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Published as doi: 10.1096/fj.06-7366lsf.
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19F-NMR detection of lacZ gene expression via the enzymic hydrolysis of 2-fluoro-4-nitrophenyl ß-D-galactopyranoside in vivo in PC3 prostate tumor xenografts in the mouse 1

Li Liu, Vikram D. Kodibagkar, Jian-Xin Yu and Ralph P. Mason2

Department of Radiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA

2Correspondence: Department of Radiology, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, Texas 75390-9058, USA. E-mail: ralph.mason{at}utsouthwestern.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gene therapy shows promise for treating prostate cancer and has been evaluated in several clinical trials. A major challenge that remains is to establish a method for verifying transgene activity in situ. The lacZ gene encoding ß-galactosidase historically has been the most popular reporter gene for molecular biology. We have designed a 19F NMR approach to reveal lacZ gene expression by assessing ß-galactosidase (ß-gal) activity in vivo. The substrate 2-fluoro-4-nitrophenyl ß-D-galactopyranoside (OFPNPG) is readily hydrolyzed by ß-gal with a corresponding decrease in the 19F-NMR signal from OFPNPG and the appearance of a new signal shifted 4–6 ppm upfield from the aglycone 2-fluoro-4-nitrophenol (OFPNP). We report proof of principle in cultures of PC3 prostate cancer cells using 19F NMR spectroscopy and 19F chemical shift imaging. More importantly, we demonstrate for the first time the ability to differentiate wild-type and lacZ-expressing prostate tumor xenografts in mice using this approach.—Liu, L., Kodibagkar, V. D., Yu, J-X., Mason, R. P. 19F-NMR detection of lacZ gene expression via the enzymic hydrolysis of 2-fluoro-4-nitrophenyl ß-D-galactopyranoside in vivo in PC3 prostate tumor.


Key Words: ß-galactosidase • 19F CSI • 19F MRS • gene reporter


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GENE THERAPY SHOWS PROMISE for treating cancer and has been tested in several clinical trials for the prostate (1 2 3 4 5 6 7 8) . A major hurdle is to establish a method of verifying transgene activity in situ; various reporter genes have been developed (9 , 10) , in some cases using a single gene as both therapeutic and reporter (e.g., thymidine kinase or cytosine deaminase) (11 12 13) . The lacZ gene encoding the enzyme ß-galactosidase (ß-gal), first described by Jacob and Monod (14) , remains a popular reporter gene in molecular biology. PCR and Western blot are the most commonly used techniques for evaluation of gene expression, and can be used for quantitation, but are highly invasive (requiring a biopsy). Multiple colorimetric reporter substrates for ß-gal have been demonstrated, and some are commercially available for histology and in vitro detection (15 16 17 18 19) .

However, current methods of detecting ß-gal activity are generally not suitable for applications in vivo. Therefore, development of reporter molecules for noninvasive in vivo detection of lacZ transgene expression would be of considerable value both for research and future clinical gene therapy trials. A characteristic of ß-gal is its extreme promiscuity (lack of substrate specificity), which can be exploited with a variety of substrate structures. Recently, Tung et al. (20) presented an optical near-infrared fluorescence approach based on 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) ß-D-galactopyranoside and detected ß-gal expression in xenografts in mice. Lee et al. (21) described a radiolabeled substrate 2-(4-[125I/123I]iodophenyl)ethyl-1-thio-ß-D-galactopyranoside, which was used to detect ß-gal activity in mice using a gamma camera. Louie et al. (22) reported a Gd(III)-based 1H MRI approach using 1-[2-(ß-D-galactopyranosyloxy)propyl]-4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane) gadolinium (III) to assess ß-gal activity in developing tadpoles.

