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Ann Thorac Surg 1997;64:216-219
© 1997 The Society of Thoracic Surgeons

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Original Articles: General Thoracic

Establishment of an Experimental Intrapulmonary Tumor Nodule Model

Thoracic Oncology Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York

Accepted for publication January 27, 1997.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Background. A pulmonary tumor model is necessary to study the biology and therapy of lung cancer. Methods to establish a solitary intrapulmonary nodule are not well defined. Two methods for solitary intrapulmonary tumor nodule development in the Fischer rat are described.

Methods. Methylcholanthrene-induced sarcoma cell suspensions were introduced into lung parenchyma of Fischer rats via limited thoracotomy and lung puncture, or instilled into a distal airway after tracheal puncture and catheterization. Intrapulmonary tumor location, implantation mortality, procedure length, and animal survival were recorded.

Results. Single pulmonary nodules developed at the implanted position in 100% (n = 320) and 95% (62/65) of animals after direct injection into the pulmonary parenchyma or via tracheal puncture and instillation. Operative mortality was 2% and 5% via lung or tracheal implantation, respectively. Less than 5 minutes was required for each implantation. Mean survival time was 24 ± 2 and 26 ± 6 days after lung or tracheal implantation in animals allowed to survive until tumor-induced death.

Conclusions. These easily performed, reproducible methods of establishing solitary intrapulmonary tumors are useful tools for lung cancer research.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Lung neoplasia is the leading cause of cancer mortality in the United States. Therapeutic advances toward lung cancer have been slow [1], prompting the investigation of new therapies. The lack of optimal animal models impedes investigation [2]. Existing experimental lung tumor models have various problems. Shortfalls include the small size and immunosuppression of animals, long tumor development time, unreliable tumor development, and imprecise intrapulmonary development of tumor [2–5].

We describe two readily performed methods for intrapulmonary tumor development that yield reproducible, accurately placed, solitary pulmonary nodules with low implantation mortality. These methods are easily learned. The open lung puncture method requires intubation and ventilation, whereas the tracheal puncture method necessitates only sedation and anesthesia. A solitary pulmonary nodule model approximates clinical lung cancer and therefore provides a useful tool for lung cancer research.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Animals and Tumor Cell Line
All experiments were performed on Fischer 344 rats weighing 200 to 250 g (Charles River, Kingston, NY). Animals were treated in accordance with the Animal Welfare Act and the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985). All animals had free access to standard laboratory rat food (Ralston-Purina Rat Chow, St. Louis, MO) and water ad libitum. Housing was temperature controlled with a 12-hour light and dark cycle.

The tumor cell line is a methylcholanthrene-induced rat sarcoma (MCA) that has been serially passed subcutaneously over many years in our laboratory. It has been extensively characterized and shown to be locally invasive and to rarely metastasize [6, 7].

Cell Suspension Preparation
Implanted tumor cells were derived after collagenase digestion of tumor tissue or cell culture. To create a cell suspension from tumor tissue, we excised approximately 5 g of fresh tumor from the flanks of passage animals and minced it into 1-mm³pieces. One percent collagenase D (Boehringer Mannheim, Indianapolis, IN) in phosphate-buffered saline solution was added to the tumor. The solution was heated in a shaking water bath for 40 minutes at 37°C. The suspension was then filtered through a 60-µm metal mesh and centrifuged at 1,000 gfor 5 minutes. The supernatant was removed and the pellet was washed two times with phosphate-buffered saline solution. The washed pellet was resuspended in cold Roswell Park Memorial Institute medium + 5% fetal bovine serum to a concentration of 3 x 10⁷ cells/mL. Tumor cell suspension viability was 100% as assessed by the trypan blue exclusion test.

Tumor Implantation by Thoracotomy and Lung Puncture
Animals were anesthetized with pentobarbital, 50 mg/kg, intraperitoneally. Under direct visualization the animals were intubated with a 16-gauge intravenous catheter placed over a guidewire and then placed on a volume ventilator (Rodent Ventilator model 683; Harvard Apparatus, South Natick, MA) [8]. Ventilation was maintained at a tidal volume of 10 mL/kg, with 100% O₂at a rate of 75 breaths/min. The left chest was shaved, prepared with a 10% povidone-iodine solution, and entered through the seventh intercostal space. The skin incision was 1 cm in length. The unilobar left lung was retracted from the thoracic cavity. A 27-gauge needle attached to a 0.5-mL insulin syringe was inserted into the lung to a depth of 0.5 cm. Tumor cells (1.5 x 10⁶) in a 50 µL volume of Roswell Park Memorial Institute medium were injected into the lung parenchyma (Fig 1). A cotton-tipped applicator was pressed on the site of puncture as the needle was withdrawn to prevent the tumor suspension from leaking out of the lung as well as to stop any bleeding caused by the puncture. To facilitate lung reexpansion a 16-gauge catheter connected to a 5-mL syringe was introduced into the left chest cavity to act as a chest tube system. The thoracotomy incision was closed. Negative pressure in the chest was created by traction on the syringe piston. When the animals were breathing spontaneously, their chest and endotracheal tubes were removed and animals were returned to their cages.

View larger version (30K): [in this window] [in a new window]   Fig 1. . Tumor implantation via lung puncture. A small thoracotomy was made and the lower lobe of the left lung was retracted from the thoracic cavity. Tumor cells (1.5 x 106)in 50 µL volume were introduced into the lung parenchyma. A cotton-tipped applicator was then pressed on the site of puncture to prevent the cell suspension leaking from the lung.

