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Ann Thorac Surg 1995;60:593-597
© 1995 The Society of Thoracic Surgeons

Original Articles: General Thoracic

Pleural-Based Mesothelioma in Immune Competent Rats: A Model to Study Adenoviral Gene Transfer

John C. Kucharczuk, MD, Ashraf A. Elshami, MD, Hong Bing Zhang, W. Roy Smythe, MD, Harry C. Hwang, MD, James S. Tomlinson, MD, Kunjlata M. Amin, PhD, Leslie A. Litzky, MD, Steven M. Albelda, MD, Larry R. Kaiser, MD

Thoracic Surgery Section, Department of Surgery, Pulmonary/Critical Care Section, Department of Medicine, Department of Pathology, and Thoracic Oncology Research Laboratory, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Despite multimodality approaches, pleural-based malignant mesothelioma remains a disease with a very poor prognosis. Novel therapeutic strategies such as gene therapy clearly are needed to improve the survival of patients with this neoplasm. To aid in the evaluation of new treatment strategies, animal models that closely mimic human disease are required. This article describes the establishment of a pleural-based model of malignant mesothelioma in immune-competent Fischer rats.

Methods.Via a modified left anterior lateral thorocotomy, a syngeneic malignant mesothelioma cell line, called II-45, was placed into the pleural cavity of Fischer rats.

Results.Placement of II-45 cells into the pleural cavity of Fischer rats results in a model of pleural mesothelioma that closely resembles the disease seen in patients and is highly reproducible, with animals dying within 1 month. We also demonstrate the feasibility of adenoviral-mediated gene transfer to normal mesothelial cells lining the pleural cavity, as well as to malignant cells deep within the substance of pleural-based malignant mesothelioma.

Conclusions.The model described here offers the opportunity to study a variety of new treatment modalities, especially somatic gene transfer, against pleural-based malignant mesothelioma in an immune competent setting.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
See also page 597 and page 598.

Malignant mesothelioma is a primary neoplasm of the mesothelial lining of the pleural and peritoneal cavities. It accounts for approximately 2,000 deaths per year in the United States. Unfortunately, no therapy currently in use is effective against this neoplasm. In fact, recent studies evaluating multimodality approaches including operation, chemotherapy and radiation therapy have failed to show significant improvements in median survival [1]. Thus, new therapeutic modalities directed against malignant mesothelioma and better model systems to evaluate their efficacy are needed.

Previously, we reported on the feasibility of gene transfer to human malignant mesothelioma using a recombinant adenoviral vector carrying the ß-galactosidase reporter gene [2]. In those studies, there was a high level of reporter gene transfer to human mesothelioma both in cell culture and in tumors xenografted into the peritoneal cavity of severe-combined immunodeficient mice. More recently, we have used a recombinant adenovirus carrying the herpes simplex thymidine kinase gene to transduce cell lines derived from human malignant mesothelioma and render them susceptible to ganciclovir administration at pharmacologic concentrations both in vitro [3] and in vivo [4]. Although the initial results are encouraging, the in vivo studies were performed in the peritoneal cavity of immune-deficient animals, a situation that does not closely mimic human disease. In fact, recent studies suggest that cells transduced in vivo by first generation adenoviral vectors are quickly cleared in animals with a competent immune system [5, 6]. Whether the immune response against gene therapy vectors will be beneficial or detrimental in the treatment of malignancy remains to be investigated. The purpose of this article is to describe the development of a new model to study gene transfer and treatment of pleural-based malignant mesothelioma in an immune competent animal.

