/N-hydroxybenzotriazole as the activating
agent was used in peptide synthesis. The purified and characterized
peptide was then attached to the iodoacetylated PGC through a
thiol-specific reaction. The NIRF dye Cy5.5 (Amersham, Arlington
Heights, IL) was subsequently conjugated to the N-terminus of the CaD
substrate peptide. Peptide and fluorochrome attachment was quantitated
by absorption measurements of FITC and Cy 5.5. Extinction coefficients
were 73 x 103
M-1cm-1
at 494 nm for FITC and 250 x 103
M-1cm-1
at 675 nm for Cy5.5. On average, each graft copolymer contained 22 NIRF
reporter groups, as determined by fluorescence measurements. The
calculated molecular weight of the entire probe is 490,000. To test the specificity of the developed probe, 0.2 µM of the CaD-sensitive probe and a nonsense scrambled control peptide-based probe [Gly-Pro-Phe-Cys(Et)-Ile-Arg-Phe-Leu-Gly-Lys(FITC)-Cys-NH2; Ref. 12 ] were treated with 1 unit of hCaD (Sigma, St. Louis, MO) in 1 ml of 50 mM glycine/hydrochloride buffer (pH 3.5) with 0.001% Brij-35. The fluorescence activation of the two probes was measured by fluorometry [excitation:emission = 675:694 nm; (U4500; Hitachi, Tokyo, Japan)].
Cell Culture.
Two rodent rat embryo tumor cell lines (3Y1) were kindly provided by
Drs. H. Rochefort and M. Garcia [Institut National de la
Santé et de la Recherche Médicale, Montpellier, France
(8
, 9) ]. One of the cell lines was stably transfected
with full-length hCaD (CaD+), whereas the other cell line was
mock-transfected with an empty expression vector, resulting in no hCaD
expression (CaD-; Fig. 2
). These rodent cell lines were initially chosen for transfection
because they neither produce nor secrete rodent CaD. Both cell lines
were grown in RPMI 1640 supplemented with 10% fetal bovine serum and
0.4 mg/ml G418 in a humidified 6% CO2 atmosphere
at 37°C (9)
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To induce solid tumors, 1.5 x 106 cells were injected s.c. into the lateral lower abdomen of nude mice (n = 5). Tumor implantations were performed 1 week apart because the CaD+ tumor demonstrated a slightly faster growth rate. Within 2–3 weeks after the last implantation, each mouse had developed both CaD+ and CaD- tumors of 5–12 mm in size.
Assays for CaD Expression.
Expression of hCaD (or lack thereof in the mock-transfected cell line)
was verified by Western blotting and RT-PCR. For the latter, previously
described primers (13)
were used at 0.2 µM
and at a cDNA equivalent of 7 ng of RNA. Combined annealing and
extension were performed at 72°C for 150 s. Control PCR
experiments for the housekeeping gene ß-actin were performed
on the same samples using commercially available primers (Clontech,
Palo Alto, CA). Cycle numbers for the individual products were 34–36
for CaD and 20–22 for ß-actin. PCR products were analyzed by
electrophoresis on 2% agarose gels. Gels were photographed using a
Kodak DC 40 system and analyzed with BioMax software (Kodak, Rochester,
NY).
For Western blotting, cells were lysed in 2 ml of buffer [20 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100]. The lysate was kept on ice for 20 min and then centrifuged for 10 min at 4°C at 16,000 x g. Protein concentration was determined by the BCA assay (Pierce, Rockford, IL). Equal amounts of protein were loaded and electrophoretically separated by SDS-PAGE in nonreducing 10% gels. After transfer onto polyvinylidene difluoride membranes, immunoblots were incubated for 30 min with an anti-hCaD polyclonal antibody (Calbiochem, San Diego, CA) for 30 min. The primary antibody was revealed with an alkaline phosphatase-conjugated goat antirabbit IgG secondary antibody (Sigma) using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate (Boehringer Mannheim, Indianapolis, IN).
In Vivo Imaging.
