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Isolated single-lung perfusion with TNF-α in a rat sarcoma lung metastases model
Isolated Single-Lung Perfusion With TNF-a in a Rat Sarcoma Lung Metastases Model Benny Weksler, MO, David Blumberg, MO, Jeffrey T. Lenert, MO, Bruce Ng, B5, Yuman Fang, MO, and Michael E. Burt, MO, PhD Thoracic Oncology Laboratory, Thoracic Service, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York
We conducted a trial of isolated lung perfusion using tumor necrosis factor (TNF) in an experimental sarcoma lung metastasis model. In an in vitro experiment, methylcholanthrene-induced sarcoma cells were incubated for 48 hours with 42 /-tg/mL of either human or murine TNF. Controls were incubated with Hank's balanced salt solution. In an in vivo experiment, 23 F344 rats were injected with 107 methylcholanthrene-induced sarcoma cells. On day 7, 4 animals were perfused with 210 /-tg of murine TNF, 5 animals were perfused with 420 /-tg of murine TNF, 10 animals underwent isolated lung perfusion with 420 /-tg of human TNF, and 4 animals were injected systemically with 420 ILg of human TNF. Animals were sacrificed on day 14 and the lung nodules counted. The cells incubated with murine TNF exhibited a 21% de-
crease in growth (p = 0.07); cells incubated with human TNF showed a 37% decrease in growth (p < 0.05). Animals perfused with 210 ILglmL of murine TNF and animals treated by systemically administered human TNF showed no tumor response. Animals perfused with 420 ILg/mL of murine TNF had 7.8 ± 14.2 nodules on the left lung and 58.5 ± 66.0 nodules on the right lung (p = 0.07). Animals perfused with 420 /-tglmL of human TNF had 21.7 ± 18.3 nodules on the left lung and 91.7 ± 66.2 nodules on the right lung (p < 0.01). On the basis of these findings, we conclude that isolated lung perfusion with TNF can be done safely in the rat and is effective in decreasing the growth of sarcoma lung metastases.
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patients, the lungs are the sole site of disease recurrence [8]. Therapy for metastatic soft tissue sarcoma to the lungs consists of complete resection, with a 5-year survival of only 25% [9]. We have shown that isolated single-lung perfusion can be performed safely in the rat, with a low operative mortality [10]. Isolated single-lung perfusion with doxorubicin has been shown to be well tolerated with minimal systemic toxicity [11]. In addition, isolated singlelung perfusion with doxorubicin has been found to be effective in eradicating sarcoma lung metastases in the rat
u m or necrosis factor-a (TNF) is a cytokine produced mainly by activated macrophages and monocytes [1], and is thought to be an important mediator of septic shock [2]. Animal experiments have demonstrated the antitumor effects of systemic TNF against human tumor xenografts [3], but human experiments have yielded disappointing findings, with low response rates and a high frequency of systemic toxicity, such as fever, chills, hypotension, and leukopenia [4]. The antitumor effect of TNF involves several mechanisms, including a direct antitumor effect [1], induction of the lymphocyte secretion of antitumor substances [5], and the selective destruction of tumor vasculature [6,7]. Because of the systemic toxicity of TNF, Lienard and colleagues [7] conducted a trial of isolated limb perfusion using high doses of TNF combined with melphalan, 'Y-interferon, and hyperthermia for the treatment of unresectable extremity melanoma and sarcoma. The isolated limb perfusion circuit allowed for the administration of higher doses of TNF with less systemic toxicity. Tumor response was observed in all patients and complete tumor response was seen in 85%. Side effects included hypotension, fever, and chills, but all patients completed therapy. The lungs are the most common site of metastases in patients with extremity soft tissue sarcoma and, in most Presented at the Thirtieth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 31-Feb 2, 1994. Address reprint requests to Dr Burt, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021.
© 1994 by The Society of Thoracic Surgeons
(Ann Thorae Surg 1994;58:328-32)
[12].
Inspired by the excellent results of the isolated limb perfusion study, we conducted a trial of isolated singlelung perfusion with TNF in an experimental model of sarcoma lung metastases in the rat.
Material and Methods Animals (male F344 rats weighing 200 to 250 g; Charles Rivers, Kingston, NY) were treated in accordance with the Animal Welfare Act and the "NIH Guide for the Care and Use of Laboratory Animals" published by the National Institute of Health (NIH publication 85-43, revised 1985). All experiments were approved by the Institutional Animal Care and Use Committee of the Memorial SloanKettering Cancer Center. All rats were allowed access to standard laboratory rat chow (Ralston-Purina Rat Chow, St. Louis, MO) and water ad libitum. Housing was temperature controlled and provided 12-hour light-dark cycles. 0003-4975/94/$7.00
WEKSLER ET AL ISOLA TED LUNG PERFUSION WITH TNF
Ann Thorae Surg 1994;58:328-32
Methylcholanthrene-Induced Sarcoma Model Tumor was harvested fresh from a tumor-bearing animal and a single tumor cell suspension was prepared by first mincing grossly viable tumor and then incubating the minced tumor in Hanks balanced salt solution with 1% collagenase (Worthington Biochemical, Freehold, NJ). This methylcholanthrene (MCA)-induced sarcoma has been serially passaged subcutaneously in our laboratory, and has been extensively characterized [13]. We have found that an intravenous injection of 107 cells results in bilateral massive pulmonary metastases at 14 days in 100% of the animals.