To accomplish these objectives, an animal model with two key features was required: (1) a well characterized syngeneic mesothelioma tumor cell line and (2) the ability to inject tumor cells and adenoviral vectors directly into the chest cavity with minimal morbidity and mortality. Craighead and associates [7] have recently described cell lines cultured from experimental asbestos-induced peritoneal mesothelioma in rats. Because the rat has a much larger pleural cavity than the mouse, we used one of the asbestos-derived mesothelioma cell lines to create an intrapleural model of malignant mesothelioma in Fischer rats and then studied the feasibility of gene transfer into the pleural space and into pleural-based tumors. Our results indicate that direct injection of tumor cells into the pleural cavity results in a model that closely resembles human disease. Furthermore, adenoviral-mediated gene transfer occurs efficiently in the rat pleural space and into the substance of established tumor nodules. These findings will allow for the study of adenoviral gene therapy and other new therapeutic approaches for malignant mesothelioma in animals with intact immune systems.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Syngeneic Malignant Mesothelioma Cell Line
A rat malignant mesothelioma cell line, called II-45, was donated by Dr Cheryl Walker at The University of Texas, M.D. Anderson Cancer Center. This cell line was established from experimentally induced mesotheliomas in Fischer 344 rats exposed to asbestos and has been extensively characterized [7]. The cells were cultured and maintained in DMEM/Ham's F12 (1:1) media supplemented with 10% fetal bovine serum, 2.5 µg/mL transferrin, 2.5 µg/mL insulin, and 2.5 ng/mL sodium selenite. The cells were grown to 80% confluence, resuspended in media by trypsinization, and counted by Coulter counter. Cells were then centrifuged at 1,000 rpm for 3 minutes, and the pellet was resuspended in unenriched RPMI medium to yield 1 x 106 cells per 300 µL in preparation for animal implantation.

Intrathoracic Implantation of the II-45 Cell Line
Fischer 344 rats (150 to 200 g) were obtained from the Charles River plant and maintained at the animal facility of The Wistar Institute of Anatomy and Biology in Philadelphia, Pennsylvania. All protocols were approved by the animal use committees of the Wistar Institute and the University of Pennsylvania in compliance with the ``Guide for the Care and Use of Laboratory Animals'' (National Institutes of Health publication 85-23, revised 1985). Surgical anesthesia was obtained by intramuscular hind limb injection of ketamine/xylazine (9:1). The left anterior-lateral thorax was shaved, and animals were positioned in a right lateral decubitus position. A 2-cm left lateral incision was made approximately 1 cm caudal to the left glenohumoral joint. Sharp dissection was carried out through the cutaneous maximus muscle, and the ventral border of the latissimus dorsi was identified. The latissimus dorsi was retracted to reveal the intercostal muscles of the fifth intercostal space. A no. 15 blade was used to gently ``sweep'' away the intercostal muscles, revealing the thin parietal pleural membrane but not violating the pleural space. The underlying lung was easily visible through the transparent pleural membrane. Under direct visualization, 300 µL of II-45 cell suspension (1 million cells) was placed into the pleural cavity with a 28-gauge tuberculin needle. The cutaneous maximus muscle was reapproximated with 5-0 nylon suture in an interrupted fashion, and the skin was closed with surgical staples. The animals were observed for 1 hour and then returned to their cages.

Characterization of the Model
Three animals were euthanized and formally autopsied on days 5, 12, 17, 20, and 24 after II-45 cell instillation to obtain aggregate tumor weight. The aggregate tumor for each animal was obtained by carefully dissecting and weighing all visible tumor encasing the intrathoracic organs and the chest wall. On days 5, 7, 12, 14, 17, and 19, one animal was sacrificed and the tissues were fixed in 10% neutral buffered formalin. After 24 hours of fixation, the tissues were paraffin embedded for histologic examination using hematoxylin and eosin staining. A sample was also fixed in gluteraldehyde for electron microscopy using standard techniques. The remaining animals (n = 12) were followed up for survival. Each animal in the survival group was autopsied at the time of death. All visible tumor was removed to assess aggregate tumor weight at the time of death.

Recombinant Adenovirus
The production of replication-deficient adenoviral vectors has been described in detail elsewhere [8]. Briefly, the vectors were constructed from an adenovirus type 5 (Ad5) mutant, which lacks most of viral sequence regions E1a and E1b and a portion of E3. By homologous recombination techniques, the Escherichia coli lacZ marker gene driven by the cytomegalovirus promoter (Ad.CMVlacZ) was inserted into the viral genome. Viral stocks were propagated in 293 cells, and titers were quantified by 293 plaque assay and spectrophotometric density.

