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Ann Thorac Surg 2002;73:1740-1746
© 2002 The Society of Thoracic Surgeons

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Original article: general thoracic

Adenoviral herpes simplex virus thymidine kinase gene therapy in an orthotopic lung cancer model

^a Cellular and Developmental Biology Division, Kurume University Research Center for Innovative Cancer Therapy, Kurume City, Fukuoka, Japan
^b Department of Surgery, Kurume University School of Medicine, Kurume City, Fukuoka, Japan
^c Department of Cardiovascular Regeneration Science, Gifu University School of Medicine, Gifu City, Gifu, Japan

Accepted for publication February 26, 2002.

* Address reprint requests to Dr Kosai, Department of Cardiovascular Regeneration Science, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu City, Gifu 500-8705, Japan
e-mail: kosai{at}cc.gifu-u.ac.jp

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. Although adenovirus-mediated herpes simplex virus thymidine kinase (HSVtk) gene therapy is a potential candidate for a novel effective therapy for lung cancer, previous studies have been performed only in a subcutaneous tumor model employing nude mice. We studied therapeutic potentials in correlation with accurate adenoviral gene transduction efficiency in a more clinically relevant orthotopic lung cancer model employing imunocompetent mice.

Methods. To analyze the cytotoxicity of adenoviral HSVtk gene transduction and ganciclovir, a cell proliferation assay was performed in vitro. A survival study was carried out in immunocompetent mice with orthotopic lung cancer, which was generated by intrapulmonary inoculation with syngeneic murine lung cancer cells that had been infected beforehand with each adenoviral vector at the predetermined gene transduction efficiencies.

Results. Tumor cells were efficiently killed by infection with adenovirus carrying the HSVtk gene with the addition of ganciclovir in vitro. In the in vivo experiment all control mice died of rapid growth of the primary lung cancer and of metastases to mediastinal lymph nodes within 26 days after tumor inoculation. In contrast 50% and 100% of mice survived more than 40 days after inoculation with adenovirally HSVtk-transfected tumor cells that moderately and highly expressed HSVtk, respectively, when followed by ganciclovir administration. Gene transduction efficiencies were 67%.

Conclusions. Adenovirus-mediated HSVtk gene therapy may be therapeutic for lung cancer when gene transduction efficiencies and sufficient expression levels of HSVtk can be achieved. Moreover, the present findings underscore the importance of the mouse orthotopic lung cancer model for studies of gene therapy.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Lung cancer is one of the leading causes of cancer death in the world [1] and its incidence continues to increase. About 80% of lung cancers are nonsmall cell lung carcinoma (NSCLC), and 60% of these are already inoperable by the time they are diagnosed. The current therapies including chemotherapy and radiotherapy are less effective for advanced NSLC for which the 5-year survival rate remains less than 20% even if the patients undergo both surgery and chemoradiotherapy [2]. This poor prognosis indicates an urgent need for the development and the establishment of innovative and novel therapies such as gene therapy for lung cancer.

An important issue in the present status of cancer gene therapy is that in vivo transfer of a therapeutic gene to every cancer cell using any of the current vector systems is impossible in human patients or animals with orthotopic cancer. In this regard transfer of the herpes simplex virus thymidine kinase (HSVtk) gene followed by administration of ganciclovir has the practical advantage of a potent bystander effect by which cytotoxicity is conferred to the neighboring nontransduced cells [3–10]. The normally nontoxic prodrug ganciclovir is phosphorylated by HSVtk, then converted to ganciclovir triphosphate, which inhibits DNA synthesis by acting as a chain terminator, leading to the death of predominantly dividing tumor cells. This putative cytotoxic mechanism may constitute another safety advantage in HSVtk gene therapy, as normal nondividing cells will theoretically have low cytotoxicity in the case of undesirable gene transduction [3].

Many aspects of new gene therapy strategies need to be tested out in animal models before use in clinical trials. However, the majority of previous studies including those on HSVtk gene therapy for lung cancer represented tumor regression as the sole therapeutic effect in subcutaneous tumor models emplying immunodificient nude mice [11]. Such data may not directly indicate the actual therapeutic potential or clinical benefit of this gene therapy strategy because some critical factors, especially limited gene transduction efficiency in the clinical situation, were not evaluated. In addition many tissue characteristics including abundant blood supply in the lung, relatively high compliance of lung tissue, tissue-specific immunocytes, and the availability of numerous endogenous factors in the pulmonary microenvironment conceivably might be responsible for the growth and the metastases of tumor cells [12]. Thus an immunocompetent orthotopic lung cancer model may be more suitable for investigation of the efficacies of new gene therapies. The present study used an orthotopic mouse model of lung cancer, which enabled us to evaluate survival prolongation in correlation with accurate gene transduction efficiency. The results suggested that adenovirus-mediated HSVtk gene therapy may be therapeutic for lung cancer when efficient gene transduction and relatively high expresion of HSVtk can be achieved.

