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[Cancer Research 64, 1579-1583, March 1, 2004]
© 2004 American Association for Cancer Research

Advances in Brief

In Vivo Imaging of ß-Galactosidase Activity Using Far Red Fluorescent Switch

Ching-Hsuan Tung, Qing Zeng, Khalid Shah, Dong-Eog Kim, Dawid Schellingerhout and Ralph Weissleder

Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
ß-Galactosidase (ß-gal) has been widely used as a transgene reporter enzyme, and several substrates are available for its in vitro detection. The ability to image ß-gal expression in living animals would further extend the use of this reporter. Here we show that DDAOG, a conjugate of ß-galactoside and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO), is not only a chromogenic ß-gal substrate but that the cleavage product has far-red fluorescence properties detectable by imaging. Importantly, the cleavage substrate shows a 50-nm red shift, enabling its specific detection in a background of intact probe, a highly desirable feature for in vivo imaging. Specifically, we show that ß-gal-expressing 9L gliomas are readily detectable by red fluorescence imaging in comparison with the native 9L gliomas. We furthermore show that herpes simplex virus amplicon-mediated LacZ gene transfer into tumors can be transiently and thus serially visualized over time. The results indicated that in vivo real-time detection of ß-gal activity is possible by fluorescence imaging technology.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
Reporter genes, such as ß-galactosidase (ß-gal), chloramphenicol acetyltransferase, luciferase, and green fluorescent protein, have been widely used to study gene expression and regulation in biological systems (1) . Most of these reporters are visualized in tissue sections or postmortem samples. The ability to monitor gene expression and regulation in living animals has therefore been of prime interest (2) , and several generic methods have been described recently. In particular, luciferases (3) , thymidine kinases (4 , 5) , and green fluorescent protein (6) have been shown recently to be detectable in vivo at different resolutions, sensitivities, costs, and abilities to quantitate (7) . Here we report an in vivo optical imaging method to directly image ß-gal activity in vivo, one of the most common reporter genes.

ß-gal (EC 3.2.1.23), expressed by the bacterial LacZ gene, is a commonly used reporter because of its ease of use and availability of different detecting methods. Both fluorogenic (8, 9, 10, 11) and chromogenic (12 , 13) substrates have been developed over the last two decades. Among the different agents, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) is routinely used for ß-gal expression staining (12) . More recently, a gadolinium-based galactosidase probe has been reported and used for magnetic resonance imaging of Xenopus larvae (14) . Given the recent advances in near infrared fluorescence (NIRF) imaging (15, 16, 17) , we hypothesized that it should be possible to detect ß-gal-converted fluorogenic compounds in vivo. We discovered that a previously described compound, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) ß-D-galactopyranoside (DDAOG), had far red fluorescence properties that could be adapted to in vivo imaging technologies (Fig. 1Citation ; Refs. 18 , 19 ). Here we demonstrate the feasibility of in vivo imaging of ß-gal expression using this red fluorescent shift reagent.



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  		Fig. 1. A, structure and reaction with galactosidase of DDAOG. B, excitation (solid line) and emission (dashed line) spectra of DDAOG and its hydrolytic product DDAO.

 

   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
In Vitro Activation.
The 9L gliosarcoma cell line was a generous gift from the University of California San Francisco Neurosurgery Tissue Bank, and the 9L-LacZ cell line was purchased from the American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM with 4.5 g/l glucose, 1% penicillin-streptomycin, and 10% fetal bovine serum.

To study cellular activation of DDAOG (Molecular Probes, Eugene, OR), different numbers of 9L and 9L-LacZ cells ranging from 50 x 103 to 300 x 103 at 50 x 103 intervals were seeded in 6-well plates for 12 h. The medium was collected from each well, and DDAOG (final concentration, 1 µM) was added into 200 µl of the collected medium in a 96-well plate and incubated for 2 h. The fluorescence signal was measured using a home-build fluorescence imaging system (20) . The excitation and emission filter sets at 615–645 and 680–720 nm, respectively, were used (Omega Optical, Brattleboro, VT).

