Cancer Research Cancer Genome no Abstract AM No Date
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Umeoka, T.
Right arrow Articles by Fujiwara, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Umeoka, T.
Right arrow Articles by Fujiwara, T.
[Cancer Research 64, 6259-6265, September 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Visualization of Intrathoracically Disseminated Solid Tumors in Mice with Optical Imaging by Telomerase-Specific Amplification of a Transferred Green Fluorescent Protein Gene

Tatsuo Umeoka1, Takeshi Kawashima1, Shunsuke Kagawa1,2, Fuminori Teraishi1, Masaki Taki1, Masahiko Nishizaki1, Satoru Kyo3, Katsuyuki Nagai4, Yasuo Urata4, Noriaki Tanaka1 and Toshiyoshi Fujiwara1,2

1 Division of Surgical Oncology, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan; 2 Center for Gene and Cell Therapy, Okayama University Hospital, Okayama, Japan; 3 Department of Obstetrics and Gynecology, Kanazawa University School of Medicine, Kanazawa, Japan; and 4 Oncolys BioPharma, Inc., Tokyo, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Currently available methods for detection of tumors in vivo such as X-ray, computed tomography, and ultrasonography are noninvasive and have been well studied; the images, however, are not specific for tumors. Direct optical imaging of tumor cells in vivo that can clearly distinguish them from surrounding normal tissues may be clinically useful. Here, we describe a new approach to visualizing tumors whose fluorescence can be detected using tumor-specific replication-competent adenovirus (OBP-301, Telomelysin) in combination with Ad-GFP, a replication-deficient adenovirus expressing green fluorescent protein (GFP). Human telomerase reverse transcriptase is the catalytic subunit of telomerase, which is highly active in cancer cells but quiescent in most normal somatic cells. We constructed an adenovirus 5 vector in which the human telomerase reverse transcriptase promoter element drives expression of E1A and E1B genes linked with an internal ribosome entry site and showed that OBP-301 replicated efficiently in human cancer cells, but not in normal cells such as human fibroblasts. When the human lung and colon cancer cell lines were infected with Ad-GFP at a low multiplicity of infection, GFP expression could not be detected under a fluorescence microscope; in the presence of OBP-301, however, Ad-GFP replicated in these tumor cells and showed strong green signals. In contrast, coinfection with OBP-301 and Ad-GFP did not show any signals in normal cells such as fibroblasts and vascular endothelial cells. We also found that established subcutaneous tumors could be visualized after intratumoral injection of OBP-301 and Ad-GFP. A549 human lung tumors and SW620 human colon tumors transplanted into BALB/c nu/nu mice were intratumorally injected with 8 x 105 plaque-forming units of Ad-GFP in combination with 8 x 106 plaque-forming units of OBP-301. Within 3 days of treatment, the fluorescence of the expressed GFP became visible by a three-chip color cooled charged-coupled device camera in these tumors, whereas intratumoral injection of Ad-GFP alone could not induce GFP fluorescence. Moreover, intrathoracic administration of Ad-GFP and OBP-301 could visualize disseminated A549 tumor nodules in mice after intrathoracic implantation. Our results indicate that intratumoral or intrathoracic injection of Ad-GFP in combination with OBP-301 might be a useful diagnostic method that provides a foundation for future clinical application.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A variety of imaging technologies are being investigated as tools for cancer diagnosis, detection, and treatment monitoring. Improvements in methods of external imaging such as computed tomography, magnetic resonance imaging, and ultrasound techniques have increased the sensitivity for visualizing tumors and metastases (1 , 2) ; a limiting factor in structural and anatomical imaging, however, is the inability to specifically identify malignant tissues. Histopathological examination is still the most effective method for the detection of neoplastic lesions. Thus, tumor-specific imaging would be of considerable value in treatment of human cancer by defining the location and area of tumors without microscopic analysis. In particular, if tumors too small for direct visual detection and therefore not detectable by direct inspection could be imaged in situ, surgeons could precisely excise tumors with appropriate surgical margins. This paradigm requires an appropriate marker that can facilitate visualization of physiological or molecular events that occur in tumor cells but not normal cells.