Isolated Single-Lung Perfusion In the rat, the left lung is unilobar with one pulmonary artery and one pulmonary vein; the right lung consists of three to five lobes. Animals underwent isolated left lung perfusion, as described in detail elsewhere [10]. Briefly, animals were anesthetized with 50 mg/kg of pentobarbital administered intraperitoneally. The animal was orotracheally intubated with a 16-gauge intravenous catheter [14]. Animals were ventilated with a volume respirator (rodent ventilator model 683; Harvard Apparatus, South Natick, MA). The left chest was opened through the fourth intercostal space, and the left pulmonary vein and artery were dissected free and clamped proximally with microvascular clamps. The pulmonary artery was cannulated with a PE 50/PE 10 catheter (Clay-Adams, Parsippany, NJ). A venotomy was made and the pulmonary vein effluent was suctioned continuously. The left lung was perfused for 10 minutes with TNF in saline at a rate of 1 mL/min and for 5 minutes with a buffered hetastarch solution. At the end of the perfusion period, the pulmonary vein and artery were repaired with 9-0 microvascular nylon suture (Ethicon, Somerville, NJ). The left chest was closed and the animal was allowed to recover from the anesthesia. When the animal was awake, it was extubated and returned to its cage.
Experimental Design The effect of murine and human TNF on the growth of MCA-induced sarcoma cells was examined during 48 hours of log phase growth to assess the direct effect of TNF on cell growth. Approximately 250,000 viable cells (identified by the trypan blue exclusion method) were plated in six-well tissue culture plates and incubated in RPMI 1640 and 5% fetal calf serum. The effect of murine and human TNF on cell growth was examined sequentially. Tumor necrosis factor was added to four wells at a concentration of 42 /-lg/mL of media, and Hanks balanced salt solution was added to four wells as a control. At 48 hours, cells were trypsinized with phosphate buffered saline, 0.125% trypsin, and 0.125% EDTA (ethylenediaminetetrascetic acid), and counted in a standard Coulter counter (Coulter Electronics, Hialeah, FL). IN VITRO EXPERIMENTS.
IN VIVO EXPERIMENT. Twenty-three rats were injected on day 0 with 107 viable sarcoma cells through the external jugular vein. On day 7, all animals underwent left isolated lung perfusion with TNF. Nine animals were perfused
329
with murine TNF (Genentech, CA), and 10 rats were perfused with human TNF (Genentech): 4 animals with 210 /-lg of murine TNF, 5 animals with 420 /-lg of murine TNF, and 10 animals with 420 /-lg of human TNF. Four animals were injected with 420 /-lg of human TNF through the external jugular vein. On day 14, all animals were sacrificed and the number of pulmonary metastases assessed by the method of Wexler [15]. Briefly, the trachea and the lungs were removed en bloc and the lungs were inflated with 10 mL of india ink. The lungs were submersed 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 number of nodules in the left lung (perfused) and the right lung (unperfused) were counted.
Tumor Necrosis Factor Assay The TNF assay in the perfusate and in the pulmonary vein effluent was performed in all animals perfused with 420 /-lg of TNF using the WEHI cell-killing assay, and the results were expressed in units of cell killing [16].
Data Analysis All data are presented as the mean ± the standard deviation. Analysis was performed by the Wilcoxon matchedpaired test for comparison of the left versus the right lung and the TNF levels in the perfusate versus the pulmonary vein effluent. The Mann-Whitney U test was used to analyze the results of the in vitro experiment.
Results In Vitro Experiment At 48 hours, cells had gone through two doubling periods. Incubation of the MCA-induced sarcoma cells with murine TNF led to a 21% decrease (p = 0.07) in the number of cells at 48 hours (control versus TNF: 1.38 X 106 ± 0.25 X 106 cells versus 1.09 X 106 ± 0.07 X 106 cells). Cells incubated with human TNF showed a 37% reduction (p < 0.05) in the number of cells at 48 hours (control versus TNF: 1.2 X 106 ± 0.19 X 106 cells versus 0.7 X 106 ± 0.17 X 106 cells).