Detailed cellular internalization and activation were monitored using a confocal microscope (Zeiss Axiovert 200, Thornwood, NY). 9L and 9L-LacZ cells were grown in a Lab-Tek II Chamber (Nalge Nunc, Naperville, IL) to ~70% confluency. The cells were incubated with DDAOG (10 µM) in Hanks’ buffer for 30 min and washed three times with Hanks’ solution before imaging. Confocal microscopy was performed with a Zeiss LSM Pascal Vario Laser Module (argon, 458/488/514 nm; HeNe, 633 nm). The argon 488-nm laser, paired with a 505-nm long-pass emission filter, was used to visualize DDAOG, and the HeNe 633-nm laser, paired with a 650-nm long-pass emission filter, was used to visualize activated product, DDAO.

In Vivo Tumor Imaging.
Two million cells (either 9L or 9L-LacZ) were injected s.c. in the mammary fat pad of athymic nude mice (nu/nu, 5–6 weeks of age, n = 6; Massachusetts General Hospital Cox breeding facility). Tumors were allowed to grow to 5 mm in size. Animals were then anesthetized by an i.p. injection of ketamine (90 mg/kg) and xylazine (10 mg/kg), and the imaging probe 0.5 mg of DDAOG in 100 µl of DMSO and PBS (1:1) was injected i.v. NIRF reflectance imaging was performed using the previously described imaging system at different time points (20) . The same excitation and emission filter sets for in vitro imaging were used for in vivo imaging. Images were acquired by 2-min exposure and analyzed using commercially available software (Kodak Digital Science 1D software, Rochester, NY). Signal and background level were measured by manually placing regions of interest (<=200 pixels) within the visible tumor margins and the adjacent skin, respectively.

Generation of LacZ Herpes Simplex Virus (HSV)-1 Amplicon Vectors.
Amplicon plasmids, pHZCX-expressing ß-gal protein under the immediate-early IE4/5 HSV promoter, were packaged as HSV amplicon vector by using a helper virus-free packaging system (21) . For this purpose, Vero 2-2 cells were transfected by using Lipofectamine (Invitrogen, Carlsbad, CA) with a mixture of the pHZCX amplicon DNA, plasmid DNA containing the ICP27 gene, and a bacterial artificial chromosome DNA that includes the entire HSV genome deleted for the sequences containing the DNA cleavage-packaging signals (ori-pac sequences) and the essential gene, ICP27. Amplicon viral vector stocks were harvested 60 h later, freeze-thawed three times, sonicated, and purified by brief centrifugation at 1000 x g for 10 min. Amplicon titers (in transducing units/ml) were determined by infecting 293T/17 cells/well in 24-well plates and then counting the LacZ-positive cells at x10 magnification 18 h after infection.

Imaging Viral Infection.
Gli36 (1.5 x 105 cells), grown in DMEM supplemented with 10% fetal bovine serum in a humidified 6% CO2 atmosphere at 37°C, were infected with 1 µl (5 x 108 transducing units/ml) of HZCX amplicon. Eighteen h after infection, the cells was detached by trypsin/EDTA solution, aspirated, and dispensed into a Lab-Tek II Chamber (Nalge Nunc). The cells were cultured for an additional 18 h, incubated with 1 µM DDAOG for 30 min, washed three times with Hanks’ buffer, and then subjected to epifluorescence microscopy as described previously.

For ß-gal gene expression correlation studies, Gli36 cells (3 x 105 cells) grown in a 24-well plate were infected with different multiplicities of infection of HZCX amplicon (0, 0.125, 0.25, 0.5, 1, and 2), and 36 h later, cells were incubated with 1 µM DDAOG for 1 h. The medium (100 µl) was collected, and the fluorescence intensity of DDAO was measured using a fluorescence plate reader (GeniosPro; Tecan, Maennedorf, Switzerland).

Gli36 cells in mid-log phase were harvested by trypsinization, and single-cell suspensions of 10 x 106 cells in 0.75 ml of DMEM were injected s.c. into six nude mice, 4–5 weeks of age. Seven to 10 days later, when solid flank tumors could be detected (5–7 mm in diameter), mice received intratumoral injections of either HZCX amplicon vector (5 x 108 transducing units/ml) or control HGCX vector expressing green fluorescent protein (4.3 x 108 transducing units/ml), in a total volume of 20 µl, with intratumoral manipulation of the needle to ensure spread of virus. Forty h after injection of virus, 0.5 mg of DDAOG in 100 µl of DMSO/PBS was injected via tail vein, and the animals were imaged at 15, 30, and 45 min. The tumors were then dissected and sectioned for histology.