Molecular and functional monitoring of cellular processes has become a reality with the development of the green fluorescent protein (GFP) technology, which was originally identified from the jellyfish Aequorea victoria (3, 4, 5) . GFP is able to specifically label biological molecules and cellular structures and can be sensitively detected by a variety of optical techniques. These labels can be detected in living tissues because of the relatively noninvasive nature of fluorescence, thus allowing transient and dynamic events to be visualized and measured (6, 7, 8, 9, 10) . In addition, because GFP is genetically encoded, cells can be labeled by introducing the exogenous GFP gene. Indeed, Yang et al. (11) have shown that GFP-expressing tumors growing and metastasizing in intact animals could be viewed externally with a whole-body optical imaging system. Moreover, transgene expression could also be monitored by using an adenovirus vector expressing the GFP gene. These observations indicate that the GFP-based approach might be feasible as well as practical for real-time tumor imaging in living mammalians.

To specifically distinguish tumors from normal tissues, an appropriate strategy to make tumor cells selectively fluorescent is required. We previously constructed the tumor-specific replication-selective adenovirus OBP-301 (Telomelysin), in which the human telomerase reverse transcriptase (hTERT) promoter element drives expression of the E1A and E1B genes linked with an internal ribosome entry site, and demonstrated the selective replication and antitumor effect of OBP-301 in human cancer cells in vitro and in vivo (12) . Because >85% of human cancer cells (but not most somatic normal cells) display telomerase activity (13 , 14) , we hypothesized that OBP-301 infection could complement E1 gene functions and selectively facilitate replication of E1-deleted replication-deficient vectors in coinfected tumor cells.

In the present study, using a replication-deficient adenovirus expressing the GFP gene in combination with OBP-301, we demonstrate a real-time fluorescence optical imaging of pleural dissemination of human non–small-cell lung cancer cells in an orthotopic murine model. This novel molecular imaging technique could be feasible for in vivo detection of disseminated small tumor nodules.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Culture Conditions.
The human non–small-cell lung cancer cell lines H1299, H358, and H226Br and the human colorectal carcinoma cell lines SW620, LoVo, and DLD-1 were routinely propagated in monolayer culture in RPMI 1640 supplemented with 10% fetal calf serum (FCS). The human non–small-cell lung cancer cell line A549 was cultured in Dulbecco’s modified Eagle’s medium containing nutrient mixture (Ham’s F-12). The transformed embryonic kidney cell line 293 was grown in Dulbecco’s modified Eagle’s medium containing high glucose (4.5 g/L) and supplemented with 10% FCS. The normal human lung diploid fibroblast cell line WI-38 (JCRB0518) was obtained from the Health Science Research Resources Bank (Osaka, Japan) and grown in Eagle’s minimal essential medium with 10% FCS. The normal human lung fibroblast cell line NHLF was purchased from TaKaRa Biomedicals (Kyoto, Japan) and cultured in the medium recommended by the manufacturer. The human umbilical vascular endothelial cell line HUVEC was kindly provided by Dr. Masayoshi Namba (Okayama University, Okayama, Japan) and propagated in monolayer culture in CSC certified medium with 100 units/ml penicillin and 100 mg/ml streptomycin.

Recombinant Adenoviruses.
The recombinant replication-selective, tumor-specific adenovirus vector was constructed and characterized previously (12) . The resultant virus was named OBP-301. The adenoviral vector containing GFP cDNA (Ad-GFP) was also used. Viral stocks were quantified by a plaque-forming assay using 293 cells and stored at –80°C.

Quantitative Real-Time Polymerase Chain Reaction Analysis.
Total RNA from the tumor samples and cultured cells was obtained using the RNeasy Mini Kit (Qiagen, Chatsworth, CA). Approximately 0.1 µg of total RNA was used for reverse transcription. Reverse transcription was performed at 22°C for 10 minutes and then at 42°C for 20 minutes. The hTERT mRNA copy number was determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) using a LightCycler instrument and a LightCycler DNA TeloTAGGG Kit (Roche Molecular Biochemicals, Indianapolis, IN). Polymerase chain reaction amplification began with a 60-second denaturation step at 95°C, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 58°C for 10 seconds, and extension at 72°C for 9 seconds.

