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Ann Thorac Surg 1995;59:205-208
© 1995 The Society of Thoracic Surgeons

Isolated Lung Perfusion With FUDR Is an Effective Treatment for Colorectal Adenocarcinoma Lung Metastases in Rats

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

Accepted for publication August 29, 1994.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Currently, the only treatment capable of significantly prolonging survival in patients with isolated pulmonary metastases from colorectal adenocarcinoma is complete resection. Systemic chemotherapy has been shown to provide little benefit. We evaluated the efficacy of high-dose, organ-specific 2`-deoxy-5-fluorouridine (FUDR) using a model of isolated single-lung perfusion (ILP) in the rat. On day 0, 28 BDIX rats were inoculated intravenously with 10⁶ viable Sp-5 colorectal adenocarcinoma cells. On day 10 after-tumor inoculation, animals were randomized into five treatment groups. Group I received a continuous intravenous infusion of FUDR (1 mg • kg^-1 • d^-1) for 7 days administered by an osmotic minipump. Group II underwent isolated left lung perfusion with a buffered Hespan solution, groups III to V underwent ILP with 3.5, 7, and 14 mg of FUDR per milliliter of the buffered Hespan solution, respectively. Animals undergoing ILP were anesthetized with pentobarbital, intubated, and ventilated, and then underwent left thoracotomy with cannulation of the pulmonary artery; the pulmonary artery and vein were clamped proximally. Groups II to V were perfused for 20 minutes at a rate of 1 mL/min, followed by a 5-minute washout with FUDR-free buffered Hespan solution. On day 26 after tumor inoculation, the animals in all groups were sacrificed and their lungs were stained and counted. Animals that underwent ILP with 14 mg of FUDR per milliliter of the buffered Hespan solution showed a significant decrease in the number of tumor nodules on the treated side versus the number on the untreated side (455.2 ± 87.3 versus 11 ± 6.4; p < 0.0001). Although the intravenous FUDR–treated animals exhibited a decreased number of pulmonary nodules compared with the number in controls, the number of left lung pulmonary nodules was significantly greater than that in the ILP treated animals (216 ± 93 versus 11 ± 6; p < 0.05). We conclude that isolated lung perfusion with high-dose FUDR is effective in the treatment of colorectal adenocarcinoma pulmonary metastases resistant to intravenous therapy in a rat model.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Colorectal adenocarcinoma remains a serious health problem, with 152,000 new cases of and 57,000 deaths resulting from colorectal carcinoma reported for 1993 [1]. Death is due primarily to metastatic disease in the liver and lung. To date, no effective chemotherapy has been available for the treatment of metastatic colorectal cancer. Systemic chemotherapy is often limited by the development of host toxicity [2]. A model of isolated lung perfusion (ILP) in the rat has been developed in our laboratory for the study of the treatment of pulmonary metastases. Isolated lung perfusion is more effective in increasing lung tissue doxorubicin levels in rats than is systemic intravenous administration; at the same time, host toxicity is minimized [3] and experimental pulmonary sarcoma metastases are eradicated [4] in the same rat model. This study was designed to compare the efficacy of ILP with 2`-deoxy-5-fluorouridine (FUDR) with the efficacy of the standard systemic administration in the treatment of colorectal adenocarcinoma lung metastases in an experimental rat model.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Twenty-eight female BDIX rats (Harlan Sprague-Dawley, Frederick, MD) weighing 180 to 225 g were used in this study. Animals were treated in accordance with the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1985). Experiments were approved by the Institutional Animal Care and Use Committee, Memorial Sloan-Kettering Cancer Center. Animals were kept in a temperature-controlled room with a 12-hour light-dark cycle. They were allowed access to standard laboratory rat chow (Purina Rat Chow, St. Louis, MO) and water ad libitum.

Tumor Model
The parent cell line, K12/TRb, is a rat colon adenocarcinoma induced by dimethylhydrazine that produces tumor in syngeneic BDIX rats. This cell line was originally established and cloned by Martin and associates [5] and has been maintained as a monolayer culture in Dulbecco's modified Eagle medium supplemented by 10% fetal calf serum and gentamycin (40 µg/mL) at 37°C in humidified 5% carbon dioxide and 95% air. The pulmonary metastatic subclone, Sp-5, was established and used in this study. The cells were prepared for injection by dispersal with 0.125% trypsin in 0.125% EDTA (ethylenediaminetetraacetic acid) after prior exposure for 5 minutes to 0.25% EDTA solution. The number of cells was counted in a Coulter counter, and cell viability was determined with a trypan blue exclusion method using a hemocytometer.