In Vivo Experiment All animals survived the surgical procedure. One animal perfused with 420 /-lg of murine TNF had a tumor in the mediastinum and was excluded from analysis. All other animals were sacrificed on day 14. Animals perfused with 210 /-lg of murine TNF showed no response to left isolated lung perfusion, and both lungs were completely replaced by tumor. Rats perfused with 420 /-lg of murine TNF had 58.5 ± 66.0 nodules on the right (untreated) lung and 7.8 ± 14.2 nodules on the left (perfused) lung (p = 0.07). Animals perfused with human TNF had 91.7 ± 66.2 nodules on the right lung and 21.7 ± 18.3 nodules on the left lung (p < 0.01). Two animals perfused with human TNF had more than 200 nodules on the right lung, and in the analysis, were considered to have 200 nodules on the right lung. One rat injected intravenously with human TNF died 24 hours after injection. The 3 other animals had massive
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WEKSLER ET AL ISOLATED LUNG PERFUSION WITH TNF
Fig 1. In vivo experiments: India ink preparation of lung blocks. Normal lung tissue appears black and metastatic nodules appear white. On the left is a normal lung; on the right is a lung obtained after the systemic administration of 420 /J-g of human tumor necrosis factor; in the middle is a block from an animal obtained after isolated left lung perfusion with 420 /J-g of human tumor necrosis factor. The left (perfused) lung exhibits markedly less nodules than does the right (untreated) lung. On the right, lungs from the animal that received tumor necrosis factor systemically are bilaterally infiltrated with tumor.
replacement of both lungs, with an uncountable number of nodules (Fig 1). The total activity of TNF in animals perfused with 420 /Lg of TNF was 561.5 ::':: 24.8 units of cell killing. The total amount of TNF recovered from the pulmonary vein effluent was 607.6 ::':: 25.5 units of cell killing. This difference did not reach statistical significance (p = 0.07).
Comment Isolated single-lung perfusion in the MCA-induced sarcoma lung metastases model is an extremely versatile technique in the evaluation of antitumor agents. It has been shown to be a safe and effective method in the treatment of experimental sarcoma metastases using doxorubicin [12]. Isolated lung perfusion allows delivery of high doses of chemotherapy but systemic toxicity is prevented. In previous studies with doxorubicin, isolated Single-lung perfusion was shown to produce a significant increase in the lung doxorubicin concentration, compared to the concentration achieved with intravenous administration; the heart concentration of doxorubicin is decreased when single-lung perfusion is used. Tumor necrosis factor is a cytokine released by activated macrophages. It is thought to mediate the response to sepsis [17] and may have a role in cancer cachexia [1]. Its role in cancer therapy has been intensively investigated [1,7,18]. Tumor necrosis factor is an ideal agent for isolated perfusion because it is cytotoxic to tumor, but the systemic toxicity associated with its use has limited its clinical usefulness [18]. Isolated single-lung perfusion with both murine and human TNF resulted in a decrease in the growth of experimental sarcoma lung metastases in the perfused left lung, compared to that seen in the untreated
Ann Thorae Surg 1994;58:328 -32
right lung. Tumor growth inhibition may be secondary to a direct cytotoxic effect of TNF or it may be mediated indirectly. Tumor necrosis factor has been shown to be directly cytotoxic to a number of cancer cell lines and its action is thought to be mediated by the generation of reactive oxygen radicals [1]. To determine if the effects of TNF on tumor growth after isolated Single-lung perfusion of the agent were directly related to a cytotoxic effect of TNF on MCA-induced sarcoma growth, we examined the effects of TNF on tumor cell growth by incubating MCAinduced sarcoma cells with a concentration of TNF identical to that used in the in vivo isolated single-lung perfusion experiment. Human recombinant TNF brought about a 37% inhibition in cell growth, and murine TNF had less effect on cell growth. Although the in vitro effect of human TNF was statistically significant, it was of small magnitude, indicating that the inhibitory effect of TNF on tumor growth in vivo may be mediated by other mechanisms as well. Tumor necrosis factor has profound stimulatory effects on peripheral blood lymphocytes and tumorinfiltrating lymphocytes [19]; activates the complement cascade and acts in concert with interleukin-I and interleukin-2 [20]; and induces damage in the tumor vasculature, causing hemorrhagic necrosis of the tumor [6, 7]. All of these actions could potentiate the direct antitumor effect of TNF. Tumor