Fluorescent Microscopy.
Human cancer cells (SW620, A549, and H1299) and normal cells (WI-38, NHLF, and HUVEC) were infected with 0.1 multiplicity of infection (MOI) of Ad-GFP alone or in combination with 1 MOI of OBP-301 in vitro. Expression of the GFP gene was assessed and photographed (x200) by an Eclipse TS-100 fluorescent microscope (Nikon, Tokyo, Japan) 24 hours after infection.

Animal Experiments.
The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Okayama University (Okayama, Japan). SW620 and A549 xenografts were produced on the back of 5-week-old female BALB/c nu/nu mice by subcutaneous injection of 5 x 106 SW620 or A549 cells in 500 µl of Hanks’ balanced salt solution. Three weeks after tumor cell inoculation, both tumors received intratumoral injection with Ad-GFP [8 x 105 plaque-forming units (pfu)/50 µl] alone or Ad-GFP (8 x 105 pfu/25 µl) plus OBP-301 (8 x 106 pfu/25 µl). Mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg) on days 1, 2, 3, 7, and 14 after virus injection and examined for GFP expression. As a control, viruses were subcutaneously injected into non–tumor-bearing mice. To generate an orthotopic model of pleural dissemination, female BALB/c nu/nu mice received intrathoracic injection with a cell suspension of A549 cells at a density of 5 x 106 cells/200 µl through a 27-gauge needle. Two weeks later, Ad-GFP (4 x 107 pfu/50 µl) and OBP-301 (4 x 107 pfu/50 µl) were injected into the thoracic space by the same technique. Five days after virus injection, mice were sacrificed, and their thoracic spaces were examined.

Cooled Charged-Coupled Device Imaging.
In vivo GFP fluorescence imaging was acquired by illuminating the animal with a Xenon 150-W lamp. The re-emitted fluorescence was collected through a long pass filter on a Hamamatsu C5810 three-chip color cooled charged-coupled device (CCD) camera (Hamamatsu Photonics Systems, Hamamatsu, Japan). High-resolution image acquisition was accomplished using an EPSON personal computer. Images were processed for contrast and brightness with the use of Adobe Photoshop 4.0.1J software.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Human Telomerase Reverse Transcriptase Levels in Human Cancer and Normal Cells.
Telomerase is a novel marker for malignant diseases (15) . To confirm the specificity of telomerase activity in human cancer cells, expression of hTERT mRNA, which plays a key role in telomerase activation (16) , was measured in a panel of human tumor and normal cell lines using a real-time RT-PCR method. All tumor cell lines, including human non–small-cell lung cancer cell lines A549, H226Br, and H1299 and human colorectal cancer cell lines SW620, LoVo, and DLD-1, expressed detectable levels of hTERT mRNA, although the levels of expression varied widely (Fig. 1A)Citation . In contrast, human fibroblast cells such as WI-38 and NHLF were negative for hTERT expression. HUVEC cells transformed with the SV40 large T-antigen gene also exhibited no hTERT mRNA expression. 293 cells are known to be telomerase positive and were used as a positive control. These results suggest that the hTERT promoter element can be used to target human cancer.



View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. A, relative hTERT mRNA expression in human tumor and normal cells determined by real-time RT-PCR analysis. The hTERT mRNA expression of A549 human lung cancer cells was considered to be 1.0, and the relative expression level of each cell line was calculated against that of A549 cells. B, schematic DNA structures of OBP-301 and Ad-GFP. OBP-301 contains the hTERT promoter sequence inserted into the E3-deleted adenovirus genome to drive transcription of the E1A and E1B bicistronic cassette linked by the internal ribosome entry site structure. The Ad-GFP vector contains GFP cDNA driven by the cytomegalovirus promoter.