	Material and methods

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Cell lines and culture
The human lung adenocarcinoma cell line A549 and the murine lung cancer cell line Lewis lung cancer (LLC) were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). Both cell lines were cultured in 5% CO₂ at 37°C in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin G, and 100 µg/mL streptomycin.

Recombinant adenoviruses
E1-deleted replication-defective adenoviral vectors encoding the HSVtk gene under the transcriptional control of rous sarcoma virus long terminal repeat (AdRSVtk) and cytomegalovirus immediate-early enhancer promoter (AdCMVtk), and a control adenoviral vector encoding the ß-galactosidase gene under the transcriptional control of the CMV promoter (AdCMVlacZ) were prepared as described previously [3]. The viral titers were determined as plaque-forming units (pfu) by plaque assays on 293 cells. The amount of infective adenoviral vector per cell (pfu/cell) in culture media was expressed as multiplicity of infection (MOI).

Animals
Female C57BL/6 mice at 6 to 8 weeks old were housed in cages in a temperature-controlled room on a 12-hour light-dark cycle and with free access to food and water. All animal studies were performed in accordance with the National Institutes of Health guidelines as dictated by the Animal Care Facility at the Kurume University School of Medicine.

Adenoviral gene transduction efficiencies in vitro
A549 and LLC cells were infected with AdCMVlacZ at MOIs of 10, 30, 100, 300, 1,000, and 3,000 in 6-well plates at 37°C for 1 hour. The media were then refreshed and the cells were incubated in 5% CO₂ at 37°C for 24 hours and sequentially fixed with 0.5% glutaraldehyde for 10 minutes. After cells had been washed twice with phosphate-buffered saline (PBS) they were stained with 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) solution in 13 mmol/L MgCl₂, 15 mmol/L NaCl, 44 mmol/L HEPES at a pH of 7.4, 300 mmol/L of potassium ferricyanide, and 300 mmol/L of potassium ferrocyanide in order to determine the percentage of X-gal positive cells.

ß-Galactosidase enzyme reporter assay
ß-Galactosidase enzyme reporter assay after infection with AdRSVlacZ or AdCMVlacZ was performed according to the manufacturer’s protocol (Promega, Tokyo, Japan). In brief, LLC cells were infected with AdCMVlacZ or AdRSVlacZ at MOI of 1,000. At 48 hours after infection cell lysates were incubated for 30 minutes with the substrate of O-nitrophenyl-ß-D-galactophyranoside and the absorbance was read at 420 nm with a spectrophotometer. The activity of ß-galactosidase was calculated according to the standard curve of the predetermined concentration of ß-galactosidase enzyme.

Cytotoxic effects of AdRSVtk in lung cancer cells in vitro
LLC cells were infected with AdRSVtk at MOI of 30, 300, and 3,000 or with AdCMVlacZ at MOI of 3,000 as a control that would allow maximal estimation of the factors of adenoviral infection and transgene. After 12 hours of incubation the media with or without 10 µg/mL ganciclovir (ganciclovir; provided by F Hoffmann-La Roche, Basel, Switzerland), respectively, were renewed daily for 5 days. At 6 days after adenoviral infection WST-1 assay was performed according to the manufacturer’s protocol (Dojindo Laboratories, Mashiki, Japan) to analyze cell viability. The cell viability after each treatment was calculated using the level of absorbance at 450 nm and expressed as the percentage of nontreated cells.

Adenovirus-mediated transgene expression in inoculated tumor cells in vivo
C57BL/6 mice were sacrificed at 3 days after the intrapulmonary injection of 1 x 10⁵ LLC cells that had been infected with AdCMVlacZ at MOI of 3,000. The whole lung tissues were excised and fixed in cold PBS containing 2% formaldehyde and 0.2% glutaraldehyde for 3 hours. In order to detect the expression of ß-galactosidase the lung was subsequently incubated with X-gal solution at 37°C for 4 hours.

