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Ann Thorac Surg 1998;66:1719-1725
© 1998 The Society of Thoracic Surgeons
a Laboratory for Experimental Surgery, University of Antwerp, Antwerp, Belgium
Address reprint requests to Dr Hendriks, Division of Thoracic and Vascular Surgery, Department of Surgery, University Hospital Antwerp (UZA), Wilrijkstraat 10, B-2650 Edegem, Belgium
e-mail: (j.hendriks{at}planetinternet.be)
Presented at the Thirty-fourth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 26–28, 1998.
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for pulmonary metastatic adenocarcinoma in the WAG/Rij rat was studied.
Methods. Survival was determined for melphalan, human tumor necrosis-. Lung, pulmonary effluent, and serum melphalan were analyzed by chromatography after isolated lung perfusion or intravenous injection. On day 0, rats were injected with 2.0 x 106 CC531S cells intravenously. On day 7, rats underwent sham thoracotomy, received melphalan intravenously, or underwent isolated left lung perfusion with saline, melphalan, tumor necrosis factor, and a combination of the latter two. On day 14, tumor nodules were counted.
Results. For the doses of 400 µg tumor necrosis factor, 1,000 µg tumor necrosis factor, or both melphalan and tumor necrosis factor (2 mg + 200 µg), survival rates after contralateral pneumonectomy were 33%, 17%, and 80%, respectively. Survival in all other groups was 100%. Left lung melphalan level was significantly higher after isolated lung perfusion compared to intravenous administration. Significantly fewer left lung nodules were found for 0.5 mg isolated lung perfusion with melphalan (28 ± 17) compared to isolated administration (200 ± 0) (p = 0.001), and for 1.0 mg intravenous lung perfusion with melphalan (16 ± 10) compared to controls (171 ± 65) (p = 0.00047). Tumor necrosis factor showed no significant effect.
Conclusions. Isolated lung perfusion with melphalan is an effective treatment for pulmonary metastases from adenocarcinoma in the rat.
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Material and methods |
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Tumor
The CC531S tumor cell line was used for all tumor experiments. This cell line was derived from a chemically-induced adenocarcinoma of the colon of a WAG rat [6]. CC531S was cultured in complete medium and maintained by serial passage. Complete medium consisted of RPMI-1640 medium, supplemented with 10% heat-activated fetal bovine serum (FBS) (both from Life Technologies, Merelbeke, Belgium), 2 mmol/L glutamine, 50 µg/mL streptomycin, and 50 U/mL penicillin.
Induction of lung metastases
CC531S cells were prepared for injection by dispersal with a solution of 0.25% ethylinediamine tetraacetic acid (EDTA) and 0.05% trypsin in Hank’s balanced salt solution (HBSS). Cell viability was determined with a tryptan blue exclusion method using a hemocytometer. To investigate the appropriate amount of cells needed for tumor induction, 36 rats were injected with 500,000, 1.0 x 106, and 2.5 x 106 CC531S single cells in 1 mL RPMI-1640 in the left femoral vein. Half of the rats were killed on day 12 and half on day 19. The number of pulmonary metastases were visualized by the method of Wexler [7]. Briefly, the trachea and the lungs were removed en bloc and the lungs were inflated with 10 mL of India ink. The lungs were submerged in sterile water for 5 minutes and then for 24 hours in Fekete’s solution. This method causes normal lung parenchyma to be stained black and metastatic nodules to be stained white. The volume of tumor in the right and left lung was determined with point grids. An average of 20 fields was measured for every case using 4 µm hematoxylin-stained slides and an image analysis system for stereologic measurements (Fenestra/Stereology, Kinetic Imaging Ltd, London, UK). Results were expressed as the percentage of lung tissue replaced by tumor. To know the optimal moment for isolated perfusion, an additional 30 rats were injected with 1.0 x 106 and 2.5 x 106 cells and killed after 3, 6, 9, 12, and 15 days, respectively. Lungs were stained with hematoxylin and eosin for pathologic examination. Metastases were scored as present or absent without further quantification.