 
Selective Visualization of Human Cancer Cells In vitro.
To selectively enhance GFP transgene expression in human cancer cells, we used a tumor- or telomerase-specific replication-selective adenovirus, OBP-301, and replication-deficient Ad-GFP. Schematic DNA structures of OBP-301 and Ad-GFP are shown in Fig. 1BCitation . SW620, A549, and H1299 human cancer cells expressed bright GFP fluorescence as early as 24 hours after coinfection with Ad-GFP (0.1 MOI) and OBP-301 (1.0 MOI) (Fig. 2)Citation . The fluorescence intensity increased gradually until approximately 3 days after infection, followed by rapid cell death due to the cytopathic effect of OBP-301 (data not shown). Inection with Ad-GFP alone at a MOI of 0.1 caused neither GFP expression nor cytotoxicity in SW620 and A549 cells. Only faint fluorescence could be detected in H1299 cells infected with 0.1 MOI of Ad-GFP because H1299 cells are highly sensitive to adenovirus infection. In contrast, normal human cells, including WI-38, NHLF, and HUVEC, were completely negative for GFP expression after coinfection with Ad-GFP and OBP-301. These results indicate that Ad-GFP can replicate exclusively in human cancer cells in the presence of OBP-301, leading to tumor cell-specific GFP fluorescence expression in vitro.



View larger version (64K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Selective bystander replication of Ad-GFP in the presence of OBP-301 in human cancer cells in vitro. Cultured human cancer [SW620 (colorectum), A549 (lung), and H1299 (lung)] and normal cells (WI-38, NHLF, and HUVEC) were infected with 0.1 MOI of Ad-GFP alone or in combination with 1.0 MOI of OBP-301, and photographed under a fluorescence microscope for GFP expression 24 hours after treatment. Magnification, x200.

 
Dose-Dependent Intensity of Fluorescence by Ad-GFP Injection into Mice.
To selectively detect tumor tissues in vivo, nonspecific GFP transgene expression in normal organs must be avoided. We first determined the optimal dose of Ad-GFP that could not induce detectable GFP fluorescence in normal tissues. Non–tumor-bearing mice received subcutaneous injection with increasing doses of Ad-GFP alone and were then monitored for fluorescence intensity over time with the CCD noninvasive imaging system. As shown in Fig. 3Citation , mice given 1.3 x 109 and 1.3 x 108 pfu of Ad-GFP displayed high and moderate GFP fluorescence signals in the skin, respectively, within 24 hours after injection. In contrast, the GFP transgene expression was not visible over a 7-day period after injection of 1.3 x 107 or 1.3 x 106 pfu of Ad-GFP. Thus, 108 pfu was considered the lowest dose of Ad-GFP that induces nonspecific GFP signals in normal tissues.



View larger version (40K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. External images of non–tumor-bearing nu/nu mice that received injection with increasing doses of Ad-GFP alone. Non–tumor-bearing mice received subcutaneous injection with 1.3 x 106, 1.3 x 107, 1.3 x 108, or 1.3 x 109 pfu of Ad-GFP alone and were photographed for GFP expression by the CCD noninvasive imaging system 24 hours after injection.

 
Selective Visualization of Subcutaneously Growing Human Tumors In vivo.
To further confirm the specificity of the GFP-based fluorescent optical detection of tumors in vivo, we performed a set of control experiments. In the control group of non–tumor-bearing animals that received subcutaneous injection with 8 x 105 pfu of Ad-GFP plus 8 x 106 pfu of OBP-301, no apparent GFP signals were detected in the skin over 7 consecutive days (Fig. 4A)Citation . Moreover, injection of Ad-GFP alone at a density of 8 x 105 pfu into subcutaneously established SW620 tumors resulted in no GFP fluorescence expression for at least 7 days after injection (Fig. 4B)Citation .