Injection of Virus Into the Pleural Space of Normal Animals
Virus was instilled using the same surgical protocol as described above for tumor cell instillation. Under direct vision, 1010 plaque-forming units (pfu) of Ad.CMVlacZ suspended in 300 µL of unmodified RPMI media was injected into the pleural space via a 28-gauge tuberculin needle. The cutaneous maximus muscle was reapproximated with 5-0 nylon suture in an interrupted fashion and the skin was closed with surgical staples. The animals were observed for 1 hour postoperatively to ensure satisfactory recovery and then returned to their cages.

Injection of Virus Into the Pleural Space of Animals and Into Established Pleural-Based Tumors
To examine the ability of recombinant adenovirus to transduce normal mesothelial cells, 3 healthy Fischer rats received 1010 pfu of Ad.CMVlacZ to the chest cavity via the technique described for tumor cell administration.

To evaluate gene transfer to an established tumor nodule, 1010 pfu of Ad.CMVlacZ was instilled into the pleural cavity of an animal that had received 1 million intrapleural II-45 cells 17 days earlier.

Evaluation of Escherichia coli lacZ Gene Expression
Two days after intrapleural injection of Ad.CMVlacZ, the rats were sacrificed by isoflurane inhalation. Chest wall and lung tissues were removed and snap-frozen in OCT embedding media (Miles Inc, Elkhart, IN) at -70°C. Five-micrometer-thick, unfixed, frozen sections were cut, placed on glass microscope slides, and stained using the following protocol: The slides were rinsed in phosphate-buffered saline solution with 1 mmol/L MgCl2 and incubated in a solution containing the ß-galactosidase substrate X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactosidase). The X-gal staining solution consisted of 5 mmol/L potassium ferricyanide, 5 mmol/L potassium ferrocyanide, 2 mmol/L MgCl2, and 1 mg/mL X-gal. After a 4-hour incubation at 37°C, the cells were postfixed in 4% formaldehyde/0.5% glutaraldehyde in phosphate-buffered saline solution. Before mounting, the sections were counterstained with hematoxylin for 30 seconds. Sections were then evaluated for lacZ gene expression by light microscopy. Expression of the gene for ß-gal was indicated by discrete cells staining an intense blue color.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Tumor Model
To evaluate the ability of the syngeneic mesothelioma cells to form pleural tumors and to evaluate the characteristics of these tumors, we injected 38 animals intrapleurally with II-45 cells. Among the 38 animals studied, there were two intraoperative deaths due to pneumothorax and no early postoperative deaths. In the animals followed up for tumor progression, small tumor nodules (2 to 3 mm) were grossly visible on the pericardial and visceral pleural surfaces by day 5. Histologic examination at this time point showed microscopic tumor on both the visceral and the parietal pleura, as well as within perithymic and paratracheal connective tissue. A single intrapulmonary nodule was also noted. The malignant cells were predominantly epithelioid, with moderate amounts of eosinophilic cytoplasm and large pleomorphic nuclei. Spindle cell areas were also identified. To further examine the ultrastructural characteristics of the tumor nodules, electron microscopy was performed on a nodule 7 days after II-45 instillation. Figure 1AGo demonstrates numerous elongated microvilli characteristic of malignant mesothelioma (electron micrograph courtesy of Giuseepe G. Peitra, MD, Department of Pathology, University of Pennsylvania). In addition, conventional histochemical staining with hematoxylin and eosin on day 7 tumor revealed epithelioid cells with extensive invasion into the skeletal muscle of the chest wall (Fig 1BGo). The histology was essentially unchanged when compared with the day 5 specimen. Occasional inflammatory cells (neutrophils, eosinophils and lymphocytes) were scattered throughout the tumor. By day 12, large tumor nodules were grossly visible on the diaphragm and the parietal pleural surfaces bilaterally. At the later time points (days 14 to 19), there was extensive tumor necrosis with increasing pulmonary parenchymal and pericardial involvement.