Study Design
On day 0, animals were inoculated with 10⁶ viable tumor cells through a right external jugular cutdown. Pulmonary metastases can be reproducibly induced by an intravenous injection of a single-cell suspension. On day 10 after tumor inoculation, animals were randomized into five groups. Group I (n = 5) received an intravenous infusion of FUDR (1 mg • kg^-1 • d^-1) for 7 days. This dosage has been shown to be well tolerated [6] and corresponds to a human dosage of 0.17 mg • kg^-1 • d^-1 [7]. Group II (the control group, n = 4) underwent isolated left lung perfusion with a buffered Hespan solution (BH); group III (n = 5) received ILP with 3.5 mg of FUDR per milliliter of BH; group IV (n = 7) received ILP with 7 mg of FUDR per milliliter of BH; and group V (n = 7) received ILP with 14 mg of FUDR per milliliter of BH. Groups II to V were perfused for 20 minutes at a rate of 1 mL/min, followed by a 5-minute washout with FUDR-free BH. On day 26 after tumor inoculation, the animals in all groups were sacrificed and their lungs were stained with India ink to identify metastases [8]. Normal lung parenchyma stains black, whereas tumor nodules remain white.

Isolated Left Lung Perfusion
Isolated left lung perfusion was performed as previously described [3]. Briefly, animals were anesthetized with 50 mg/kg of pentobarbital delivered intraperitoneally and were intubated orotracheally. Animals were ventilated with 100% oxygen at 70 strokes per minute with a tidal volume of 10 mL/kg. Anesthesia was supplemented with 0.5% halothane as needed. A left thoracotomy was performed through the fourth intercostal space, and the pulmonary artery and vein were dissected free. Using an operating microscope (x16 magnification; OpMi-1; Karl Zeiss, Thornweed, NY), a PE-10 catheter (Becton Dickinson, Parsippany, NJ) was placed in the pulmonary artery. The pulmonary artery and vein were clamped proximally, preventing the perfusate from leaking into the systemic circulation. The left lung was then perfused for 20 minutes at a rate of 1 mL/min, followed by a 5-minute drug-free washout with BH, and the pulmonary vein effluent was collected through a venotomy. At the end of the perfusion, the pulmonary arteriotomy and venotomy were repaired with 9-0 nylon suture and pulmonary circulation was restored. A 16-gauge catheter was introduced through a separate puncture wound in the left chest cavity and connected to a 2-cm water seal pleural trap and suction device. The chest was then closed in three layers. The endotracheal and chest tubes were removed when the animal was breathing spontaneously.

Intravenous Administration of 2`-Deoxy-5-fluorouridine
The evening before placement, pumps were loaded with FUDR, heparin (500 units), and a 0.9% sodium chloride solution, and were connected to PE-50 polyethylene catheters (inner diameter, 0.58 mm; outer diameter, 0.965 mm; Becton Dickinson, Parsippany, NJ). The pumps were then primed overnight by placing them in a 0.9% saline solution bath and incubating them at 37°C. On day 10, the animals were anesthetized with pentobarbital (50 mg/kg intraperitoneally). Five hundred units of aqueous penicillin were administered subcutaneously. Pumps were then placed in the subcutaneous tissue of the anterior abdominal wall, and the catheter was brought through a subcutaneous tunnel to a right neck incision, where it was inserted into the right external jugular vein. The incisions were irrigated with 500 units of aqueous penicillin and closed with clips (Becton Dickinson). On day 17, the pumps were removed and the reservoirs were aspirated to ensure that the drug had been delivered.

Statistical Analysis
Data are presented as the mean ± the standard deviation. Analysis was performed using analysis of variance and, when significance was found, individual groups were compared using the Mann-Whitney U test. Significance is defined as a p value of less than 0.05.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
The changes in the daily weights of the animals during the study period are shown in Figure 1. After undergoing treatment on day 10, all animals in all groups lost weight for 2 to 3 days and then recovered. On day 26, the body weight of the group V animals was significantly higher than that for all the other groups (p < 0.01).