View larger version (45K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. A, external images of non–tumor-bearing mice that received injected with Ad-GFP plus OBP-301. As a control, non–tumor-bearing mice received subcutaneous injection with 8 x 105 pfu of Ad-GFP in combination with 8 x 106 pfu of OBP-301 and were assessed for GFP fluorescence 24 hours after administration. As expected, no GFP signals were obtained. Top left panel, macroscopic appearance of mouse. B, external images of SW620 tumor-bearing nu/nu mice that received injection with Ad-GFP alone. Three weeks after subcutaneous inoculation of SW620 tumor cells (5 x 106 cells/mouse), mice received intratumoral injection of Ad-GFP at a density of 8 x 105 pfu alone. As expected, no GFP fluorescence expression was detected for at least 7 days after injection. Left panels, macroscopic appearance of SW620 tumors; right panels, fluorescence detection.

 
We next examined the kinetics of GFP transgene expression in SW620 and A549 tumors after intratumoral injection of 8 x 105 pfu of Ad-GFP and 8 x 106 pfu of OBP-301. Treated mice displayed detectable intratumoral signals within 24 hours after local delivery of viruses (Fig. 5)Citation . Whole-body images of mice showed light spots in the tumor area, although complete tumor images were not observed. The fluorescence intensity reached maximum levels 3 to 7 days after injection and gradually decreased to background fluorescence by 14 days after treatment. When mice were sacrificed 7 days after intratumoral injection of viruses, GFP expression was confined to the tumor; no GFP fluorescence was detected in various normal organs such as the liver, kidney, spleen, small intestine, brain, heart, pancreas, ovary, and adrenal gland (data not shown).



View larger version (69K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Time course of external images of subcutaneous SW620 and A549 tumors after intratumoral injection of Ad-GFP plus OBP-301. Three weeks after subcutaneous inoculation of SW620 and A549 tumor cells (5 x 106 cells/mouse), Ad-GFP and OBP-301 at concentrations of 8 x 105 pfu and 8 x 106 pfu, respectively, were injected directly into established tumors. The GFP fluorescence intensity was monitored for 14 consecutive days under the CCD noninvasive imaging system. Left panels, macroscopic appearance of subcutaneous tumors; right panels, fluorescence detection.

 
Selective Visualization of Pleural Dissemination of Human Lung Cancer Cells In vivo.
We also evaluated the potential of GFP-based visualization of A549 human non–small-cell lung cancer cells growing intrathoracically in athymic nu/nu mice. We reported previously (17) that when A549 cells were inoculated into the thoracic space, dissemination of tumors appeared in all mice 14 days after tumor injection. As shown in Fig. 6Citation , the optical CCD imaging of the thoracic cavity after a midsternal thoracotomy demonstrated that disseminated tumor nodules in the visceral pleura, parietal pleura, diaphragmatic pleura, and mediastinum could be detected as light-emitting spots by intrathoracic coinjection of 4 x 107 pfu of Ad-GFP and 4 x 107 pfu of OBP-301. The large tumors were labeled in spots with GFP fluorescence; small nodules, however, could be imaged entirely as intensive GFP signals. Histological analysis confirmed the presence of disseminated tumor cells in the sites of fluorescence emission (Fig. 6)Citation . The thoracic cavity of non–tumor-bearing mice was negative for GFP transgene expression (data not shown).



View larger version (56K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Internal images of pleural dissemination visualized by intrathoracic injection of Ad-GFP and OBP-301. Female BALB/c nu/nu mice received intrathoracic implant with 5 x 106 A549 tumor cells. Two weeks later, 4 x 107 pfu/50 µl each of Ad-GFP and OBP-301 were injected into the thoracic space. Five days after virus injection, mice were sacrificed, and their thoracic spaces were examined. Three representative mice are shown. Left panels, fluorescent detection; right panels, gross appearance of A549 tumors grown orthotopically in the thoracic spaces. Arrows, disseminated tiny tumor. Bottom left panel, H&E staining of the lesion of fluorescence emission. Magnification, x100.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Gene-based therapeutic strategies, including replacement of defective tumor suppressors, enhancement of immune-mediated tumor surveillance, and tumor-selective virotherapy, have emerged as promising adjuvants to conventional modalities for human cancer (18, 19, 20, 21) . We previously reported (12) the development of a tumor-specific replication-selective adenovirus, OBP-301, which is capable of replicating exclusively in telomerase-positive human cancer cells. In the current study, we investigated the potential of OBP-301 to visualize human tumors by facilitating replication of a GFP-expressing replication-deficient adenovirus, Ad-GFP. As reported previously (8) , for real-time imaging of growing tumors in live mice, GFP appears to be an extremely effective tumor cell marker to identify tumor tissues at a single-cell level. A major advantage of GFP labeling is that it requires no preparative procedures such as contrast agents, substrates, and light-tight boxes, unlike other imaging techniques.