We previously presented the successful synthesis and evaluation of fluoro-nitro-phenyl-galactopyranosides to detect ß-gal activity in vitro by 19F NMR spectroscopy and imaging (23 24 25 26 27 28) . In particular, 2-fluoro-4-nitrophenyl ß-D-galactopyranoside (OFPNPG) is highly responsive to the action of ß-gal, and cleavage to form the aglycone 2-fluoro-4-nitrophenol (OFPNP) results in a pH-dependent chemical shift of 4–6 ppm for the fluorine resonance. Our previous investigations focused on breast cancer or transiently transfected prostate cancer cells. We now report application to stably transfected human PC3 prostate tumor cells; most importantly, we demonstrate in vivo application of OFPNPG to assess lacZ expression in PC3 xenografts in mice.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Stably transfected PC3 cell line
Escherichia coli lacZ gene (EC 3.2.1.23 from pSV-ß-gal vector; Promega, Madison, WI, USA) was inserted into high-expression human cytomegalovirus (CMV), immediate-early enhancer/promoter vector phCMV (Gene Therapy Systems, San Diego, CA, USA), giving a recombinant vector phCMV/lacZ, which was used to transfect PC3 cells using GenePORTER2 (Gene Therapy Systems). Cells were grown in DMEM (Dulbecco’s modification of Eagle’s medium, Mediatech, Inc., Herndon, VA, USA), 10% FBS (fetal bovine serum, Hyclone, Logan, UT, USA) with 1% penicillin-streptomycin solution (Mediatech). The highest ß-gal-expressing colony was selected using G-418 disulfate (aminoglycoside antibiotic, Research Products International Corp., Mt. Prospect, IL, USA) (800 µg/ml), which was also included for routine culture (200 µg/ml).

X-gal and S-gal staining for ß-gal
Cells were fixed in PBS plus 0.5% glutaraldehyde (5 min) and rinsed in PBS prior to staining. Staining was performed using standard procedures for 2 h at 37°C in PBS plus 1 mg/ml X-gal (Sigma, St. Louis, MO, USA), 1 mM MgCl2, 5 mM K3Fe(CN)6, and 5 mM K4Fe(CN)6, or with 1.5 mg/ml S-gal (Sigma) and 2.5 mg/ml ferric ammonium citrate.

ß-gal assay
The ß-gal activity of tumor cells and tissues in mice was measured using the ß-gal assay kit (Promega) with yellow o-nitrophenyl ß-D-galactopyranoside (ONPG). The extracted protein was quantified by a protein assay (Bio-Rad, Hercules, CA, USA) based on the Bradford method (29) . The enzyme activity is expressed as units/mg protein, where one unit corresponds to the hydrolysis of 1.0 µmol ONPG/min).

Western blot
Protein was extracted from tumors and other normal organs, and quantified using the Bradford method. Each well was loaded with 30 µg protein, separated by 10% SDS-PAGE (Nu-PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane. Primary monoclonal anti-ß-gal antibody (Promega) and anti-actin antibody (Sigma) were used as probes at a dilution of 1:5000, and reacting protein was detected using a horseradish peroxidase-conjugated secondary antibody and ECL detection (Amersham, Piscataway, NJ, USA).

MRS/MRI studies
19F MRS measurements were performed with a Varian Unity INOVA 4.7 T (188.2 MHz for 19F) or 9.4 T (376.4 MHz for 19F) system. Since no 1H coupling has been observed at either field, no decoupling was applied. T1 was measured for both substrate and product as a mixture in saline at 37°C at 9.4 T using a standard saturation recovery sequence. For in vitro cell studies at 9.4 T, OFPNPG (2.9 mg, 9 mmol) in 100 µl PBS solution was added to a suspension of 2 x 107 cells in 600 µl PBS in a 5 mm NMR tube, and NMR data were acquired immediately at 37°C. Each spectrum was acquired in 102 s (single pulse-acquire, 10 µs pulse, 55° flip angle, TR=1 s, spectral width=50 kHz, 30k time domain points, 64 transients) and the conversion of OFPNPG to OFPNP was assessed over a period of 31 min for stably transfected PC3-lacZ human prostate tumor cells. All studies at 4.7 T used a 2 cm, single-turn volume coil. In vitro imaging studies at 4.7 T used a standard spin-echo chemical shift imaging (CSI) sequence to image the conversion of OFPNPG (15.6 mg, 48 mmol) to OFPNP by PC3-lacZ cells in a 1 cm diameter Eppendorf tube at ~16°C with field of view = 30 x 30 mm, spectral width = 70 ppm, one slice with thickness = 10 mm, data matrix = 16 x 16 voxels, TR/TE = 1000/12 ms, 4 averages. Each CSI data set required ~5 min. NMR data were reconstructed and analyzed with home-built programs written in the MATLAB programming language. For in vivo studies, 2 x 106 PC3 wild-type or PC3-lacZ cells were implanted in the flank of nude mice (n=3 each) and tumors were allowed to grow to a volume of ~1 cm3. A solution of OFPNPG [0.24 M, as 3.9 mg in 50 µl, of 1:1 aqueous dimethyl sulfoxide (DMSO)], with 10 mg/ml sodium trifluoroacetate as chemical shift reference, was injected intratumorally. For in vivo experiments, a simple pulse-acquire sequence was used with a 2 cm single-turn volume coil to acquire sequential spectra over a period of 1 h (40 µs pulse width, 70° flip angle, TR=1 s, spectral width=19 kHz, 5k time domain points, 128 transients).

