Noninvasive Imaging of Human Telomerase Reverse Transcriptase (hTERT) Messenger RNA with 99mTc-Radiolabeled Antisense Probes in Malignant Tumors

Meng Liu; Rong Fu Wang; Chun Li Zhang; Ping Yan; Ming Ming Yu; Li Juan Di; Hong Jie Liu; Feng Qin Guo

doi:10.2967/jnumed.107.042622

Abstract

The expression of human telomerase reverse transcriptase (hTERT) is present in most malignant cells (>85%) but is undetectable in most normal somatic cells. Visualization of hTERT expression using radionuclide targeting can provide important diagnostic information in malignant tumors. The overall aim of this study was to evaluate whether ^99mTc-radiolabeled antisense oligonucleotide (ASON) targeting hTERT messenger RNA (mRNA) can be used for imaging of hTERT expression in vivo. Methods: One 18-mer antisense or sense uniformly phosphorothioate-modified oligonucleotide targeting hTERT mRNA was radiolabeled with ^99mTc through the bifunctional chelator N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (S-acetyl NHS-MAG3). Then the radiolabeled probe was characterized in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to assay the mRNA level after proliferating cells had been incubated with the antisense and sense probes. ^99mTc-MAG3-ASON or ^99mTc-MAG3-SON was injected intravenously in mammary tumor-bearing BALB/c nude mice. Biodistribution and in vivo imaging was performed periodically. All data were analyzed by statistical software. Results: The labeling efficiencies of ^99mTc-MAG3-ASON/SON reached 76% ± 5% (n = 5) within 15–30 min at room temperature, the specific activity was up to 1,850 kBq/μg, and the radiochemical purity was >96% after purification. ^99mTc-MAG3-ASON showed complete stability at room temperature and in fresh 37°C human serum. In comparison with ^99mTc-MAG3-SON, ^99mTc-MAG3-ASON preserved the capacity to bind living hTERT-expressing cells specifically and to inhibit the expression of hTERT mRNA significantly as well as ASON. In nude mice bearing hTERT-expressing MCF-7 xenografts, tumor radioactivity uptake of ^99mTc-MAG3-ASON after injection was significantly higher than that of ^99mTc-MAG3-SON after injection (P < 0.05). The hTERT-expressing xenografts were clearly imaged at 4–8 h noninvasively after injection of ^99mTc-MAG3-ASON, whereas the xenografts were not imaged at any time after injection of ^99mTc-MAG3-SON. Conclusion: This in vivo study provides evidence that ^99mTc-MAG3-ASON targeting hTERT mRNA can be used as a potential candidate for visualization of hTERT expression in carcinomas.

Telomerase, a hallmark of malignant tumors, is minimally composed of the telomerase RNA (hTR), the telomerase reverse transcriptase (hTERT), and telomerase-associated proteins (1,2). hTERT represents the major determinant of telomerase activity. In contrast to hTR, which is commonly expressed in both normal tissues (without telomerase activity) and cancers, hTERT expression is found solely in tumor cells. Consequently, hTERT is one of the most attractive molecular markers available, promising a target spectrum of approximately 85% of all tumors (3,4). In addition, hTERT expression enhances the chemoresistance of cancer cells, and effective therapies are associated with decreased expression of hTERT, so imaging of hTERT expression also has prognostic value (5,6).

Although it has potential clinical significance, the measurement of hTERT expression has predominantly been by immunohistochemistry or by reverse transcription-polymerase chain reaction (RT-PCR), which requires the destruction of the tissues or cells examined. At present, reporter genes have provided new methods for noninvasive imaging, combining molecular biology with techniques such as fluorescent imaging, bioluminescent imaging, and radionuclide imaging. This indirect imaging paradigm usually includes a “marker/reporter gene”, a “marker/reporter probe,” and imaging technology that can visualize the spatial distribution of a probe (7,8). Examples using radionuclide or trimodality fusion imaging of hTERT promoter activity were recently published, and the results were encouraging (9,10).