Elevated levels of telomerase activity are found in the majority of malignancies (Fig. 1A)Citation and believed to play a critical role in tumorigenesis (22) . Therefore, telomerase-specific OBP-301 was able to selectively compensate the E1 gene products for Ad-GFP that lacks E1 proteins in human tumor cells, but not in normal cells. However, because the recombinant adenovirus vectors can efficiently infect a wide variety of dividing and nondividing cells and transfer exogenous genes into mammalian cells, infection with Ad-GFP alone at high concentrations may induce GFP fluorescence nonselectively in both tumor and normal cells. Our preliminary experiments demonstrated that infection with Ad-GFP alone at <1.0 MOI was insufficient to induce GFP expression under fluorescence microscopy in most tumor and normal cells in vitro (data not shown). We confirmed that coinfection with 0.1 MOI of Ad-GFP and 1.0 MOI of OBP-301 could express GFP fluorescence selectively in tumor cells, although detectable green signals were evident with Ad-GFP infection alone in cells sensitive to adenovirus infection, such as H1299 cells (Fig. 2)Citation . However, the fact that most normal cells are relatively resistant to adenovirus infection suggests that the false-positive detection of normal cells is likely to be less frequent.

It has been reported that 1.6 x 109 pfu of Ad-GFP delivered to various organs induced high-intensity GFP expression that could be visualized by whole-body imaging (11) ; therefore, we determined 107 pfu of Ad-GFP as a suboptimal dose for GFP detection in vivo by the dose titration study (Fig. 3)Citation . Indeed, intratumoral injection of 8 x 105 pfu of Ad-GFP plus 8 x 106 pfu of OBP-301 resulted in intense GFP expression in tumors, whereas no GFP signals were detectable by subcutaneous administration of these vectors into normal areas (Figs. 4Citation and 5)Citation . CCD imaging demonstrated the heterogeneous pattern of GFP expression only in tumors in mice that received intratumoral injection of 50 µl of the vector solution, suggesting that the vectors might not be able to spread throughout the tumor tissues (Fig. 5)Citation . Moreover, the green signals in tumors disappeared within 14 days after intratumoral injection of Ad-GFP and OBP-301, presumably on dilution of the vectors with tumor cell proliferation (Fig. 5)Citation . We showed previously (12) that intratumoral injection of OBP-301 was effective for suppressing the growth of H1299 human lung tumors subcutaneously transplanted into nu/nu mice; 8 x 106 pfu of OBP-301, however, might be insufficient for growth inhibition of SW620 and A549 tumors. Additional studies are required to determine the optimal doses of vectors to obtain complete and prolonged GFP labeling of solid tumors.