Isolated lung perfusion
Isolated left lung perfusion was performed according to the technique described by Wang and coworkers of Memorial Sloan-Kettering Cancer Center, New York [8], which was modified in our laboratory. Briefly, anesthesia was induced with isoflurane (Forene, Abbott Louvain-la-Neuve, Belgium) in a mixture of nitrous oxide (N2O) and oxygen (O2). Isoflurane was administered in a concentration of 4%; N2O:O2 ratio was 3:1. After 5 minutes, rats were intubated by translaryngeal illumination with a 16-gauge Insyte-W catheter. Once connected to the ventilator, the N2O/O2-ratio was set to 1:1 (0.5 L/min) and isoflurane titrated between 0.5% and 1.5% according to muscle relaxation, heart rate, and pupil size. Ventilation was accomplished with a volume-controlled ventilator (Rodent Ventilator model 683; Harvard Apparatus, South Natick, MA) at a rate of 75 strokes/min and a tidal volume of 10 mL/kg. Instead of posterior luxation, retractor and lung were positioned anteriorly and the hilus was dissected free from the posterior side. After clamping the pulmonary artery and vein with curved microclips, a 16-G Angiocath was placed through the chest wall. The PE-10 perfusion catheter (Becton Dickinson, Bornem, Belgium) was introduced into the chest through the Angiocath and secured by a 4/0 silk tie after insertion into the pulmonary artery. Perfusate was delivered through this catheter. A pulmonary venotomy was performed and the effluent collected by a suction catheter placed in proximity to the venotomy. At the completion of the perfusion, the catheters were removed and the arteriotomy was repaired with a single simple suture (10/0 Ethilon, Ethicon, Dilbeek, Belgium). The microvascular clamps were removed and the left lung was returned to its anatomic position. Pressure was applied with gauze placed over the lung to control the bleeding from the venotomy. Through a separate puncture wound, a 16-gauge catheter connected to a 10-mL syringe was introduced into the left side of the chest cavity to facilitate lung reexpansion. The thoracotomy incision was closed in three layers with 4-0 vicryl and the animal was supplied with room air only. When the animals were alert and breathing spontaneously, the chest and endotracheal tubes were removed.
In all experiments, animals were perfused on the left side for 25 minutes at a rate of 0.5 mL/min. Both for the toxicity and efficacy studies rats underwent a 5-minute washout with buffered hetastarch (BHE) [9] at 0.5 mL/minute, whereas for the pharmacokinetics study rats underwent a 15-minute washout with BHE. Melphalan (Alkeran, 50 mg/vial, Wellcome, Waterloo, Belgium) perfusate solutions were prepared by reconstituting lyophilized powder in the supplied diluent and performing appropriate dilutions with BHE. Human tumor necrosis factor- (TNF-) was kindly provided by Boehringer (Ingelheim, Germany) with a specific activity of 5 x 107 U/mg as determined in the murine L-M cell assay. Endotoxin levels were less than 1.25 EU/mg protein. Human tumor necrosis factor- was delivered in 1.0-mL vials in a concentration of 2.0 mg/mL.
Experiment 1: toxicity study
On day 0, 44 male WAG/Rij rats (240 to 356 g) were randomized into eight groups. Group 1 (n = 6) had left ILuP with 1,000 µg of TNF, group 2 (n = 6) with 400 µg of TNF, group 3 (n = 4) with 200 µg of TNF, and group 4 (n = 4) with 100 µg of TNF. Group 5 (n = 5) had ILuP with 2 mg of MN and group 6 (n = 5) with 2 mg MN + 200 µg TNF. Group 7 (n = 10) had no ILuP (sham thoracotomy), whereas group 8 (n = 4) had ILuP with BHE. Groups 7 and 8 served as controls for right pneumonectomy and ILuP with BHE, respectively. All animals underwent right contralateral pneumonectomy (CPn) on day 21 to assess left lung function. Animals were killed 5 days later, and the lungs retrieved and stained with hematoxylin and eosin for pathologic examination.