The imaging system used in this study consisted of a light-tight
imaging chamber equipped with a 150 W halogen white light filtered with
an excitation bandpass filter (610–650 nm; Omega Optical, Brattleboro,
VT; Ref. 14
) to excite Cy5.5. Fiberoptic cables and light
diffusers resulted in a relatively spatially homogenous photon source
over the imaging area. Fluorescence was detected by a 12-bit monochrome
CCD camera (Kodak) equipped with a f/1.2 12.5–75 mm zoom lens and an
emission long pass filter at 700 nm (Omega Optical). Images were
digitally acquired and saved as 16-bit Tiff files.
Anesthetized mice (n = 5) were imaged 24 h after i.v. injection of 2.5 nmol of fluorochrome (corresponding to 2.5 mg/kg probe). To further analyze in vivo imaging findings, each tumor pair excised from a single animal was also imaged after excision. Image analysis was performed using commercially available software (Kodak Digital Science 1D software, Rochester, NY). Elliptical regions of interest were selected manually for tumor center and tumor periphery for CaD+ and for entire CaD- tumors, as well as for representative adjacent nontarget tissue (adjacent thigh). The mean and SD of SI of pixel values was recorded. CNRs were calculated for each animal. Significance of differences among groups was determined using a one-tailed Student’s t test.
Histology.
Harvested tumors were snap frozen in liquid nitrogen and cut into
8–20-µm sections. Immunohistochemistry was performed using a primary
rabbit polyclonal antibody against hCaD (Dako, Carpinteria, CA). The
primary antibody was revealed with a goat antirabbit antibody
conjugated to alkaline phosphatase. After a heat step (65°C, 30 min),
phosphatase activity was visualized using nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate, and
sections were counterstained with nuclear fast red. CaD expression was
also assessed on nonfixed 20-µm sections by NIRF microscopy using
appropriate excitation and emission filters (650/700 nm; Omega
Optical). Sections were viewed in phase-contrast or NIRF mode using an
inverted epifluorescence microscope (Zeiss Axiovert, Thornwood, NY),
and images were obtained with a cooled CCD camera (Sensys;
Photometrics, Tuscon, AZ) interfaced to a Macintosh computer.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The mock-transfected control cell line (CaD-) and
whole CaD- tumors were negative for mRNA expression. Western blotting
of the CaD+ cells showed a faint band at
Mr 52,000 (proCaD) and a pronounced
band at Mr 34,000 (activated CaD; Fig. 2b
).
In vitro, the synthesized CaD probe was essentially
optically "silent," generating <1 AU at 675 nm excitation and 694
nm emission, whereas the CaD activated probe generated over 60-fold
higher fluorescence. To test for the specificity of activation, we also
compared the probe with a scrambled peptide containing the same amino
acids but in a different sequence. As shown in Fig. 3a
, there was essentially no NIRF activation with the control
probe when incubated with purified hCaD. To test for the linearity of
NIRF signal generation, the probe was also incubated with different
amounts of CaD+ cells (Fig. 3b)
. The measured NIRF signal
was linear over a range of at least 4 orders of magnitude
(r2 = 0.99; Fig. 3b
). When
CaD+ cells incubated with the probe for 1 h were observed by
confocal fluorescence microscopy, a NIRF signal could be seen
originating from vesicular structures within cells, presumably
representing the lysosomal compartment (Fig. 3, c and d)
. No detectable signal was observed when control probes
containing nonsense peptide spacers were used. Likewise, no signal was
observed when CaD- cells were used.
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. The CaD+ tumors always emitted much higher fluorescence than the
matched CaD- tumors. Mean signal intensities and CNRs are summarized
in Table 1
. The mean CNR for tumor periphery CaD+ tumors compared with CaD-
tumors was 8.6, whereas the center of CaD+ tumors compared with CaD-
tumors resulted in a CNR of 6.0. The CNR of CaD+ periphery and center
compared to nontarget tissue was 22.8 and 16.2, respectively. The
differences between mean SI for tumor periphery of CaD+ compared with
entire CaD- tumors and for tumor center of CaD+ compared with entire
CaD- tumors were highly significant (P < 0.001 and P < 0.005, respectively), as was
the comparison of CaD+ tumor periphery and center with background
tissue (P < 0.001 for both).