Progression of non–small-cell lung cancer is characterized by the dissemination of malignant pulmonary epithelial cells (23) . To overcome the poor prognosis of lung cancer involving the pleura, innovative methodology for early diagnosis as well as treatment is needed. We reported previously (17) that intrathoracic injection of replication-deficient adenovirus expressing antisense against heparanase, which is associated with metastatic potential, inhibited pleural dissemination of human lung cancer cells in an orthotopic murine model. These observations suggest that intrathoracically administered adenovirus is capable of infecting disseminated neoplastic nodules in the thoracic cavity. Obstacles to effective diagnosis and therapy for pleural dissemination include the fact that tumors that are either too small or hidden in an internal body location could not be visually detected. Our optical CCD imaging was able to visualize tiny foci of pleural dissemination of A549 human lung cancer cells with GFP fluorescence after intrathoracic injection of OBP-301 and Ad-GFP, although these lesions could not be macroscopically identified (Fig. 6)Citation . Several studies have demonstrated the utility of CCD imaging to track metastasis and/or dissemination of tumor cells that were stably transfected with the GFP gene in small animals (6, 7, 8, 9, 10) ; the vector-based strategy in the present study, however, is considered more convenient and clinically relevant. If target lesions can be identified during thoracotomy, surgeons will be capable of removing disseminated tumors. Moreover, a future option may be to develop robotic laser therapy for pleural dissemination with an automatic detection tool that chases GFP fluorescence under thoracoscopy.

The approach is considered to be promising; however, it also suffers from some limitations. For example, tumors existing within organs could not be detected because detection requires exposing the tumors to a Xenon lamp. Because the entire tumors were not fluorescent (except for very tiny tumors), the margins of invasive tumors that are not clearly encapsulated might go unseen. In addition, the distant metastatic tumors could not be visualized by intratumorally or regionally injected OBP-301 and Ad-GFP. As a means to circumvent this limitation, development of the tumor-specific replication-selective adenovirus OBP-401, in which the GFP gene was inserted, has allowed systemic administration, leading to the whole-body distribution of the vector, thereby detecting distant metastatic lesions with telomerase-specific GFP expression. Preclinical studies using OBP-401 are currently under way in our laboratory.

In conclusion, our data presented here indicate that locoregional injection of Ad-GFP plus OBP-301 in combination with highly sensitive CCD imaging might be a useful diagnostic strategy for real-time visualization of macroscopically invisible tumor tissues. Although the sensitivity and specificity of the method must be assessed by additional experiments, this technology provides a foundation for future clinical application.


   FOOTNOTES
 
Grant support: Grants from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare, Japan.

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: Toshiyoshi Fujiwara, Center for Gene and Cell Therapy, Okayama University Hospital, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail: toshi_f{at}md.okayama-u.ac.jp