necrosis factor is known to increase its own endogenous production. Although we could not clearly demonstrate that TNF levels in the pulmonary vein effluent were increased, there was a strong trend toward this reaching statistical significance. It is possible that, by perfusing the pulmonary artery and collecting the pulmonary vein effluent for a longer period, the increase in endogenous TNF production could be documented. A trial of isolated limb perfusion with high-dose TNF, y-interferon, and melphalan, in combination with hyperthermia, was recently reported on for 23 patients with extremity sarcoma or melanoma. All patients experienced fever and chills and several experienced hematologic toxic reactions. There was no local toxic reaction attributed to TNF and 21 patients (91%) experienced partial or complete tumor response [7]. The striking antitumor effects of this perfusion regimen may be attributed to the combination of the four agents used. It is apparent that the combination of y-interferon and TNF enhances the antitumor effects of the latter by increasing the number of TNF receptors on tumor cells [21]. Other possible mechanisms of TNF's antitumor effects include vascular damage and direct effects on tumor cells. The vascular hypothesis explaining the tumor action of TNF was reinforced in this study by our finding of destruction of tumor neovasculature, as shown by angiograms obtained after isolated limb perfusion with TNF [7]. When administered systemically, the main toxic effect of TNF is hypotension and pulmonary toxicity resembling the adult respiratory distress syndrome [22]. We expected to see more toxicity in our in vivo experiment. Of the animals receiving systemically administered TNF, only 1
WEKSLER ET AL ISOLATED LUNG PERFUSION WITH TNF
Ann Thorac Surg 1994;58:328-32
animal died. Although we did not evaluate lung toxicity directly, it is reasonable to assume that lung toxicity would be manifested by the appearance of unilateral lung edema and adult respiratory distress syndrome, which would have caused shunting, hypoxia, and death. As no death occurred in the animals undergoing isolated lung perfusion with TNF, we believe that TNF may not have a direct pulmonary toxic effect. This is corroborated by Chang and colleagues [23], who showed that, when it is given systemically, TNF causes an increase in the lung wet-to-dry ratio (a marker of pulmonary edema) but it does not increase the lung wet-to-dry ratio after isolated lung perfusion in an ex vivo model. It is possible that lung injury is not mediated by TNF directly, but by some other cytokine or biologic response modifier. After isolated lung perfusion with TNF, a significant antitumor effect was observed. The lung perfused with TNF exhibited approximately five times less tumor than did the unperfused lung. The tumor response observed can probably be further enhanced by increasing the dose of TNF and by pretreatment with -y-interferon. The combination of TNF with -y-interferon pretreatment [21] or with hyperthermia [24] may significantly augment the antitumor effect in this model. In the present study, when we administered TNF systemically in the same doses as those used in the isolated perfusion experiments, we saw no antitumor effects, with no decrease in the pulmonary metastases in animals receiving TNF systemically. In summary, we have demonstrated that isolated lung perfusion with TNF is safe and is associated with a surprisingly low mortality. This treatment modality was highly effective in decreasing the growth of experimental sarcoma lung metastases in the rat. The systemic administration of TNF was not effective in decreasing tumor growth. Although a direct cytotoxic effect of TNF was shown, its magnitude was small and it is possible that other factors are important in the antitumor action of TNF. More experiments involving escalating doses of TNF and with the combination of TNF, interferon, and hyperthermia are needed to fully elucidate the potential role of isolated lung perfusion with TNF in the management of pulmonary metastases.
References 1. Vassalli P. The pathophysiology of tumor necrosis factors. Annu Rev Immunol 1992;10:411-52. 2. Tracey K, Beutler B, Lowry SF, et al. Shock and tissue injury induced by recombinant human cachectin. Science 1986;234: 470-4. 3. Balkwill FR, Lee A, Aldam G, et al. Human tumor xenografts treated with human tumor necrosis factor along or in combination with interferons. Cancer Res 1986;46:3990-3. 4. Schiller JH, Witt PL, Storer B, et al. Clinical and biologic effects of combination therapy with gamma-interferon and tumor necrosis factor. Cancer 1992;69:562-71. 5. Ramila P, Epstein LB. Tumor necrosis factor as immunomodu-
6. 7.
8. 9.
10. 11. 12.
13. 14. 15. 16. 17. 18.
19.
20.
21. 22. 23. 24.