HSVtk gene therapy for orthotopic lung cancer in mice
LLC cells were infected with AdRSVtk at MOI of 300 (n = 4) and 3,000 (n = 4), and with AdCMVtk at MOI of 3,000 (n = 6). For the control, LLC cells were infected with AdCMVlacZ at MOI of 3,000 (n = 8) or treated with a vehicle (n = 6) instead of with AdRSVtk and AdCMVtk. After 48 hours of incubation mice received a percutaneous and intrapulmonary inoculation of the LLC cells as previously described with modification in terms of cell numbers [13]. In brief after a small incision had been made in the skin on the left chest wall, a 29-gauge needle attached to a syringe was directly inserted into the left lung at a depth of 5 mm while the animal was under the ether anesthesia. Subsequently, 1 x 10⁵ adenovirally infected LLC cells mixed with matrigel basement membrane matrix (Collaborative Biochemical Products, Bedford, MA) were injected into the left lung. At 3 days after tumor inoculation all mice, including those receiving control LLC cells, were treated with an intraperitoneal injection of 50 mg/kg ganciclovir twice a day for 14 consecutive days. Subsequently a long-term survival study was performed after each treatment.

Hematoxylin and eosin staining in histology sections
For histopathologic analysis lung tissues were fixed with 10% formalin and embedded in paraffin. Sections were cut at thickness of 4 µm and stained with hematoxylin and eosin.

Statistical analysis
All of the values of cytotoxicity in vitro are presented as the mean ± SD. Differences among groups were compared utilizing the Turkey-Kramer test. The Kaplan-Meier survival data were examined using Mantel-Cox log-rank analysis. All p values of less than 0.05 were considered statistically significant.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Adenoviral gene transduction efficiencies in vitro
Adenoviral gene transduction efficiencies (AGTE) in the murine LLC cells were much lower than those in the human A549 cells at each MOI in vitro (Fig 1A), while AGTE in both cells were increased as higher MOI were used. Adenoviral gene transduction efficiencies in LLC cells at MOI of 300 and 3,000 were 23% and 67%, respectively.

View larger version (21K): [in this window] [in a new window]   Fig 1. (A) Adenoviral gene transduction efficiencies in murine Lewis lung cancer (LLC) and human A549 lung cancer cell lines. A549 and LLC cells were infected with AdCMVlacZ at various multiplicities of infection and subsequently stained with X-gal. Adenoviral gene transduction efficiencies in both cell lines were increased in a dose-dependent manner. (B) Cytotoxic effects of AdRSVtk with and without ganciclovir in LLC cells in vitro, respectively. The cytotoxic effects of AdRSVtk/ganciclovir were significantly higher than those of AdRSVtk/PBS at each multiplicity of infection ([MOI] p < 0.0001). Nearly all cells were killed with AdRSVtk/ganciclovir at MOI of 3,000. Treatment with AdRSVtk/PBS at MOI of 3,000 also showed significant cytotoxicity compared with treatment with AdCMVlacZ/PBS at MOI of 3,000 (p < 0.0001). (GCV = ganciclovir; PBS = phosphate-buffered saline.)

The activities of the ß-galactosidase enzyme were 23 and 50 mU after AdRSVlacZ and AdCMVlacZ infection in LLC cells, respectively. Thus expression in LLC cells after infection with the adenoviral vector encoding the transgene under the transcriptional control of the CMV promoter was significantly higher than that of the RSV promoter.

Cytotoxic effects with potent bystander effects of AdRSVtk/ganciclovir in lung cancer cells in vitro
Cytotoxic effects of AdRSVtk/ganciclovir were significantly higher than those of AdRSVtk/PBS in LLC cells at each MOI. Nearly all cells were killed by treatment with AdRSVtk/ganciclovir at MOI of 3,000 (0.04% viability of nontreated cells; Fig 1B). Interestingly such a cytotoxic effect was relatively and significantly strong even at MOI of 30 and 300, which represented only 9% and 23% of gene transduction efficiencies, respectively. Thus after AdRSVtk infection, ganciclovir-dependent cytotoxicity and its bystander effect were comparatively strong. On the other hand treatment with AdRSVtk/PBS at MOI of 3,000 also showed significant cytotoxicity in contrast to no apparent cytotoxicity observed after infection with AdCMVlacZ at MOI of 3,000 regardless of addition of ganciclovir, suggesting that the HSVtk expression conferred some ganciclovir-independent cytotoxicity in LLC cells when the adenoviral transduction efficiency of HSVtk gene was relatively high.