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  		Fig 1. . (A) Electron microscopy (original magnification, x135,000) of day 7 tumor. (B) Conventional staining (original magnification, x200) of day 7 tumor. (C) Day 19 radiograph (anteroposterior view). (D) Autopsy photograph on day 19 confirming radiographic findings. (E) Distribution of virally transduced cells 48 hours after intrapleural administration of Ad.CMVlacZ. (F) Distribution of virally transduced cells (arrows) within established tumor nodule 48 hours after intrapleural administration of Ad.CMVlacZ. (L = lungs; T = tumor.)

 
A chest radiograph (anteroposterior view) obtained on a day 19 animal is shown in Figure 1CGo. This radiograph demonstrates opacification of both lungs, loss of the cardiac silhouette, and a mass deformity of left chest wall. The radiographic findings are confirmed on the autopsy photograph of this animal, shown in Figure 1DGo. This photograph demonstrates massive tumor with encasement of the lungs and the mediastinal organs, as well as invasion into the chest wall. Tumor progression, as assessed by increasing aggregate tumor weight as a function of time, is shown in Figure 2Go (n = 3 at each time point).



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  		Fig 2. . Intrathoracic progression of II-45 tumors in Fischer rats by tumor weight. Animals were injected with 1 x 106 tumor cells on day 0, and 3 animals were sacrificed at each time point. At autopsy, all visible tumor was removed and weighed. Bars represent standard error of the mean.

 
In the 12 animals followed up for survival, the median survival was 26.5 ± 1.5 days. As shown in Figure 3Go, most animals died between day 25 and day 27. At autopsy, these animals had massive tumors encasing the lungs, the heart, the mediastinum, and the chest wall. Animals had an aggregate tumor weight of 6.3 ± 0.6 g at the time of death.



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  		Fig 3. . Fischer rat survival curve after intrapleural administration of one million II-45 cells. Twelve animals were injected with 1 x 106 tumor cells on day 0 and followed up for survival. All animals died by day 31. Tumor weight at the time of death was 6.3 ± 0.6 g.

 
Gene Transfer
To test the ability of adenovirus to transduce pleural mesothelium, 1010 pfu of Ad.CMVlacZ was instilled into the chest cavity of 3 healthy Fischer rats. As shown in Figure 1EGo, this concentration of virus resulted in highly efficient gene transfer to mesothelial cells of the visceral pleura (as evidenced by the uniform blue staining). Significantly, no gene transfer was noted in underlying lung tissues. Similar results were observed on the parietal pleura of the chest wall and diaphragm.

To assess the ability of recombinant adenovirus to transduce tumor cells within established tumors, 1010 pfu of Ad.CMVlacZ was instilled into the pleural cavity of an animal that received 1 million intrapleural II-45 cells 17 days earlier. In contrast to the apparent barrier provided by the normal mesothelial cells, the marker gene was visible in many cells deep within the tumor nodule (Fig 1FGo).


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
Several characteristics of malignant mesothelioma make it an attractive target for initial attempts at somatic gene therapy. First, the tumor grows in the potential space of the pleural cavity and is easily accessible for direct administration of therapeutic vectors carrying genetic material. Second, the morbidity and mortality associated with malignant mesothelioma is due almost exclusively to local rather than metastatic disease, suggesting that local control may markedly improve survival. Although we have demonstrated the feasibility of using adenoviral vectors to deliver genes to the mesothelial cells of the peritoneum of immunodeficient animals [2, 3], these studies have not addressed the important issues of delivering adenovirus to the pleural space of immunocompetent animals with malignant mesothelioma.

The first major finding of this study is that injection of syngeneic II-45 cells into the pleural cavity of Fischer rats results in a model of pleural mesothelioma that closely resembled the disease seen in patients. Although several subcutaneous [9], intraperitoneal, and intrapleural [10] animal models for malignant mesothelioma using xenografted human mesothelioma cell lines have been reported previously, these models do not represent the clinically relevant situation of pleural-based disease in an animal with an intact immune system. The approach described in this study has advantages over previously described models in that it (1) closely resembles the histologic appearance of pleural disease in humans, (2) allows for the study of the immune response and immunotherapeutic modalities, (3) enables precise quantification of tumor mass, and (4) is highly reproducible, with untreated animals dying within 1 month.