331
lator and mediator of monocyte cytotoxicity induced by itself, gamma interferon and interleukin-1. Nature 1986;323:86-9. Watanabe N, Yoshiro N, Umeno H, et al. Toxic effect of tumor necrosis factor on tumor vasculature in mice. Cancer Res 1988;48:2179-83. Lienard 0, Ewalenko P, Delmotte Renard N, Lejeune FJ. High-dose recombinant tumor necrosis factor alpha in combination with interferon gamma and melphalan in isolation perfusion of the limbs for melanoma and sarcoma. J Clin Oncol 1992;10:52-60. Potter DA, Glenn J, Kinsella T. Patterns of recurrence in patients with high grade soft-tissue sarcomas. J Clin Oncol 1985;3:353-66. [ablons 0, Steinberg SM, Roth J, Pittaluga S, Rosenberg SA, Pass HI. Metastasectomy for soft tissue sarcoma. Further evidence for efficacy and prognostic indicators. J Thorac Cardiovasc Surg 1989;97:695-705. Weksler B, Schneider A, Ng B, Burt ME. Isolated single lung perfusion in the rat: an experimental model. J Appl Physiol 1993;74:2736-9. Weksler B, Ng B, Lenert J, Burt M. Isolated single lung perfusion with doxorubicin is pharmacokinetically superior to systemic administration. Ann Thorac Surg 1993;56:209-14. Weksler B, Lenert J, Ng B, Burt M. Isolated single lung perfusion with doxorubicin is effective in eradicating softtissue sarcoma lung metastases in the rat. J Thorac Cardiovasc Surg 1994;107:50-4. Burt ME, Lowry SF, Gorschboth CM, Brennan MF. Metabolic alteration in a noncachectic animal tumor system. Cancer 1981;47:2138-46. Weksler B, Ng B, Lenert J, Burt M. A simple method for endotracheal intubation in the rat. J Appl Physiol 1994;76: 1823-5. Wexler H. Accurate identification of experimental pulmonary metastasis. J Nat! Cancer Inst 1966;36:641-5. Debs RJ, Fuchs HJ, Philip R, et al. Immunomodulatory and toxic effects of free and liposome-encapsulated tumor necrosis factor alpha in rats. Cancer Res 1990;50:375-80. Tracey KJ, Fong Y, Hesse DG, et al. Anti-cachectin/TNF antibodies prevent septic shock during lethal bacteremia. Nature 1987;330:662-4. Abbruzzese JL, Levin B, Ajani JA, et al. Phase I trial of recombinant human gamma-interferon and recombinant human tumor necrosis factor in patients with advanced gastrointestinal cancer. Cancer Res 1989;49:4057-61. Stolfi VM, Milson JW, Finke JH, Fazio VW, Fiocchi C. Tumor necrosis factor alpha selectively enhances growth and cytotoxic activity of tumor infiltrating lymphocytes from human colorectal cancer. J Surg Res 1992;52:39-45. Fong Y, Lowry SF. Cytokines and the cellular response to injury and infection. In: Wilmore OW, Brennan MF, Harkin 0, Holcroft JW, Meakins JL, eds. American College of Surgeons: care of the surgical patient. Baltimore: Scientific America, 1992:1-17. Aggarwal BB, Eessalu TE, Hass PE. Characterization of receptors for human tumor necrosis factor and their regulation by interferon-gamma. Nature 1985;318:665-7. Ferrari-Baliviera E, Mealy K, Smith RJ, Wilmore OW. Tumor necrosis factor induces adult respiratory distress syndrome in rats. Arch Surg 1989;124:1400-5. Chang SW, Ohara N, Kuo G, Voelkel NF. Tumor necrosis factor-induced lung injury is not mediated by plateletactivating factor. Am J Physiol 1989;257:L232-9. Niitsu Y, Watanabe N, Umeno H, et al. Synergistic effects of recombinant human tumor necrosis factor and hyperthermia on in vitro cytotoxicity and artificial metastasis. Cancer Res 1988;48:654-7.
n,
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DISCUSSION DR HARVEY I. PASS (Baltimore, MD): For 1 year, we have been conducting an ongoing phase I dose-escalating trial of tumor necrosis factor (TNF), interferon, and hyperthermia in human patients using isolated lung perfusion. We treat rather large nodules in human patients using TNF, and the TNF may actually be working on the microvasculature, but the nodules in your animals are small, and still you are seeing very good effects. Do you think that the TNF mechanism of action is going to be critically important as far as the vasculature? In other words, do you think that large nodules are going to be more responsive than small nodules in the human patient? DR WEKSLER: The mechanism of TNF's antitumor effects is definitely complex. The study by Lienard and colleagues using isolated limb perfusion revealed the remarkable vascular toxicity of TNF to thrombose vessels to the tumor, and this has also been shown in other animal experimental models. But, the other mechanism of TNF, which I think will playa larger role in our model, is the induction of the cytokine cascade, complement, stimulation of lymphocytes, and injury to the cells brought about by oxygen free radicals. lt has been shown that cells that are very sensitive to TNF have less protection against this action of TNF, and this is actually independent of the number of TNF receptors on the cell surface. So, I think that, in terms of larger nodules, as in your experiment and in the experiment of Lienard's group, the vascular effect will be the predominant one. I suspect that all other antineoplastic effects of TNF in the smaller nodules will be more important,
although I am sure that this will have to be sorted out in carefully designed experiments. DR FRANK A. BACIEWICZ (Detroit, MI): We have been conducting clinical studies in a small number of patients with isolated lung perfusion using Adriamycin (doxorubicin). One of my concerns with the cannula position, and the fact that the lung is being inflated and deflated during the operation, is whether there are equal levels of drug in all lobes. In this one lobe model, is the tumor eradicated in all the different areas of the lobe or are there certain parts that are preferentially perfused using your technique? Second, and this is similar to the question asked by Dr Pass, when you operate on the animal 7 days after administering the tumor cells, are the larger lesions affected the same as the smaller ones? DR WEKSLER: In regard to your first question, when we set up this model of isolated lung perfusion, we tested those lungs with methylene blue, and we perfused at more than one rate using methylene blue in the perfusate. We found there was an even distribution of the blue dye in the lung. We did not measure the TNF lung tissue levels. When we did our doxorubicin experiments, we also did not measure the doxorubicin levels in several areas of the lung. But, in our feasibility experiments, we saw an even distribution of tumor eradication. Regarding your second question, if we inject sarcoma cells on day 0, both lungs are massively infiltrated with tumor by day 14. On day 7, tumors are present, but barely visible.