Adenovirus-mediated transgene expression in inoculated tumor cells in vivo
To confirm the formation of the tumor nodule and in vivo transgene expression in adenovirally gene-transduced tumor cells in the lung, at the time of starting ganciclovir treatment in vivo X-gal staining was performed on the lung inoculated with tumor cells that had been infected with AdCMVlacZ beforehand (Fig 2).

View larger version (76K): [in this window] [in a new window]   Fig 2. Macroscopic pictures of intrapulmonary inoculated tumor and transgene expression at the initiation of ganciclovir treatment. (A) The low-power view. The arrow indicates the tumor nodule at 3 days after inoculation of Lewis lung cancer cells that had been infected with AdCMVlacZ beforehand. (B) The high-power view of the tumor lesion. The white tumor nodule was not completely circumscribed and some tumor cells were invading the surrounding lung parenchyma. X-gal staining revealed transgene (ß-galactosidase) expression.

AdRSVtk/ganciclovir and AdCMVtk/ganciclovir treatments prolong survival in orthotopic lung cancer in mice
All control mice given vehicle-treated or AdCMVlacZ-infected tumor cells and subsequent administration of ganciclovir died of rapid growth of primary lung cancer and metastases to the mediastinal lymph nodes within 26 days after tumor inoculation (Fig 3A–C, Fig 4). In contrast, mice receiving AdRSVtk- or AdCMVtk-infected tumor cells and subsequent administration of ganciclovir survived significantly longer than control mice.

View larger version (136K): [in this window] [in a new window]   Fig 3. Macroscopic and microscopic pictures of the lung of a mouse that had received AdCMVlacZ- or AdCMVtk-infected tumor cells. (A–C) The lung at the time of death of a mouse that had received AdCMVlacZ-infected Lewis lung cancer (LLC) cells at multiplicity of infection (MOI) of 3,000 and subsequent ganciclovir administration. Large tumor nodules and mediastinal metastases are prominent. (D–F) The mouse lung at 40 days after intrapulmonary inoculation of AdRSVtk-infected LLC cells at MOI of 3,000 and subsequent ganciclovir administration. Tumor nodules were not microscopically detected in the lung although histologic examination revealed a very small tumor nodule. Magnification (hematoxylin and eosin staining) is x6 (B and E) and x40 (C and F).

View larger version (20K): [in this window] [in a new window]   Fig 4. Survival prolongation after intrapulmonary inoculation with adenovirally infected Lewis lung cancer (LLC) cells and subsequent administration of ganciclovir. LLC cells were infected with each adenoviral vector at each multiplicity of infection (MOI), as shown, or with a vehicle instead of an adenoviral vector. After 48 hours of incubation LLC cells were percutaneously and intrapulmonarily inoculated into the left lung in mice. Three days later all mice including control mice were given intraperitoneal injections of ganciclovir twice a day for 14 consecutive days. Survival advantages, which were seen in AdRSVtk and AdCMVtk groups, became more significant as higher adenoviral gene transduction efficiencies and higher transgene (HSVtk gene) expressions were used.

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Findings in the present study showed the potent therapeutic effect of adenovirus-mediated HSVtk gene therapy for lung cancer in an orothotopic lung cancer model employing immunocompetent mouse. The most encouraging finding from a preclinical viewpoint is that all mice receiving intrapulmonary inoculated tumor cells, 67% of which expressed HSVtk under the transcription control of the CMV promoter and subsequent ganciclovir administration were observed to be cured of lung cancer. In addition the survival of mice receiving AdRSVtk-infected tumor cells and subsequent ganciclovir administration was prolonged more significantly at higher MOI than that at lower MOI despite the fact that these mice still had some remaining tumor cells. These findings taken together with the fact that the CMV promoter is stronger than the RSV promoter indicate that adenovirus-mediated HSVtk gene therapy may be clinically therapeutic and useful for lung cancer and importantly that sufficient gene transduction efficiencies and a relatively high expression level of HSVtk (ie, the use of relatively strong promoter) are critical factors for successful clinical trials.

The present results also generally suggest the importance of assessing therapeutic potentials of gene therapy approaches in clinically relevant models before clinical trials. For some gene therapy approaches, the discrepancy between promising data in experiments and a somewhat disappointing outcome (ie, little clinical benefit) in actual clinical trials may be due at least in part to insufficient analyses or unsuitable animal models. The majority of such studies presented in vitro cytotoxicity or tumor regression as a therapeutic effect in a very simple animal model of subcutaneous tumor [11]. However, such data do not directly represent therapeutic potentials or guarantee clinical benefit; some critical factors especially gene transduction efficiencies and tissue characteristics that potentially influence tumor growth and therapeutic potentials were not well evaluated in such experiments.