However, direct inhibition of telomerase activity in cancer cells, a significant strategy, has been proposed. Compared with indirect imaging, imaging endogenous expression directly is more important, despite its greater difficulty, because it is directed at the genotypic basis of the normal phenotypic function of cells and at the alterations at gene expression that can initiate disease (11). Thus, antisense imaging is one of the most promising tools of molecular imaging, whose strategies exploit the exquisite specificity of nucleic acid base-pair binding. Although there is much controversy, the use of antisense imaging as an in vivo diagnostic method is still attractive in that it would bring molecular imaging to the level of gene expression, especially for the early detection of tumor.

In present study, we tried to use antisense oligonucleotide (ASON) to noninvasively measure hTERT messenger RNA (mRNA) expression directly. One 18-mer antisense uniformly phosphorothioate-modified oligonucleotide targeting hTERT mRNA was radiolabeled with ^99mTc. We evaluated the characteristics of this antisense probe in vitro and observed whether it could be used for imaging of hTERT expression in malignant tumors in vivo.

MATERIALS AND METHODS

All oligonucleotides were purchased from the same source as the uniform phosphorothioates (Sangon Biotechnologies), purified with polyacrylamide gel electrophoresis (PAGE), and used as supplied. The sequence of ASON is 5′-TAGAGACGTGGCTCTTGA-3′, and that of sense oligonucleotide (SON) is 5′-TCAAGAGCCACGTCTCTA-3′, both with a primary amine on the terminal 3′ phosphate group via a 6-member methylene carbon spacer, which was designed for covalent conjugation with the chelator. The molecular weight of chain was about 5.5 kDa each. The amine ASON/SON was generally handled under sterile conditions. The chelator of the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (S-acetyl NHS-MAG3) was kindly provided by Donald J. Hnatowich (University of Massachusetts Medical School). Sephadex G25 (Amersham) was purchased and used as received. All other chemicals were reagent grade and were used without purification. The fresh ^99mTc-pertechnetate generator eluate was obtained from a ⁹⁹Mo-^99mTc radionuclide generator (China Institute of Atomic Energy).

Oligonucleotide Conjugation and Radiolabeling

ASON with a primary amine on the terminal 3′ phosphate group was conjugated with S-acetyl NHS-MAG3 as described (12,13). In brief, a solution of ASON was prepared at a concentration of 5 μg/μL in 0.1 M N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) buffer, pH 8.0. A fresh 20 mg/mL solution of S-acetyl-NHS-MAG3 in dry N,N-dimethylformamide (DMF) was added dropwise with agitation to a final MAG3-to-ASON molar ratio of 20:1. This reaction mixture was incubated at room temperature for 60–120 min. Then the conjugated ASON was purified on a 0.7 × 20 cm Sephadex G25 gel-filtration column using sterile 50 mM phosphate buffer, pH 7.2, as eluant. The peak fractions were pooled, and the DNA concentration was quantitated by a recording ultraviolet visible spectrophotometer (SMA3000; Beijing Genomics Institute, China) at 260 nm (optical density, 33 μg/mL). The preparation of MAG3-ASON was dispensed at 10 μg per vial, lyophilized, and stored at −20°C for future use. SON was treated as described for ASON.

A fresh 50 mg/mL solution of sodium tartrate was prepared in sterile 0.5 M sodium bicarbonate, 0.25 M ammonium acetate, 0.175 M ammonium hydroxide, pH 9.2. The high pH of the tartrate solution was necessary so that the final pH would be approximately 7.6. In addition, a 1 mg/mL solution of SnCl₂·2H₂O in 10 mM hydrochloric acid was prepared just before use. At room temperature, the conjugated and purified MAG3-ASON (10 μg) was typically dissolved at a concentration of 500 μg/mL in 0.25 M ammonium acetate buffer, pH 5.2. To the solution of MAG3-ASON, sodium tartrate was added as a transchelator to a final concentration of about 7 μg/μL, and the labeling pH reached 7.6. Then sufficient ^99mTc-pertechnetate generator eluate (10 μL) was added to this solution to provide about 3,700 kBq/μg of ASON. The stannous ion solution was added immediately thereafter such that 1 μg of SnCl₂·2H₂O was added for each 10 μg of ASON. After 15–30 min, the labeled MAG3-ASON preparation was purified on a 0.7 × 20 cm Sephadex G25 gel-filtration column with 50 mM phosphate buffer, pH 7.2, eluant. Fractions were collected to detect the radioactivity and absorbance at 260 nm. SON was radiolabeled identically with ^99mTc.