Histology
The tumors were excised after imaging, embedded in Tissue-Tek OCT (Miles Laboratory, Elkhart, IN, USA), and frozen in liquid nitrogen. Cryostat sections (8 µm) were collected on gelatin-coated glass slides and stained with X-gal and eosin (Sigma) for ß-gal activity.

Toxicity
PC3-WT and -lacZ cells were seeded in 24-well plates (Costar, Corning, NY, USA) at a density of 2 x 104 cells in 1 ml of medium per well and maintained at 37°C (5% CO2). Each treatment condition was assessed in quadruplicate. After 24 h at 37°C, the medium was aspirated and 1 ml new medium containing different concentrations of OFPNP and OFPNPG, respectively was added. Plates were incubated at 37°C for the indicated time, then the total number of cells was determined using a crystal violet assay. Briefly, the medium was aspirated and 1% glutaraldehyde (500 µl per well, Sigma, St. Louis, MO, USA) in PBS was added for a 15 min incubation at room temperature (RT). After removal of glutaraldehyde, 0.5% crystal violet (500 µl per well, Sigma) solution was added for a further 15 min incubation at RT. Plates were then washed three times in water and left to dry at RT. To the dried plates 500 µl of Sorenson’s solution (9 g trisodium citrate in 305 ml of distilled water, 195 ml 0.1 N HCl, 500 ml 90% ethanol) was added to elute the crystal violet. After 30 min, 100 µl of the resulting solution in each test well was transferred into a 96-well plate and read at 540 nm using an ELX800 microplate reader (Bio-Tek Instruments, Winooski, VT, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The phCMV/lacZ plasmid (Fig. 1 A) was successfully created and used to transfect cells. High-expressing cells were selected and evaluated, and the strongest expressing clones were designated PC3-lacZ and PC3-lacZ1 on the basis of Western blot (Fig. 1B ). The PC3-lacZ clone showed the highest lacZ expression and was used for all further investigations. When stably transfected PC3-lacZ cells and wild-type PC3 counterparts were stained using X-gal or S-gal, >90% of PC3-lacZ cells stained blue or black, respectively, after 30 passages in culture; the PC3 wild-type cells did not stain (Fig. 1C ).


Figure 1
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  		Figure 1. Generation of PC3 cells stably expressing of ß-gal. A) Map of recombinant lacZ vector (phCMV/lacZ), B) Western blot: cell extracts of two transfected lines PC3-lacZ1 (lane 1) and PC3-lacZ (lane 3), together with PC3-WT (lanes 2 and 4), were examined. C) PC3 wild-type and PC3-lacZ cells were stained using X-gal and S-gal: >90% of PC3-lacZ cells were stained blue or black, respectively, whereas the PC3 wild type cells did not stain.

OFPNPG has a single sharp resonance at –54.93 ppm with respect to dilute sodium trifluoroacetate. The spin-lattice relaxation time T1 = 0.74 ± 0.03 s at 9.4 T. Cleavage by ß-gal releases the OFPNP, which has a pH-sensitive chemical shift of –61 ppm at pH 7.4 (range {delta}(acid) –58.77 ppm, {delta}(base)=–61.01 and pKa=6.03) and a spin-lattice relaxation time T1 = 2.33 ± 0.04 s, which is considerably longer than for the substrate.

Addition of OFPNPG to a suspension of PC3-lacZ cells at 37°C led to rapid cleavage of the galactoside and release of the yellow aglycone OFPNP. Both substrate and product were detectable by 19F-MRS, and a time course is shown in Fig. 2 . Loss of substrate signal is accompanied by the appearance of the algycone product 4–6 ppm upfield. The ultimate intensity of the product is less than that of the substrate due to partial spectral saturation, since T1 of the algycone is about three times longer. Since OFPNP could act as a toxic ionophore, both the substrate and aglycone were tested for toxicity. At 0.5 mM, OFPNP showed considerable toxicity toward both lacZ and WT cells, with a 60 to 80% survival after 96 h exposure and only 5% survival for 2 mM. The substrate was much less toxic with essentially no toxicity after 96 h for 0.5 mM. At 2 mM there was an ~80% survival after 96 h for WT cells, whereas considerable cell loss was observed after 96 h exposure to concentrations of >1 mM for the lacZ cells with only 10% survival at 2 mM. Conversion of OFPNPG to OFPNP by stably transfected human prostate cancer PC3-lacZ cells could also be detected by 19F CSI (Fig. 3 ). The addition of 107 PC3-lacZ cells to a 70 mM solution of OFPNPG resulted in ~40% conversion to OFPNP after 4 h.