Histology.
Tumors were excised 45 min after DDAOG injection for the highest fluorescence signal, embedded in Tissue-Tek OCT (Sakura Finetechnical, Torrance, CA), frozen in liquid nitrogen, and sectioned into 10-µm slices. The frozen sections were divided into two sets. One set was costained with X-gal and H&E. The X-gal stain was the mixture of 0.1 M sodium phosphate buffer containing 10 mM KCl, 1 M MgCl2, 100 mM K4Fe(CN)6, 100 mM K3Fe(CN)6, 10% Triton X-100 (pH 7.5), and 2% X-gal (Bioscience, La Jolla, CA) in dimethyl formamide. The slides were incubated at 37°C in a moisture chamber for 4 h, and the reaction was stopped by adding 0.5 mM EDTA in PBS. The sections were then rinsed in PBS and stained with H&E (Fisher, Fair Lawn, NJ). The adjacent sections of the X-gal and H&E-stained sections were fixed only with 0.5% glutaraldehyde for 1 min and washed with PBS. The histological sections were then viewed in phase contrast or fluorescence mode using an inverted epifluorescence microscope (Zeiss Axiovert). Excitation wavelengths were 600–620 nm. A cooled CCD camera (Sensys; Photometrics, Tucson, AZ) adapted with a narrow-bandpass, 650–690-nm filter was used for image capture.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
DDAOG, a conjugate of ß-galactoside and DDAO and its analogs were originally reported as chromogenic substrates (Fig. 1ACitation ; Refs. 18 , 19 ). However, we noticed that this compound also has fluorescence properties, which may enable in vivo imaging of ß-gal activity. In vivo fluorescence imaging has been reported recently by reflectance (20) or tomographic (16) methods, generally using far red (600–700 nm) and NIR (700–900 nm) wavelengths for more efficient tissue penetration. DDAOG has an excitation/emission at 465/608 nm in PBS; however, interestingly, its hydrolyzed product, DDAO, has red-shifted excitation and emission peaks at 646 and 659 nm, respectively (Fig. 1B)Citation . This dramatic bathochromic shift permits: (a) more efficient tissue penetration; and (b) the detection of the hydrolytic product, even in the presence of large amounts of substrate when coupled with appropriate excitation and emission filters. Indeed, excitation >600 nm excites only the hydrolytic product but not the intact DDAOG precursor (Fig. 1)Citation .

To determine the utility of DDAOG as an imaging probe, we first tested it with 9L (LacZ-negative) and 9L-LacZ-positive glioma cell lines (22) . Fig. 2ACitation shows considerable higher fluorescence with 9L-LacZ cells when compared with control cells. Furthermore, there was a near linear dose-response curve over the concentrations tested (50 to 300 x 103 cells/well; Fig. 2ACitation ). Cellular distribution of the probes was further examined by confocal microscopy using a 488-nm argon laser and a 633-nm HeNe laser as well as with 505-nm and 650-nm long-pass emission filters for DDAOG and DDAO, respectively. In 30 min, DDAOG and activated DDAO signals were found localized in the cytoplasm of 9L-LacZ cells but not in the nuclei (Fig. 2B)Citation . In 9L cells, only the intact DDAOG precursor was found but not the hydrolyzed red-shifted DDAO product.



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  		Fig. 2. A, cellular activation of the DDAOG with 9L and 9L-LacZ cells. Bars, SD. B, confocal microscopy images of DDAOG, DDAO, and merged images in 9L and 9L-LacZ cells. The cells were incubated with DDAOG (1 µM) for 30 min. Note the difference in the DDAO images.

 
In the next set of experiments, we wanted to determine whether similar DDAOG activation could be observed in intact tumors in vivo. For each experimental animal, we co-implanted both tumors into the chest wall of nude mice. When tumors reached ~5 mm in diameter, DDAOG (0.5 mg) was injected though the tail vein, and animals were imaged serially for 2 h using a previously described reflectance imaging system (20) . Visually, a much higher fluorescence signal was observed from all 9L-LacZ tumors when compared with co-implanted 9L tumors (Fig. 3A)Citation . Serial image analysis is shown in Fig. 3BCitation and shows a 4-fold higher fluorescent signal (P < 0.0001) emanating from 9L-LacZ tumors compared with control tumors 45 min after DDAOG injection. Serial imaging showed that the highest signal to peak, at 40–60 min, appeared as early as 10 min after injection and completely abated by 5 h, enabling serial imaging. To verify the imaging results, we subsequently performed NIRF imaging of unstained sections, as well as H&E and X-gal staining (Fig. 3C)Citation . As expected, intensive blue X-gal staining and concomitant DDAO fluorescence was found only in 9L-LacZ tumors but not in control tumors.