Experiment 2: pharmacokinetics
Ten male WAG/Rij rats (240 to 260 g) were randomized into four groups. Group 1 (n = 2) had left ILuP with 0.5 mg of MN, whereas group 2 (n = 2) had ILuP with 1.0 mg of MN. Groups 3 (n = 3) and 4 (n = 3) received 0.5 mg and 1 mg of MN via femoral venous injection. The ILuP groups had a PE-90 catheter inserted into the pulmonary vein for collection of pulmonary effluent samples. The pulmonary effluent samples were collected at 5-minute intervals throughout the perfusion. At the completion of a 25-minute MN perfusion and 15-minute BHE washout, blood samples were collected by cardiac puncture and both lungs were removed and frozen at -70°C for later MN analysis. Animals receiving iv MN underwent laparatomy 30 minutes after injection and blood samples were drawn from the abdominal aorta. Subsequently, the animals were killed and both lungs were removed for measurement of MN tissue levels.
Melphalan processing and measurement
Melphalan in serum and lung tissue was measured by gas chromatography-mass spectrometry based on the method described by De Boeck and associates [10]. P-[Bis(2-chloroethyl)amino]-phenylacetic acid methyl ester was used as an internal standard. Samples were extracted over trifunctional C18 silica columns.
Experiment 3: efficacy study 1
Thirty-five male WAG/Rij rats (216 to 278 g) were randomized into six groups. On day 0, all groups underwent an iv injection of 2.5 x 106 CC531S adenocarcinoma cells via the left femoral vein. On day 7 group 1 (n = 6) underwent a left thoracotomy without perfusion, group 2 (n = 5) had left ILuP with BHE, group 3 (n = 6) received 1 mg of MN intravenously, group 4 (n = 6) underwent left ILuP with 100 µg of TNF, group 5 (n = 6) underwent left ILuP with 1 mg of MN and group 6 (n = 6) underwent left ILuP with 100 µg TNF + 1 mg MN. ILuP was given for 25 minutes in groups 2, 4, 5, and 6.
Efficacy study 2
Twenty-three male WAG/Rij rats underwent a 25-minute perfusion. Group 7 (n = 5) underwent left ILuP with 300 µg TNF and group 8 (n = 5) with 0.5 mg MN, whereas group 9 (n = 6) underwent left ILuP with the combination of 300 µg TNF and 0.5 mg MN. Group 10 (n = 7) received 0.5 mg MN intravenously. In this second efficacy study, a lung biopsy was taken at day 7 before perfusion to confirm growth of pulmonary metastases. Hemoclips were placed at the edge of the lung in such a way that a wedge resection could be performed.
On day 14, rats of both efficacy studies were sacrificed and their lungs were stained with India ink according to the method of Wexler for identification of the number of pulmonary carcinoma nodules [7].
Statistical analysis
All data are presented as mean ± standard deviation (SD). Pharmacokinetic data were analyzed by analysis of variance (ANOVA). Efficacy data within groups between left and right lung were analyzed with a Mann-Whitney U-test. Multiple comparisons were tested by a Kruskal-Wallis test. Significance was defined as p < 0.05.
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Experiment 2: pharmacokinetics
Lung MN levels were measured for all groups receiving MN. Left lung MN levels were significantly higher in the MN ILuP group than in the iv treatment groups. No significant difference in left lung MN levels could be detected between animals perfused with 0.5 mg MN and 1.0 mg MN (Table 2). Melphalan levels in the pulmonary venous effluent in the ILP group were high after 5 minutes and were almost constant throughout the 25 minutes of perfusion. At the end of the washout, MN levels of the venous effluent dropped to zero (Fig 1). Serum MN levels were measured for the same groups. No MN was detected in the serum of animals that received ILuP. The serum MN level of the animals receiving 0.5 mg were significantly lower than the group receiving 1.0 mg MN intravenously (Table 2).
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Discussion |
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Colorectal adenocarcinoma remains a serious health problem, with 133,500 new cases and 54,900 deaths reported for 1996 in the United States. It accounts for a significant number of isolated lung metastases, even though the incidence of pulmonary metastases is only 2% to 4% [1].
In an attempt to address the clinical problem of metastatic pulmonary carcinoma, our laboratory developed a model of pulmonary metastases from colorectal carcinoma. ILuP in the WAG/Rij rat under pentobarbital anesthesia with the catheterization technique according to Wang and colleagues [8] resulted in a high mortality rate, and total operative time was unpredictable. Therefore, techniques of intubation, anesthesia, and arterial cannulation were changed. This enabled us to perform ILuP in the WAG/Rij rat without mortality.