Therefore the present study, which carefully examined the therapeutic potentials of adenovirus-mediated HSVtk gene therapy (adenovirally transduced HSVtk gene and subsequent ganciclovir administration), correlating its effects with accurate transduction efficiencies of HSVtk gene in a model of orthotopic lung cancer employing immunocompetent mice substantially provides more clinically relevant and important information than that from any previous studies of HSVtk gene therapy for lung cancer in a subcutaneous tumor model employing nude mice.

In actuality the results of in vitro cytotoxicity in the present study were better than those of survival prolongation in the orthotopic lung cancer model. For example in vitro cytotoxicity of AdRSVtk at MOI of 300 with ganciclovir was strong and the majority of tumor cells were killed. In contrast, all mice receiving AdRSVtk-infected tumor cells at MOI of 300 and subsequent ganciclovir administration finally died of primary lung cancer and metastases to the mediastinum lymph nodes despite the fact that survival prolongation was an apparent clinical benefit. Such discrepancies are possibly due to differences in environment between artificial in vitro culture and the native orthotopic region by which some mixed factors may influence cytotoxicity or tumor growth, although the exact mechanisms remain to be resolved.

Obviously direct in vivo gene transfer into preestablished tumor in the lung would be a more clinically relevant procedure if such a procedure could be reproducibly and reliably performed in animals. Inoculation of tumor cells into the mouse lung successfully led to the formation of small nodules in the lung, as described in the present study. However, our early trials of direct, accurate in vivo injection of each adenoviral vector into this preestablished small tumor nodule in the mouse lung were fruitless. First, a direct injection under thoracotomy or percutaneously without thoracotomy was hampered because mice cannot tolerate thoracotomy without special equipment and a system set up to function as a mouse respirator and because such a small tumor nodule could not be visualized or detected by chest roentgenogram. In addition even if such trials could be performed despite these drawbacks the resulting data on whether injection of adenoviral vector into the very small nodule in the mouse lung could be performed accurately would have little bearing on whether such a procedure could be performed on a much larger tumor nodule in human patients under the fully equipped condition.

Formation of tumor nodule and transgene expression before ganciclovir administration in the present study indicated that the present experimental system may mimic some clinical situations and may be very suitable, at least for the purpose of assessing therapeutic effects. Moreover the present experimental system has the additional advantage of facilitating the assessment of therapeutic effect in correlation with accurate gene transduction efficiencies in orthotopic lung cancer, which may be a more meaningful and attractive area of investigation. Potential side effects and toxicity, which could not be estimated in the present model, should be scrupulously and extensively investigated in another study and depending on results it may be necessary to newly generate other appropriate animal models and experimental systems focusing on whatever adverse side effects have been observed.

From a clinical viewpoint not only a complete cure of lung cancer but also significant prolongation of survival appeared to be a potential clinical benefit of the gene therapy strategy investigated, especially for NSCLC patients for whom current therapies have been shown to be less effective [14]. In addition, some combination therapies of adenovirus-mediated HSVtk gene therapy with current therapies or with other gene therapy approaches may enhance therapeutic potential. For example, combination gene therapy with HSVtk and cytokine genes largely and synergically enhanced the therapeutic potential for treating metastatic colon cancer in the mouse liver, for which adenovirus-mediated HSVtk gene therapy alone resulted in very limited survival prolongation [4]. In addition, tumor-specific replication-competent adenoviruses, which have been recently reported, may also increase gene transduction efficiencies [15]. Such combination trials may be studied step by step using the present orothotopic lung cancer model in future studies.

In conclusion, adenovirus-mediated HSVtk gene therapy may be therapeutic and clinically useful for lung cancer to the extent that sufficient gene transduction efficiencies can be achieved and a relatively strong promoter is used. Moreover the present findings emphasize the importance of the mouse orthotopic lung cancer model for gene therapy study.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
We thank Dr Eisuke Mekada (Research Institute for Microbial Diseases, Osaka University), Dr Yasuhiro Terazaki (Department of Surgery, Kurume University) for help and discussion, and Masako Uenosono for technical assistance.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fukunaga, M. Articles by Kosai, K.-i. Search for Related Content PubMed PubMed Citation Articles by Fukunaga, M. Articles by Kosai, K.-i.