For routine quality control of labeling, the labeling efficiency and radiochemical purity of radiolabeled probes were calculated by paper chromatography on Xinhua no. 1 filter paper with acetone and normal saline as the mobile phase.

In Vitro Stability Analysis

To evaluate the labeling stability, the radiochemical purity of ^99mTc-MAG3-ASON at room temperature, as well as that in normal saline at room temperature and in fresh 37°C human serum at 0.01 μg/μL, was confirmed by paper chromatography periodically over 24 h.

PAGE was performed to assess the stability of ^99mTc-MAG3-ASON in serum by comparing the electrophoretic mobility. Incubation with fresh 37°C human serum for 1, 2, 4, and 6 h later, ^99mTc-MAG3-ASON was extracted by a phenol/chloroform method. After ethanol precipitation at −20°C overnight, the samples were centrifugated (12,000g/min) for 10 min. The precipitation was dried naturally and redissolved in distilled water. Then the samples in each time point, including the control, were resolved on 20% PAGE and stained with silver nitrate. The resulting gels were finally scanned using an ultraviolet transilluminator (AlphaImager 2200; Alpha Innotech Corp.) and photographed.

Cell Culture

The HepG2 cell line was a generous gift from the Department of Infectious Disease, Peking University First Hospital, and was maintained in the media recommended by them. The cells were grown in Dulbecco's modified Eagle medium (DMEM) containing l-glutamine (2 mM), sodium bicarbonate (1.5 mg/L), nonessential amino acids (0.1 mM), and sodium pyruvate (1.2 mM), supplemented with 10% fetal bovine serum (FBS) and 100 mg/mL of penicillin–streptomycin (GIBCO). Cells were cultivated under standard conditions (37°C, humidified atmosphere containing 5% CO₂). Cells between passages 4 and 12 were used and were harvested by trypsin treatment (0.25% trypsin/0.02% ethylenediaminetetraacetic acid, 5 min, 37°C). The cell growth status was monitored by inverted microscopy with phase contrast, and cell viability was assessed by trypan blue exclusion.

Cell Transfections

Before transfection, the cells were seeded in appropriate culture plates and grown for 2 d. On the third day, the cells were transfected once, with a mixture consisting of molecular probe and liposome (ratio, 1:3 w/w, diluted in serum-free OptiMEM) according to the manufacturer's instructions (Lipofectamine 2000; Invitrogen). After incubation under standard conditions for 4 h, the cells were washed with phosphate-buffered saline (PBS) and incubated in fresh culture medium (DMEM with 10% FBS) to maintain growth, until additional analyses.

RT-PCR

After transfection with ^99mTc-MAG3-ASON, ^99mTc-MAG3-SON, and ASON, respectively, the cells were trypsinized and washed twice with PBS. The total cellular RNA was extracted from the HepG2 cells by Trizol 1-step method (TRIZOL reagent; Bio Basic Inc.). For reverse transcription, 2 μg of total RNA were subjected to reverse transcription primed by oligodeoxythymidine with avian myeloblastosis virus reverse transcriptase according to the recommendations of the First Strand cDNA Synthesis Kit (Bio Basic Inc.). Then the complementary DNA (cDNA) was applied to perform PCR using the PCR Kit (Sangon Biotechnologies). As an internal control for cDNA synthesis, the housekeeping gene of β-actin was amplified too. PCR primers were as follows: $\text{[math]}$ $\text{[math]}$ $\text{[math]}$ $\text{[math]}$ $\text{[math]}$ $\text{[math]}$

PCR amplification of hTERT cDNA was performed with an initial heating at 94°C for 4 min, followed by 34 cycles consisting of denaturing for 30 s at 94°C, annealing for 30 s at 61°C, and elongation for 30 s at 72°C, and a final extension period of 5 min at 72°C. PCR amplification of β-actin was performed as described. Amplified products were size-fractionated by 2% agarose gel electrophoresis. The gel was stained with ethidium bromide (1 μg/mL), visualized by an ultraviolet transilluminator, and photographed. The density of each PCR band was measured and analyzed by video documentation system software (AlphaImager version 5.5; Alpha Innotech Corp.). The amount of mRNA products for hTERT was expressed as the ratio to β-actin mRNA product, which was conserved as the internal control.