The present study evaluated MN and TNF as anticarcinoma agents delivered via ILuP. Experimental studies have revealed an antitumor response of isolated liver perfusion with MN against liver metastases. Addition of TNF to MN was explored based on the excellent synergistic effects of MN and TNF with isolated limb perfusion for the treatment of melanoma [5].
The effectiveness of MN against colorectal cancer via isolated liver perfusion [3] and the fact that isolated lung perfusion with MN in the treatment of sarcomatous pulmonary metastases in the rat could be performed safely [15], encouraged us to explore MN ILuP for the treatment of pulmonary colorectal carcinoma metastases. Although ILuP with FUDR proved to be effective in a model of lung metastases from adenocarcinoma [16], isolated liver perfusion revealed superior antitumor effects of MN above FUra [3, 4].
Toxicity studies determined a tolerated dose of MN in this rat ILuP model of 2 mg (8 mg/kg) and a LD50 of TNF after CPn of approximately 400 µg (1,600 µg/kg). Combined doses up to 2 mg MN with 200 µg TNF have been tested and demonstrated an 80% survival after CPn. Because systemic administration of TNF in the clinic is limited by severe toxicity, we did not perform iv dose-effect studies in the rat.
Lung levels of MN in the MN ILuP group were 50 times as high as in the iv group, for both 0.5 and 1.0 mg of MN. The elevated concentration of lung MN in the left perfused lung as compared with the right lung is consistent with results seen in the study of Nawata and colleagues [15]. Intravenous lung perfusion is capable of delivering higher levels of chemotherapeutic agents than does administration, even for the nonlethal systemic doses, whereas systemic exposure is avoided. In our study, no significant elevation of tissue levels of MN could be detected when the dose was doubled from 0.5 mg to 1.0 mg. The concentration curves were nearly identical and the decrease in metastatic nodules similar between the two groups. Although this observation needs to be investigated in a larger number of animals, it may suggest that lower doses of MN can be used. It would not change the toxicity but could be a better dose for combination with other agents.
An antitumor effect against lung metastases from colon adenocarcinoma was evident in the left lungs of animals treated with MN ILuP, as a single therapy or combined with TNF. However, because MN as single therapy yielded excellent results, no enhanced macroscopic antitumor effect of TNF in a dose of 300 µg could be demonstrated. Isolated lung perfusion with TNF alone could not demonstrate any consistent antitumor effect in this rat model during a 25-minute perfusion for a dose of 100 µg whereas increasing the dose to 300 µg resulted in a partial response in only 2 of 6 animals. Regarding the varied response to single TNF, it should be kept in mind that anoxia in the tumor may be the critical determinant for its propensity to respond to TNF [17]. Because the bronchus and bronchial circulation are open during perfusion, no real anoxic conditions were present in this ILuP model. Also, the involvement of the immune system is considered to be important in the TNF tumor response, and invasion of polymorphonuclear cells and stimulation of inflammatory cells may play an important role [18]. The latter is only described in immunogenic tumors, which can explain the lack of antitumor response in our weakly immunogenic CC531S adenocarcinoma pulmonary metastases model. The cytotoxic effect of TNF has also been shown to be dose- and temperature-dependent. Van der Zee and coworkers have reported hyperthermic enhancement of TNF toxicity in WAG/Rij rats [19]. Weksler and coworkers demonstrated TNF to be effective in a sarcoma metastases model in a dose of 400 µg (1,600 µg/kg), whereas lower doses failed to eradicate tumor [20].
In conclusion, ILuP with MN is safe and effective in the treatment of lung metastases from adenocarcinoma in the rat. Antitumor effects with TNF were not consistent, whereas enhancement of MN effects by TNF could not be demonstrated in this adenocarcinoma pulmonary metastases model. Clinical trials evaluating MN ILuP in patients with isolated pulmonary metastases from carcinoma and sarcoma are warranted.
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Acknowledgments |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionAcknowledgmentsReferences |
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References |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionAcknowledgmentsReferences |
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in combination with interferon-gamma and melphalan in isolation perfusion. World J Surg 1992;16:234-240.[Medline]
) in isolated limb perfusion in the rat. Reg Cancer Treat 1994;7:155-159.
) given by continuous i.v. infusion as compared to repeated i.v. injections in a rat liver metastasis model. Int J Cancer 1989;43:905-909.[Medline]
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