Figure 2
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  		Figure 2. Conversion of OFPNPG to OFPNP by stably transfected PC3-lacZ cells. Stacked plot series showing the conversion of OFPNPG (2.9 mg; 9 mmol) by PC3-lacZ prostate cancer cells (2x107 in PBS, pH=7.4 at 37°C) to the aglycone OFPNP, which resonates ~5 ppm upfield of the substrate (OFPNPG, gray traces, OFPNP black traces). Sequential spectra were acquired in 102 s each over a period of 31 min.


Figure 3
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  		Figure 3. Conversion of OFPNPG to OFPNP by PC3-lacZ cells detected using 19F-CSI at 4.7 T. PC3-lacZ cells (107) were added to a 1 cm diameter vial containing OFPNPG (15.6 mg; 48 mmol (70 mM) and imaged over a period of 4 h at the ambient temperature of the magnet bore (~16°C): field of view = 30 x 30 mm, spectral width = 70 ppm, one slice with thickness = 10 mm, data matrix = 16 x 16 voxels, TR/TE = 1000/12 ms, 4 averages. A) Bulk spectra and B) chemical shift images each acquired in 5 min.

Direct injection of OFPNPG (3.9 mg in 50 µl aqueous DMSO) into a tumor (~1.7 cm3, providing a concentration of ~6.5 mM) yielded a 19F-MRS signal, which was readily detected with a signal-to-noise ratio of ~20–30 in 4 min; OFPNPG and OFPNP signals were easily distinguishable at 4.7 T (Fig. 4 ). Over a period of 30 min, the PC3-lacZ tumor converted ~80% of OFPNPG to OFPNP demonstrating ß-gal activity (Fig. 4A ). No visible OFPNP signal was detected over the same period in PC3-WT tumor (Fig. 4B ). After MRS, histology and protein analysis were performed on excised tissue. Histological sections from PC3-lacZ showed >90% of tissue stained blue with X-gal for ß-gal (Fig. 4C ), whereas PC3-WT tumor showed little or no blue stain (Fig. 4D ). The ß-gal assay and Western blot were performed on tissue from liver, heart, lung, kidney, spleen, PC3-lacZ tumor, wild-type tumor, muscle, and bone. High ß-gal activity was detected in PC3-lacZ tumor alone with little activity in wild-type tumor or normal tissues (Fig. 4E, F ).


Figure 4
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  		Figure 4. In vivo detection of ß-gal expression. Time courses for 19F-MRS spectra obtained from solid tumors in vivo (4.7 T) using a 2 cm single-turn volume coil, following direct intratumoral injection of OFPNPG (3.9 mg in 50 µl aqueous DMSO). A) PC3-lacZ tumor (dimensions 1.4 cmx1.5 cmx0.8 cm). B) PC3-WT tumor (dimension 1.3 cmx1 cmx0.6 cm) and X-gal staining for C) PC3-lacZ and D) PC3-WT tumor sections, respectively. Tumors and tissues (liver, heart, lung, kidney, spleen, PC3-lacZ tumor, PC3-WT tumor, muscle, and bone) were also examined using E) Western blot showing bands for ß-gal and actin (as control) and F) ß-gal assay.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We previously demonstrated that fluorophenyl ß-D-galactopyranosides could be used to detect ß-gal activity by 19F-MRS of cells and identified OFPNPG as the best candidate among several simple analogs (24) . Upon cleavage by ß-gal, the substrate OFPNPG releases the aglycone product OFPNP, which has a pH-dependent 19F chemical shift of {Delta}{delta}= –4 to –6 ppm relative to the substrate (24) . The pKa of the aglycone is 6.0 and the {Delta}{delta} at pH 7 is ~–5.0 ppm, so that the signal is well separated from that of the substrate, allowing spectroscopic resolution and chemical shift imaging of each species at 4.7 T with no overlap between the two peaks. Our new results indicate that the chemical shift difference is sufficient to observe ß-gal activity by 19F CSI in stably transfected PC3-lacZ cells, though currently the signal-to-noise obtained at 4.7 T with a 2 cm resonator does not permit in vivo imaging.