We conducted a trial of isolated lung perfusion using tumor necrosis factor (TNF) in an experimental sarcoma lung metastasis model. In an in vitro experiment, methylcholanthrene-induced sarcoma cells were incubated for 48 hours with 42 /-tg/mL of either human or murine TNF. Controls were incubated with Hank's balanced salt solution. In an in vivo experiment, 23 F344 rats were injected with 107 methylcholanthrene-induced sarcoma cells. On day 7, 4 animals were perfused with 210 /-tg of murine TNF, 5 animals were perfused with 420 /-tg of murine TNF, 10 animals underwent isolated lung perfusion with 420 /-tg of human TNF, and 4 animals were injected systemically with 420 ILg of human TNF. Animals were sacrificed on day 14 and the lung nodules counted. The cells incubated with murine TNF exhibited a 21% de-
crease in growth (p = 0.07); cells incubated with human TNF showed a 37% decrease in growth (p < 0.05). Animals perfused with 210 ILglmL of murine TNF and animals treated by systemically administered human TNF showed no tumor response. Animals perfused with 420 ILg/mL of murine TNF had 7.8 ± 14.2 nodules on the left lung and 58.5 ± 66.0 nodules on the right lung (p = 0.07). Animals perfused with 420 /-tglmL of human TNF had 21.7 ± 18.3 nodules on the left lung and 91.7 ± 66.2 nodules on the right lung (p < 0.01). On the basis of these findings, we conclude that isolated lung perfusion with TNF can be done safely in the rat and is effective in decreasing the growth of sarcoma lung metastases.
T
patients, the lungs are the sole site of disease recurrence [8]. Therapy for metastatic soft tissue sarcoma to the lungs consists of complete resection, with a 5-year survival of only 25% [9]. We have shown that isolated single-lung perfusion can be performed safely in the rat, with a low operative mortality [10]. Isolated single-lung perfusion with doxorubicin has been shown to be well tolerated with minimal systemic toxicity [11]. In addition, isolated singlelung perfusion with doxorubicin has been found to be effective in eradicating sarcoma lung metastases in the rat
u m or necrosis factor-a (TNF) is a cytokine produced mainly by activated macrophages and monocytes [1], and is thought to be an important mediator of septic shock [2]. Animal experiments have demonstrated the antitumor effects of systemic TNF against human tumor xenografts [3], but human experiments have yielded disappointing findings, with low response rates and a high frequency of systemic toxicity, such as fever, chills, hypotension, and leukopenia [4]. The antitumor effect of TNF involves several mechanisms, including a direct antitumor effect [1], induction of the lymphocyte secretion of antitumor substances [5], and the selective destruction of tumor vasculature [6,7]. Because of the systemic toxicity of TNF, Lienard and colleagues [7] conducted a trial of isolated limb perfusion using high doses of TNF combined with melphalan, 'Y-interferon, and hyperthermia for the treatment of unresectable extremity melanoma and sarcoma. The isolated limb perfusion circuit allowed for the administration of higher doses of TNF with less systemic toxicity. Tumor response was observed in all patients and complete tumor response was seen in 85%. Side effects included hypotension, fever, and chills, but all patients completed therapy. The lungs are the most common site of metastases in patients with extremity soft tissue sarcoma and, in most Presented at the Thirtieth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 31-Feb 2, 1994. Address reprint requests to Dr Burt, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021.
© 1994 by The Society of Thoracic Surgeons
(Ann Thorae Surg 1994;58:328-32)
[12].
Inspired by the excellent results of the isolated limb perfusion study, we conducted a trial of isolated singlelung perfusion with TNF in an experimental model of sarcoma lung metastases in the rat.