The second major finding in this study is that intrapleural administration of adenovirus carrying a lacZ marker gene efficiently transduces the majority of surface mesothelial cells on both the visceral and parietal surfaces of the rat pleura. In addition, the pleural mesothelium appears to form a tight barrier, preventing gene transfer to tissues adjacent to the transduced mesothelium. No gene expression was observed in either the lung or the skeletal muscle of the chest wall, suggesting that administration of virus might be safely confined to the thorax. These findings are similar to those in the peritoneum and support the idea that direct intrapleural injection will be feasible in the treatment of animals with mesothelioma. Importantly, these studies, like those in the severe-combined immunodeficient mouse model of human mesothelioma [2], show that recombinant adenovirus has the ability to transduce cells deep within an established tumor mass. This feature is likely to be critical for effective tumor treatments using current gene therapy approaches, such as the herpes simplex virus thymidine kinase/ganciclovir system, where it may be necessary to transduce 5% to 10% of tumor cells [11].

In summary, the Fischer rat model for malignant mesothelioma described here offers the opportunity to study various new treatment modalities. Studies are currently underway to evalute the therapeutic efficacy of recombinant adenoviral transfer of herpes simplex virus thymidine kinase, as well as transfer of immunotherapeutic genes such as IL-2 and GM-CSF in this model.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
Supported by grants from the University of Pennsylvania Cancer Center, National Cancer Institute (NCI PO1 66726), and American Lung Association (ALA 94-488).


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Rusch VW. Pleurectomy/decortication and adjuvant therapy for malignant mesothelioma. Chest 1993;103:382S–4S.[Medline]
  2. Smythe WR, Kaiser LR, Amin KM, et al. Successful adenovirus mediated gene transfer in an in vivo model of human malignant mesothelioma. Ann Thorac Surg 1994;57: 1395–401.[Abstract]
  3. Smythe WR, Hwang HC, Amin KM, et al. Use of recombinant adenovirus to transfer the herpes simplex virus thymidine kinase gene to thoracic neoplasms: an effective in vitro drug sensitization system. Cancer Res 1994;54:2055–9.[Abstract/Free Full Text]
  4. Smythe WR, Hwang HC, Elshami AA, et al. Treatment of experimental human mesothelioma using adenovirus transfer of the herpes simplex thymidine kinase gene. Ann Surg 1995;222:78–86.[Medline]
  5. Yang Y, Nunes FA, Berencsi E, Gonczol E, Engelhardt JF, Wilson JM. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nature Genet 1994;7:362–9.[Medline]
  6. Engelhardt JF, Xuehai Y, Doranz B, Wilson JW. Ablation of E2a in recombinant adenoviruses improves transgene persistance and decreases inflammatory response in mouse liver. Proc Natl Acad Sci USA 1994;91:6196–200.[Abstract/Free Full Text]
  7. Craighead JE, Akley NJ, Gould LB, Libbus BG. Characteristics of tumors and tumor cells cultured from experimental asbestos-induced mesotheliomas in rats. Am J Pathol 1987;129:448–62.[Abstract]
  8. Englehardt JF, Yang Y, Stratford-Perricaudet LD, et al. Direct gene transfer of human CFTR into human bronchial epithelia of xenographs with E1-deleted adenoviruses. Nature Genet 1993;3:27–34.
  9. Linden CJ, Johansson L. Progressive growth of a human pleural mesothelioma xenografted to athymic rats and mice. Br J Cancer 1988;58:614–8.[Medline]
  10. Prewitt TW, Lubensky IA, Pogrebniak H, Pass HI. Orthotopic implantation of mesothelioma in the pneumonectomized immune-deficient rat: a model for innovative therapies. Int J Cancer 1993;55:877–80.[Medline]
  11. Moolten FL. Drug sensitivity (``suicide'') genes for selective cancer therapy. Cancer Gene Ther 1994;1:279–87.[Medline]

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