The 19F-MRS approach is shown to be feasible in vivo for prostate tumor xenografts with a volume of > 1 ml growing in the mouse. Here, we achieved an SNR in the range 20–30 in 4 min based on an injection of 3.9 mg of substrate. Assuming uniform distribution of reporter throughout the tumor this represents a concentration of ~6.5 mM, which is expected to be cytotoxic. However, one could expect to achieve an SNR of ~5–8 with 0.5 mg substrate (<1 mM) in 16 min causing little or no toxicity. Further improvement in SNR is possible with the use of size-matched surface coils. The product aglycone line width is broader than the substrate in vivo. This may be attributed to the pH dependency of the chemical shift and would be influenced by the expected extra- plus intracellular distribution and pH heterogeneity of the tumor. Ultimately, substrate and product would be expected to wash out of the tumors, but here the substrate intensity was found to be quite stable in wild-type tumor over a period of 25 min. Most important, the comparison between lacZ transfected and wild-type PC3 xenografts by 19F-MRS of OFPNPG matches the histological results for ß-gal staining.

19F-MRS is becoming increasingly popular for in vivo investigations because the 19F nucleus exhibits an exceptionally large, structure-dependent chemical shift range and because there is essentially no endogenous background signal (30) . Here, we exploited OFPNPG with the fluorine substituent at the ortho position relative to the glycosidic linkage. However, we believe superior molecules can be developed. We have tested the analog with a CF3 moiety in place of the single fluorine atom and found the expected enhanced signal-to-noise ratio. However, the chemical shift response to bond cleavage was much smaller ({Delta}{delta}<1.2 ppm) (26) . 19F CSI was achieved by deconvolution for cultured cells, but the small {Delta}{delta} may be a problem for in vivo imaging. We were able to reduce molecular toxicity by using a fluoropyridoxol aglycone reporter in place of the fluoronitrophenol, but the synthesis is more complex; the substrate has poor water solubility and the enzyme-catalyzed reaction was much slower (25) . These issues could be addressed by polyglycosylation (27) , and we are investigating such complex substrates.

A particular problem with the OFPNPG/OFPNP approach is the need for direct intratumoral injection. We are currently addressing this issue by developing agents that release trapped or precipitated aglycone products (e.g., 2-[(ß-D-galactopyranosyl)oxy]-3-fluorocatechol; ref. 31 ), with the goal of accumulating the diagnostic product by analogy with nuclear medicine techniques that detect trapped phosphorylated agents (10) . While intratumoral injection is not ideal, we believe it represents progress for NMR reporter approaches to ß-gal. The previous approach of Louie et al. (22) required direct intracellular injection, with the trapped molecule being used to follow cell lineage in tadpole development.

Since 19F NMR has been used to monitor the conversion of 5-fluorocytosine to 5-fluorouracil (5-FU) in cells (11) and tumors (12) , one might question the need for a reporter gene approach to lacZ. However, lacZ remains one of the most widely used reporter genes in molecular biology, and it may be useful to exploit a gene that does not generate a cytotoxic product (viz. 5-FU). Therefore, we continue to seek reporters with lower toxicity. As a corollary, our approach could stimulate new approaches for detecting gene-directed enzyme prodrug therapy or antibody-directed prodrug therapy (32 , 33) . Instead of seeking to minimize product toxicity, we may seek to generate a highly toxic product that is liberated locally by ß-gal activity.

We believe that noninvasive in vivo detection of gene reporter molecules will become increasingly important in biomedicine and that it will be important to have diverse agents, genes, and modalities for specific applications. Fluorophenyl ß-D-galactosides offer a novel approach for detecting ß-gal activity. This study provides the first evidence for the utility of OFPNPG as a gene reporter molecule for investigations in animals. It demonstrates a novel approach and increases the diversity of tools for potential evaluation of gene therapy.


   ACKNOWLEDGMENTS
 
Supported in part by DOD Prostate Cancer Initiative postdoctoral fellowship DAMD17–03-1–0101 PC031075 (L.L.), and the Cancer Imaging Program, NCI Pre-ICMIC P20 CA086354. NMR experiments were conducted at the Advanced Imaging Research Center, which is supported by an NIH BTRP facility #P41-RR02584. We also recognize valuable advice and access to facilities provided by Dr. Steve L. Brown (Henry Ford Health System, Detroit, MI, USA) and Drs. Jer-Tong Hsieh, Zhengwang Zhang, and Jinhai Fan (Department of Urology, UTSW). Jennifer McAnally provided outstanding technical assistance and microscopy was undertaken in the Live Cell Imaging Core facility (Dr. Kate Luby-Phelps, Director).


   FOOTNOTES
 
1 Presented in part at the 97th annual meeting of the American Association for Cancer Research, Washington, DC 2006, USA.

Received for publication September 19, 2006. Accepted for publication January 25, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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