Biodistribution of ^99mTc-MAG3-ASON and ^99mTc-MAG3-SON in MCF-7 Xenograft-Bearing Nude Mice After Systemic Injection

BALB/c nu/nu mice (female, 20 ± 3 g, 4- to 6-wk-old; Department of Laboratory Animal Science, Peking University Health Science Center) were used in this study. The mice were inoculated with 1 × 10⁷ MCF-7 mammary tumor cells (obtained from Department of Laboratory Animal Science, Peking University Health Science Center) in the right upper limbs, and the tumors were allowed to grow to a diameter of 1.0 cm. The mice were maintained using a standard diet, bedding, and environment, with free access to food and drinking water.

Fifty BALB/c nu/nu mice with MCF-7 xenografts were randomly divided into 10 groups of 5 mice each (5 groups for injection of antisense probe, another 5 groups for injection of sense probe). Immediately after Sephadex G25 gel-filtration column purification, 1 μg (1,850 kBq) of ^99mTc-MAG3-ASON (or ^99mTc-MAG3-SON) in 100 μL of eluant (50 mM phosphate buffer, pH 7.2) was injected into each mouse via a tail vein. All injections were tolerated well. At 0.5, 1, 2, 4, and 6 h after injection, the mice were sacrificed by cervical dislocation. Then the mice were dissected and tissues of interest (heart, liver, spleen, lung, kidney, stomach, small intestine, bladder, skeletal muscle, bone marrow, blood, and tumor) were weighed, and their radioactivity was measured using a γ-well counter along with a standard of the injection. Radioactivity results were recorded as the percentage injected activity per gram (%ID/g) of tissue corrected for background and decay.

γ-Camera Imaging

Ten BALB/c nu/nu mice with MCF-7 xenografts were randomly divided into 2 groups of 5 mice each (one group for injection of antisense probe, the other for injection of sense probe). Immediately after Sephadex G25 gel-filtration column purification, 4 μg (7,400 kBq) of the antisense or sense probe in 100 μL of eluant (50 mM phosphate buffer, pH 7.2) were injected into each mouse via a tail vein. All injections were tolerated well. At 0.5, 1, 2, 4, 6, and 8 h after injection, imaging was performed in the Department of Nuclear Medicine, Peking University First Hospital, using SPECT (SPR SPECT; GE Healthcare, Inc.) equipped with a low-energy, high-resolution, parallel-hole collimator. Static images (100,000 counts), obtained with a zoom factor of 2.0, were digitally stored in a 256 × 256 matrix.

Statistical Analysis

The variables are expressed as average ± SD ( $\text{[math]}$ ). The variables of RT-PCR were analyzed using 1-way ANOVA, and other statistical comparisons of average values were performed with the Student t test. P < 0.05 was considered significant.

RESULTS

Oligonucleotide Conjugation and Radiolabeling

Under the set of conditions, average labeling efficiencies of 76% ± 5% (n = 5) were achieved within 15–30 min at room temperature, specific activities of up to 1,850 kBq/μg were obtained, and radiochemical purity was >96% after purification. Control labeling of unmodified ASON under the same conditions showed only about 5% ± 1.6% (n = 5) binding, indicating that radiolabeling was largely specific and mediated by the chelator.

The labeling efficiency and radiochemical purity of radiolabeled agent were calculated by the paper chromatography on Xinhua no. 1 filter paper with acetone and normal saline as the mobile phase. With acetone as the mobile phase, only ^99mTc-pertechnetate migrated with the solvent, whereas ^99mTc-MAG3-ASON and other labeled colloids remained at the origin. Otherwise, with the normal saline as the mobile phase, ^99mTc-pertechnetate and ^99mTc-MAG3-ASON migrated with the solvent, whereas labeled colloids remained at the origin (Table 1).

View this table:

TABLE 1

R_f Value in 2 Kinds of Developing Solvents