View larger version (26K): [in this window] [in a new window]   Fig 2. . Tumor implantation via tracheal puncture. The tissues surrounding the trachea were separated and the trachea exposed. A 16-gauge Angiocath catheter was inserted into the trachea and through this a PE-50 catheter was inserted. Tumor cells (1.5 x 106)in 50 µL volume were introduced into the distal bronchus through the PE-50 catheter.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Since 1993, 320 F344 rats have undergone tumor implantation by the open lung puncture method and 65 via the tracheal puncture and instillation method. Less than 5 minutes of implantation time was required for each animal. Surgical mortality was 2% in the open implantation group and 5% in the tracheal puncture group. Postprocedure grooming habits were unaltered. Tumors developed at the site of direct injection in 100% (320/320) of animals undergoing open lung injection. Tumors developed in 95% (62/65) of animals undergoing tumor suspension placement via tracheal puncture, catheterization, and instillation. The tumors of these animals developed in a distal bronchus. Animals had tumor implantation for varied experiments requiring solitary pulmonary nodule development.

Of the animals in which tumors were implanted for this descriptive article, mean survival time was 24 days (range, 20 to 32 days) after open lung implantation and 26 days (range, 9 to 40 days) after tracheal instillation (Fig 3). Animals died of respiratory failure due to overwhelming pulmonary tumor burden. Body weights were not different between the two groups in the week after tumor implantation (Fig 4). Tumor sizes were approximately 3.8 x 1.8 and 5.4 x 2.5 mm via lung implantation and 1.9 x 1.3 and 2.5 x 1.6 mm via trachea implantation on day 7 or day 10 after tumor implantation. Tumor sizes were measured on the samples fixed in 10% buffered formalin acetate (Fig 5).

View larger version (13K): [in this window] [in a new window]   Fig 3. . Animal survival curves after tumor implantation. All animals died of tumor growth. Mean survival time was 24 days in the lung implantation group (squares), 26 days in the tracheal implantation group (circles) (Student's t test, p = not significant; n = 10/group).

View larger version (14K): [in this window] [in a new window]   Fig 4. . Daily weights of animals after tumor implantation. No significant difference in body weight was seen between the lung implantation (squares) and tracheal implantation (circles) groups (p = not significant, t test).

View larger version (79K): [in this window] [in a new window]   Fig 5. . Pulmonary histology on day 7 (A) and 10 (B) after lung implantation and on day 7 (C) and 10 (D) after tracheal instillation. Arrows indicate the location of the solitary pulmonary nodule.

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment References

Potential difficulties with the open lung method are the requirement of endotracheal intubation and the development of thoracic adhesions possibly complicating experiments requiring repeat thoracotomy. We found intubation easy to master, and adhesions encountered at reoperation could be taken down with gentle traction on a cotton-tipped swab.

Intubation and thoracotomy are not required for the tracheal puncture and instillation method. Initial difficulty with this method involved the animals' inability to tolerate a large-volume tumor suspension instillation. Animals tolerated the 50-µL volume without difficulty.

The ability to dictate the intrapulmonary placement of tumor can be used to great advantage by the investigator. Localization is specific as to position in a lobe. Studies in our laboratory require mobilization of the hilar pulmonary vessels, and this region can be consistently spared from tumor involvement with both the pulmonary injection and tracheal instillation methods. Tumors can be visualized by the naked eye in approximately 5 days. Unexpected growth into the mediastinum and chest wall was not seen.

Previously we attempted to create solitary pulmonary nodules via percutaneous tumor cell suspension injection. Using percutaneous injection we encountered significant spillage of tumor suspension into the thoracic cavity, which led to extrapulmonary tumor growth. As our laboratory's studies necessitated tumors to be isolated to the lung, we developed the methods described in this article.

Many histologic tumor types can be used with our methods. Tumor development times would vary by line, but personal experience reveals our methods are not limited to MCA sarcoma. It is probable that other species of rat would tolerate either method of tumor development. We hope these methods will facilitate the investigation into the biologic basis for and the treatment of lung cancer.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Address reprint requests to Dr Burt, Department of Surgery, Thoracic Service, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 

1. Travis WD, Travis LB, Devesa SS. Lung cancer. Cancer1995;75:191–202.[Medline]
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3. Witschi HP, Hakkinen PL, Kehrer JP. Modification of lung tumor development in A/L mice. Toxicology1981;21:37–45.[Medline]
4. Reznik-Schuller HM, Gregg M. Pathogenesis of lung tumors induced by N-nitrosoheptamethyleneimine in F344 rats.Virchows Arch Pathol Anat1981;333:333–41.
5. Howard RB, Chu H, Zeligman BE, et al. Irradiated nude rat model for orthotopic human lung cancers. Cancer Res1991;51:3274–80.[Abstract/Free Full Text]
6. Burt M, Lowry SF, Gorschboth CM, Brennan MF. Metabolic alterations in a noncachectic animal tumor system. Cancer1981;47:2138–46.[Medline]
7. Nagashima A, Yasumoto K, Nakahashi H, Furukawa T, Inokuchi K, Nomoto K. Establishment and characterization of high and low-metastatic clones derived from a methylcholanthrene-induced rat fibrosarcoma. Cancer Res1986;46:4420–4.[Abstract/Free Full Text]
8. Weksler B, Ng B, Lenert J, Burt M. A simplified method for endotracheal intubation in the rat. J Appl Physiol1994;76:1823–5.[Abstract/Free Full Text]

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This Article Abstract 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 Author home page(s): Michael E. Burt Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, H.-Y. Articles by Burt, M. E. Search for Related Content PubMed PubMed Citation Articles by Wang, H.-Y. Articles by Burt, M. E.