Material and Methods Animals (male F344 rats weighing 200 to 250 g; Charles Rivers, Kingston, NY) were treated in accordance with the Animal Welfare Act and the "NIH Guide for the Care and Use of Laboratory Animals" published by the National Institute of Health (NIH publication 85-43, revised 1985). All experiments were approved by the Institutional Animal Care and Use Committee of the Memorial SloanKettering Cancer Center. All rats were allowed access to standard laboratory rat chow (Ralston-Purina Rat Chow, St. Louis, MO) and water ad libitum. Housing was temperature controlled and provided 12-hour light-dark cycles. 0003-4975/94/$7.00
WEKSLER ET AL ISOLA TED LUNG PERFUSION WITH TNF
Ann Thorae Surg 1994;58:328-32
Methylcholanthrene-Induced Sarcoma Model Tumor was harvested fresh from a tumor-bearing animal and a single tumor cell suspension was prepared by first mincing grossly viable tumor and then incubating the minced tumor in Hanks balanced salt solution with 1% collagenase (Worthington Biochemical, Freehold, NJ). This methylcholanthrene (MCA)-induced sarcoma has been serially passaged subcutaneously in our laboratory, and has been extensively characterized [13]. We have found that an intravenous injection of 107 cells results in bilateral massive pulmonary metastases at 14 days in 100% of the animals.
Isolated Single-Lung Perfusion In the rat, the left lung is unilobar with one pulmonary artery and one pulmonary vein; the right lung consists of three to five lobes. Animals underwent isolated left lung perfusion, as described in detail elsewhere [10]. Briefly, animals were anesthetized with 50 mg/kg of pentobarbital administered intraperitoneally. The animal was orotracheally intubated with a 16-gauge intravenous catheter [14]. Animals were ventilated with a volume respirator (rodent ventilator model 683; Harvard Apparatus, South Natick, MA). The left chest was opened through the fourth intercostal space, and the left pulmonary vein and artery were dissected free and clamped proximally with microvascular clamps. The pulmonary artery was cannulated with a PE 50/PE 10 catheter (Clay-Adams, Parsippany, NJ). A venotomy was made and the pulmonary vein effluent was suctioned continuously. The left lung was perfused for 10 minutes with TNF in saline at a rate of 1 mL/min and for 5 minutes with a buffered hetastarch solution. At the end of the perfusion period, the pulmonary vein and artery were repaired with 9-0 microvascular nylon suture (Ethicon, Somerville, NJ). The left chest was closed and the animal was allowed to recover from the anesthesia. When the animal was awake, it was extubated and returned to its cage.
Experimental Design The effect of murine and human TNF on the growth of MCA-induced sarcoma cells was examined during 48 hours of log phase growth to assess the direct effect of TNF on cell growth. Approximately 250,000 viable cells (identified by the trypan blue exclusion method) were plated in six-well tissue culture plates and incubated in RPMI 1640 and 5% fetal calf serum. The effect of murine and human TNF on cell growth was examined sequentially. Tumor necrosis factor was added to four wells at a concentration of 42 /-lg/mL of media, and Hanks balanced salt solution was added to four wells as a control. At 48 hours, cells were trypsinized with phosphate buffered saline, 0.125% trypsin, and 0.125% EDTA (ethylenediaminetetrascetic acid), and counted in a standard Coulter counter (Coulter Electronics, Hialeah, FL). IN VITRO EXPERIMENTS.
IN VIVO EXPERIMENT. Twenty-three rats were injected on day 0 with 107 viable sarcoma cells through the external jugular vein. On day 7, all animals underwent left isolated lung perfusion with TNF. Nine animals were perfused
329
with murine TNF (Genentech, CA), and 10 rats were perfused with human TNF (Genentech): 4 animals with 210 /-lg of murine TNF, 5 animals with 420 /-lg of murine TNF, and 10 animals with 420 /-lg of human TNF. Four animals were injected with 420 /-lg of human TNF through the external jugular vein. On day 14, all animals were sacrificed and the number of pulmonary metastases assessed by the method of Wexler [15]. Briefly, the trachea and the lungs were removed en bloc and the lungs were inflated with 10 mL of india ink. The lungs were submersed 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 number of nodules in the left lung (perfused) and the right lung (unperfused) were counted.
Tumor Necrosis Factor Assay The TNF assay in the perfusate and in the pulmonary vein effluent was performed in all animals perfused with 420 /-lg of TNF using the WEHI cell-killing assay, and the results were expressed in units of cell killing [16].
Data Analysis All data are presented as the mean ± the standard deviation. Analysis was performed by the Wilcoxon matchedpaired test for comparison of the left versus the right lung and the TNF levels in the perfusate versus the pulmonary vein effluent. The Mann-Whitney U test was used to analyze the results of the in vitro experiment.