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  		Fig. 3. In vivo imaging of ß-gal expression. A, whole-body images of 9L (left) and 9L-LacZ (right) tumors at 45 min after i.v. injection of 0.5 mg of DDAOG. B, time course of in vivo activation of DDAOG in 9L and 9L-LacZ tumors (n = 6; P < 0.0001)). Bars, SD. C, histology and fluorescence microscope of 9L and 9L-LacZ tumor sections.

 
The method was further tested to determine whether transient gene expression of LacZ mediated through a viral vector could be detected. We used an HZCX-HSV amplicon vector containing the LacZ gene under the immediate-early IE4/5 HSV promoter to infect human glioma Gli36 tumor cells. Fig. 4ACitation demonstrates that HZCX-HSV amplicon-infected cells were able to activate the DDAOG. The correlation of gene expression and fluorescence intensity was also quantitated by adding various amounts of virus. As seen in Fig. 4BCitation , a near linear correlation (r = 0.99) was observed. In vivo transduction experiments of the amplicon vector into s.c. implanted Gli-36 tumors were subsequently carried out. Forty h after tumoral injection of the vector, animals received a single dose of DDAOG via tail vein injection. HSV amplicon-injected tumors showed a 3-fold higher fluorescence signal compared with noninfected tumor (Fig. 4, C and D)Citation . The expression of LacZ gene in tumors was corroborated by histological staining and fluorescence imaging on tumor sections (Fig. 4E)Citation . When a negative control, green fluorescent protein-expressing HSV amplicon was injected to tumor, no increased fluorescence signal was observed (data not shown).



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  		Fig. 4. In vitro infection of Gli36 cells with HSV virus containing LacZ (A) and the correlation of gene expression and fluorescence signal (B). Gli36 cells (300 xE3) were infected with various amounts of HSV-LacZ for 40 h. Bars, SD. C–E, in vivo gene therapy of Gli36 tumor with HSV-LacZ. The viruses were injected into the left tumor, and DDAOG was injected systemically 40 h later. C, whole-body imaging of gene expression (45 min). D, fluorescence intensity 45 min after probe injection (n = 6). Bars, SD. E, histology.

 
The above experiments indicate that far red and potentially NIRF shift reagents represent a novel and useful approach to image enzymes such as galactosidase in living animals. Because the nonconverted probe has different fluorescent properties, enzymatic conversion (and hence presence of an enzyme) can be detected reliably. Conversion of the probe into fluorescent species thus represents one form of inherent signal amplification by reducing background. We hypothesize that the current approach could be extended to a variety of other enzyme systems and may be particularly suited to image endogenous hydrolytic and proteolytic enzymes. Although the current system appeared to be robust, one could envision several strategies to further improve the utility of the system:

(a) By exchanging DDAO for a more red-shifted molecule, imaging could be performed in the NIR (e.g., 800 nm), which usually results in better target to background ratios because of improved tissue penetration, reduced scattering, and lower autofluorescence (17) .

(b) Sensitivity will be considerably improved by applying fluorochromes with better separated excitation and emission spectra. The currently used DDAO has a narrow emission spectrum and it seriously overlaps with the excitation spectrum (Fig. 1B)Citation ; thus, the sensitivity was significantly cut back.

(c) Use of tomographic rather than reflectance imaging could be used to quantitatively measure enzyme conversion in deeper tissues such as in brain, lung, or orthopic tumors (16) .

(d) It is conceivable to design reporter moieties with specificity for other biologically relevant enzymes.


   ACKNOWLEDGMENTS
 
We thank Dr. Xandra Breakefield for technical support and the University of California San Francisco Neurosurgery Tissue Bank for the kind gift of 9L gliosarcoma cells.


   FOOTNOTES
 
Grant support: NIH P50-CA86355 and RO1 CA99385.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Ching-Hsuan Tung, Center for Molecular Imaging Research, Massachusetts General Hospital, 149 13th Street, Room 5406, Charlestown, MA 02129. Phone: (617) 726-5779; Fax: (617) 726-5708; E-mail: tung{at}helix.mgh.harvard.edu

Received 10/14/03. Revised 12/12/03. Accepted 1/ 6/04.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

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