Received 4/15/04. Revised 6/16/04. Accepted 7/ 7/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Tearney GJ, Brezinski ME, Bouma BE, et al In vivo endoscopic optical biopsy with optical coherence tomography. Science (Wash DC), 276: 2037-9, 1997.[Abstract/Free Full Text]
  2. MacDonald SL, Hansell DM. Staging of non-small cell lung cancer: imaging of intrathoracic disease. Eur J Radiol, 45: 18-30, 2003.[CrossRef][Medline]
  3. Misteli T, Spector DL. Applications of the green fluorescent protein in cell biology and biotechnology. Nat Biotechnol, 15: 961-4, 1997.[CrossRef][Medline]
  4. van Roessel P, Brand AH. Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins. Nat Cell Biol, 4: E15-20, 2002.[CrossRef][Medline]
  5. Ehrhardt D. GFP technology for live cell imaging. Curr Opin Plant Biol, 6: 622-8, 2003.[CrossRef][Medline]
  6. Rashidi B, Yang M, Jiang P, et al A highly metastatic Lewis lung carcinoma orthotopic green fluorescent protein model. Clin Exp Metastasis, 18: 57-60, 2000.[CrossRef][Medline]
  7. Yang M, Baranov E, Jiang P, et al Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases. Proc Natl Acad Sci USA, 97: 1206-11, 2000.[Abstract/Free Full Text]
  8. Hoffman RM. Visualization of GFP-expressing tumors and metastasis in vivo. Biotechniques, 30: 1016-22, 10246, 2001.[Medline]
  9. Bouvet M, Wang J, Nardin SR, et al Real-time optical imaging of primary tumor growth and multiple metastatic events in a pancreatic cancer orthotopic model. Cancer Res, 62: 1534-40, 2002.[Abstract/Free Full Text]
  10. Yamamoto N, Yang M, Jiang P, et al Real-time GFP imaging of spontaneous HT-1080 fibrosarcoma lung metastases. Clin Exp Metastasis, 20: 181-5, 2003.[CrossRef][Medline]
  11. Yang M, Baranov E, Moossa AR, Penman S, Hoffman RM. Visualizing gene expression by whole-body fluorescence imaging. Proc Natl Acad Sci USA, 97: 12278-82, 2000.[Abstract/Free Full Text]
  12. Kawashima T, Kagawa S, Kobayashi N, et al Telomerase-specific replication-selective virotherapy for human cancer. Clin Cancer Res, 10: 285-92, 2004.[Abstract/Free Full Text]
  13. Kim NW, Piatyszek MA, Prowse KR, et al Specific association of human telomerase activity with immortal cells and cancer. Science (Wash DC), 266: 2011-5, 1994.[Abstract/Free Full Text]
  14. Shay JW, Wright WE. Telomerase activity in human cancer. Curr Opin Oncol, 8: 66-71, 1996.[Medline]
  15. Shay JW, Wright WE. Telomerase: a target for cancer therapeutics. Cancer Cell, 2: 257-65, 2002.[CrossRef][Medline]
  16. Nakayama J, Tahara H, Tahara E, et al Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet, 18: 65-8, 1998.[CrossRef][Medline]
  17. Uno F, Fujiwara T, Takata Y, et al Antisense-mediated suppression of human heparanase gene expression inhibits pleural dissemination of human cancer cells. Cancer Res, 61: 7855-60, 2001.[Abstract/Free Full Text]
  18. Shao J, Fujiwara T, Kadowaki Y, et al Overexpression of the wild-type p53 gene inhibits NF-kB activity and synergizes with aspirin to induce apoptosis in human colon cancer cells. Oncogene, 19: 726-36, 2000.[CrossRef][Medline]
  19. Waku T, Fujiwara T, Shao J, et al Contribution of CD95 ligand-induced neutrophil infiltration to the bystander effect in p53 gene therapy for human cancer. J Immunol, 165: 5884-90, 2000.[Abstract/Free Full Text]
  20. Itoshima T, Fujiwara T, Waku T, et al Induction of apoptosis in human esophageal cancer cells by sequential transfer of the wild-type p53 and E2F-1 genes: involvement of p53 accumulation via ARF-mediated MDM2 down-regulation. Clin Cancer Res, 6: 2851-9, 2000.[Abstract/Free Full Text]
  21. Ohtani S, Kagawa S, Tango Y, et al Quantitative analysis of p53-targeted gene expression and visualization of p53 transcriptional activity following intratumoral administration of adenoviral p53 in vivo. Mol Cancer Ther, 3: 93-100, 2004.[Abstract/Free Full Text]
  22. Masutomi K, Hahn WC. Telomerase and tumorigenesis. Cancer Lett, 194: 163-72, 2003.[CrossRef][Medline]
  23. Fukuse T, Hirata T, Tanaka F, Wada H. The prognostic significance of malignant pleural effusion at the time of thoracotomy in patients with non-small cell lung cancer. Lung Cancer, 34: 75-81, 2001.[Medline]



This article has been cited by other articles:


Home page
 JNMHome page
P. T. Winnard Jr., A. P. Pathak, S. Dhara, S. Y. Cho, V. Raman, and M. G. Pomper
Molecular Imaging of Metastatic Potential
J. Nucl. Med., June 1, 2008; 49(Suppl_2): 96S - 112S.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
K. Guse, T. Ranki, M. Ala-Opas, P. Bono, M. Sarkioja, M. Rajecki, A. Kanerva, T. Hakkarainen, and A. Hemminki
Treatment of metastatic renal cancer with capsid-modified oncolytic adenoviruses
Mol. Cancer Ther., October 1, 2007; 6(10): 2728 - 2736.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Umeoka, T.
Right arrow Articles by Fujiwara, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Umeoka, T.
Right arrow Articles by Fujiwara, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online