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confirmed the marked differences in probe activation between CaD+ and
CaD- tumors. Heterogeneity of tumoral NIRF signal was observed
in vivo, in excised tumor specimen (Fig. 5)
, and also in histological sections (Fig. 6)
. CaD expression was
commonly highest in the periphery of tumors, in intratumoral
septae, and in focal nodular areas with high growth rates. A similar
finding has also been observed previously for other proteases when
studied in tumor chamber models by intravital microscopy (data not
shown). The morphological appearance of the tumors and their vascular
density were similar by histology.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The primary goal of the current study was to investigate whether
a given single protease could be detected noninvasively by in
vivo imaging. Although the model system used in this study may be
somewhat artificial (overexpression of a transgene), our results
clearly confirm the feasibility of the approach. CaD, an aspartic
lysosomal protease, was chosen as a target protease because of the
availability of CaD+ and CaD- tumor models (8
, 9)
, the
fact that the enzyme is primarily localized in the lysosomal
compartment of cells (8)
into which the graft copolymer is
internalized (7
, 11)
, and because of the potential role of
the enzyme in metastasis formation (8)
and local
invasiveness (27)
. Early detectable NIRF signals emanated
from the CaD+ tumors both in vivo and ex vivo and
by microscopy. Interestingly, in vivo CaD expression in
experimental tumors was somewhat heterogeneous and preferentially
occurred in the tumor periphery, well-vascularized septae traversing
the tumor, and/or focal areas with high cell turnover. Although it is
conceivable that the CaD+ and CaD- tumors may have slightly different
vascular permeabilities, prior research has shown that the developed
graft copolymers distribute evenly in the steady state, unless VEGF is
markedly up-regulated, for example, such as in transfected tumor models
(28)
. Even if local accumulation of the nonactivated probe
in the interstitium was heterogeneous, this would be undetectable by
NIRF imaging because the probe has not yet been activated (note also
the absence of NIRF signal in the blood, where probe concentration is
relatively high; Fig. 6
).
The developed protease reporter probe was based on a synthetic graft copolymer (partially methoxy-polyethylene glycol-modified poly-L-lysine) originally developed as a long-circulating drug carrier with circulation times in excess of 36 h in rodents and 20 h in humans (6 , 11 , 29) . The graft copolymer is slowly internalized into proliferating tumor cells (7 , 11) and has thus been used for chemotherapeutic drug delivery (30) . The CaD-specific peptide (5) used in this study consisted of the Gly-Pro-Ile-Cys(Et)-Phe-Phe-Arg-Leu-Gly-Lys(FITC)-Cys-NH2 sequence. Given the modular design of the reporter probe, the peptide sequence could easily be replaced with sequences specific for other proteases. For example, fairly specific peptide substrates have been described for caspases (31) , MMPs (32) , viral proteases (33) , or prostate specific antigen, an intracellular serine protease (34) . Future developments of imaging probes against these and other targets would be of considerable clinical value.
The NIRF fluorochrome chosen for this study was an indocyanine compound (Cy5.5) with an emission maximum at 694 nm. The NIR wavelength of excitation and emission was chosen to improve tissue penetration compared with fluorochromes active in the visible light range (35) . The use a number of other fluorochromes with excitations/emissions at even longer wavelengths (700–1000 nm) to further decrease attenuation by tissues and potentially enable multichannel imaging in the NIR spectrum is conceivable. The second reason for using indocyanine fluorochromes was their generic ability to efficiently quench when in close spatial proximity to each other, thus avoiding the need for dedicated quenchers such as 4-(4'-dimethylaminophenylazo)benzoic acid used in other molecular beacon approaches (1) . In prior research, we had optimized the number of fluorochromes per PGC for maximum quenching to occur and shown that at least 10 molecules are required (4) . In the current study, 22 fluorochromes were positioned on peptide stalks on each PGC, which is the reason for the extremely low signal of the reporter in its native state and the intense fluorescence in the activated state (12) .