View larger version (22K): [in this window] [in a new window]   Fig 1. . Daily weights during study period: On day 0, all groups were inoculated with 106 viable Sp-5 colorectal adenocarcinoma cells. On day 10, group I received a continuous intravenous infusion of 2`-deoxy-5-fluorouridine for 7 days via an osmotic pump. Groups III to V underwent isolated lung perfusion with 2`-deoxy-5-fluorouridine. Group II served as the control group and underwent isolated lung perfusion with a buffered Hespan solution.

View larger version (105K): [in this window] [in a new window]   Fig 2. . Posterior view of the lungs of four rats. Shown from left to right are normal control lung, lung after left isolated lung perfusion with buffered Hespan solution, lung after the continuous intravenous infusion of 2`-deoxy-5-fluorouridine (FUDR), and lung after left isolated perfusion with 14 mg of FUDR per milliliter of the buffered Hespan solution. In the last group of animals, as seen here, the left lung had significantly fewer tumor nodules, whereas the right lung showed massive tumor infiltration. The intravenous administration of FUDR produced a moderate tumor response.

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment References

Systemic chemotherapy has offered little benefit in terms of prolonging survival in patients with metastatic colorectal carcinoma, even in those with radiographically undetectable disease [17]. Overall response rates of approximately 20% with a median survival of only 8 to 10 months can be expected [2]. Systemic chemotherapy in doses high enough to achieve a possible improvement in survival is limited by the development of host toxicity [18]. Fluorinated pyrimidines, FUDR and 5-fluorouracil, are among the most active agents and the most used agents for the treatment of colorectal adenocarcinoma. These drugs, however, often precipitate myelosuppression, gastrointestinal mucositis, and diarrhea, which can be dose limiting. In human beings, there appears to be a definite dose response, with higher doses of drug leading to higher response rates [18]. In our laboratory, we developed a model of isolated single-lung perfusion in the rat to allow us to study the locoregional delivery of high-dose chemotherapy that otherwise would be too toxic for systemic delivery. We have shown that there is a significant elevation in the doxorubicin level in the lung using this technique, compared with the level brought about by systemic intravenous administration, while minimizing the cardiac uptake of doxorubicin [3]. In addition, we were able to eradicate experimental pulmonary sarcoma metastases with high-dose doxorubicin in 90% of the perfused lungs [4]. Recently, we demonstrated ILP with tumor necrosis factor is safe and possesses significant antineoplastic activity in the same rat model.

Given the initial success of ILP with doxorubicin and tumor necrosis factor in our sarcoma model, we believed this technique could ultimately prove useful in the therapy for colorectal carcinoma metastatic to the lungs. We have shown that lungs perfused with high doses of FUDR have significantly higher lung tissue levels of the agent than those achieved with intravenous administration [19]. Animals that underwent ILP with FUDR showed a 15-fold increase in the total 5-fluorouracil and FUDR lung tissue levels than did those treated with systemic administration. Histologic analysis of the perfused lung revealed only mild to moderate pleural thickening and no severe lung damage. Additionally, there are pharmacokinetic advantages to ILP with FUDR. Unlike liver parenchyma, normal lung tissue extracts FUDR inefficiently and is not able to detoxify it [20]. Therefore, the amount of perfused FUDR tumor cells would be exposed to increases and the tumor is more likely to extract more FUDR. Theoretically, this makes ILP delivery of high-dose FUDR more likely to be effective.

A significant improvement in response and local control has been demonstrated in patients who undergo locoregional perfusion with FUDR for the control of colorectal adenocarcinoma hepatic metastases [21]. However, Kemeny and associates [21] did not demonstrate a survival benefit; the cause of death was often attributed to progressive pulmonary disease. In the present study, we demonstrated that isolated single-lung perfusion with FUDR (14 mg per milliliter of BH) is effective in the treatment of colorectal adenocarcinoma pulmonary metastases resistant to systemic therapy. Lungs perfused with high-dose FUDR had significantly fewer tumor nodules than did the untreated lungs, and significantly fewer than the group receiving intravenous FUDR. The role of ILP with antineoplastic agents as an adjunct to surgical resection for the management of colorectal adenocarcinoma pulmonary metastases warrants further studies.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment References

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

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 

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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 Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ng, B. Articles by Burt, M. E. Search for Related Content PubMed PubMed Citation Articles by Ng, B. Articles by Burt, M. E.