NIRF signal generation in this study was detected by using CCD technology, either in the form of microscopy or whole animal imaging (14) . The latter was done with a planar imaging system, using a continuous excitation source and a long pass filter to record emission at >700 nm. Because we used a long pass rather than a bandpass filter to record generated fluorescence, the native background signal was high from both reflected excitation photons not filtered due to the slight overlap of filter frequency cutoff tails and other native fluorochromes with emission >700 nm. The more recent use of narrow bandpass filters (705–715 nm) and the simultaneous use of polarization filters have further decreased nonspecific background. Although this prototype imaging system is useful for fast, high-resolution screening of superficial events, it generally does not allow adequate detection of photons from deep tissues (i.e., >10 mm deep). If the latter is required (as in the clinical scenario, unless endoscopic imaging is performed), different NIR optical detection systems have to be used. Such systems can be based on phase modulation signal (36 , 37) , diffuse optical tomography (38) , or ultrafast imaging of ballistic and/or early arriving photons (39) . As these innovative imaging approaches enter the realm of clinical testing, it will become apparent that there will be sufficient SI from deep tissues when used with the developed probes (40) .
The current study indicates that the autoquenched probe is specific in vivo. Although the detection threshold of the probe was not directly determined in this study, Cy5.5 concentrations in the nanomolar range have been previously detectable (4) . With a more sensitive detection system such as phase array technology (36) , the detection threshold is expected to be in the picomolar range in deep tissue. Recalculating these detection thresholds, it is entirely feasible to reach locally high enough substrate concentrations at physiologically acceptable doses of fluorochromes. Although the dose used in this rodent study would scale up to 175 mg of drug substance in a human, it is at least 2–3 orders of magnitude above the theoretical detection threshold. Fluorochromes such as indocyanine green are used routinely in cardiology, hepatology, ophthalmology, and, most recently, clinical diffuse optical tomography imaging (40) at i.v. doses of 25–50 mg.
Although the current study used CaD as a model protease, a variety of other proteases are highly relevant in oncological and cardiovascular imaging (18 , 19) . The next logical step will be to identify a clinically relevant protease and design a similar probe for clinical testing. Ideally, the probe would be tested in a protease inhibitor trial to monitor real-time drug efficacy at the molecular level. To this extent, we have synthesized a MMP-2-sensitive probe and shown that MMP-2 inhibitor treatment can indeed be monitored by this approach.
In summary, the use of reporter probes to noninvasively image enzyme activity has been demonstrated in vivo. One of the main utilities of the technique is expected to lie in the evaluation of the large number of metalloprotease and HIV protease inhibitors currently under development and/or in clinical use. As we move forward to design and build imaging probes with molecular specificity, new applications are likely to occur such as in vivo imaging of transgenes or screening of potential drug candidates.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by National Institute of
Diabetes and Digestive and Kidney Diseases Grant R21-DK55713-01, the
Massachusetts General Hospital-Center for Molecular Imaging Research
Development Fund, and a grant from the Massachusetts General Hospital
Center for Innovative Minimally Invasive Therapy. 
2 To whom requests for reprints should be
addressed, at Center for Molecular Imaging Research, Massachusetts
General Hospital, Building 149, Room 5403, Charlestown, MA 02129.
Phone: (617) 726-8226; Fax: (617) 726-5708; E-mail: weissleder{at}helix.mgh.harvard.edu
3 The abbreviations used are: NIR, near-infrared;
NIRF, NIR fluorescence; PGC,
poly-L-lysine/methoxypolyethylene glycol graft copolymer;
CaD, cathepsin D; hCaD, human CaD; RT-PCR, reverse transcription-PCR;
CCD, charge-coupled device; CNR, contrast:noise ratio; MMP, matrix
metalloproteinase; SI, signal intensity.
Received 3/29/00. Accepted 6/27/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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