Results In Vitro Experiment At 48 hours, cells had gone through two doubling periods. Incubation of the MCA-induced sarcoma cells with murine TNF led to a 21% decrease (p = 0.07) in the number of cells at 48 hours (control versus TNF: 1.38 X 106 ± 0.25 X 106 cells versus 1.09 X 106 ± 0.07 X 106 cells). Cells incubated with human TNF showed a 37% reduction (p < 0.05) in the number of cells at 48 hours (control versus TNF: 1.2 X 106 ± 0.19 X 106 cells versus 0.7 X 106 ± 0.17 X 106 cells).
In Vivo Experiment All animals survived the surgical procedure. One animal perfused with 420 /-lg of murine TNF had a tumor in the mediastinum and was excluded from analysis. All other animals were sacrificed on day 14. Animals perfused with 210 /-lg of murine TNF showed no response to left isolated lung perfusion, and both lungs were completely replaced by tumor. Rats perfused with 420 /-lg of murine TNF had 58.5 ± 66.0 nodules on the right (untreated) lung and 7.8 ± 14.2 nodules on the left (perfused) lung (p = 0.07). Animals perfused with human TNF had 91.7 ± 66.2 nodules on the right lung and 21.7 ± 18.3 nodules on the left lung (p < 0.01). Two animals perfused with human TNF had more than 200 nodules on the right lung, and in the analysis, were considered to have 200 nodules on the right lung. One rat injected intravenously with human TNF died 24 hours after injection. The 3 other animals had massive
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Fig 1. In vivo experiments: India ink preparation of lung blocks. Normal lung tissue appears black and metastatic nodules appear white. On the left is a normal lung; on the right is a lung obtained after the systemic administration of 420 /J-g of human tumor necrosis factor; in the middle is a block from an animal obtained after isolated left lung perfusion with 420 /J-g of human tumor necrosis factor. The left (perfused) lung exhibits markedly less nodules than does the right (untreated) lung. On the right, lungs from the animal that received tumor necrosis factor systemically are bilaterally infiltrated with tumor.
replacement of both lungs, with an uncountable number of nodules (Fig 1). The total activity of TNF in animals perfused with 420 /Lg of TNF was 561.5 ::':: 24.8 units of cell killing. The total amount of TNF recovered from the pulmonary vein effluent was 607.6 ::':: 25.5 units of cell killing. This difference did not reach statistical significance (p = 0.07).
Comment Isolated single-lung perfusion in the MCA-induced sarcoma lung metastases model is an extremely versatile technique in the evaluation of antitumor agents. It has been shown to be a safe and effective method in the treatment of experimental sarcoma metastases using doxorubicin [12]. Isolated lung perfusion allows delivery of high doses of chemotherapy but systemic toxicity is prevented. In previous studies with doxorubicin, isolated Single-lung perfusion was shown to produce a significant increase in the lung doxorubicin concentration, compared to the concentration achieved with intravenous administration; the heart concentration of doxorubicin is decreased when single-lung perfusion is used. Tumor necrosis factor is a cytokine released by activated macrophages. It is thought to mediate the response to sepsis [17] and may have a role in cancer cachexia [1]. Its role in cancer therapy has been intensively investigated [1,7,18]. Tumor necrosis factor is an ideal agent for isolated perfusion because it is cytotoxic to tumor, but the systemic toxicity associated with its use has limited its clinical usefulness [18]. Isolated single-lung perfusion with both murine and human TNF resulted in a decrease in the growth of experimental sarcoma lung metastases in the perfused left lung, compared to that seen in the untreated
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right lung. Tumor growth inhibition may be secondary to a direct cytotoxic effect of TNF or it may be mediated indirectly. Tumor necrosis factor has been shown to be directly cytotoxic to a number of cancer cell lines and its action is thought to be mediated by the generation of reactive oxygen radicals [1]. To determine if the effects of TNF on tumor growth after isolated Single-lung perfusion of the agent were directly related to a cytotoxic effect of TNF on MCA-induced sarcoma growth, we examined the effects of TNF on tumor cell growth by incubating MCAinduced sarcoma cells with a concentration of TNF identical to that used in the in vivo isolated single-lung perfusion experiment. Human recombinant TNF brought about a 37% inhibition in cell growth, and murine TNF had less effect on cell growth. Although the in vitro effect of human TNF was statistically significant, it was of small magnitude, indicating that the inhibitory effect of TNF on tumor growth in vivo may be mediated by other mechanisms as well. Tumor necrosis factor has profound stimulatory effects on peripheral blood lymphocytes and tumorinfiltrating lymphocytes [19]; activates the complement cascade and acts in concert with interleukin-I and interleukin-2 [20]; and induces damage in the tumor vasculature, causing hemorrhagic necrosis of the tumor [6, 7]. All of these actions could potentiate the direct antitumor effect of TNF. Tumor necrosis factor is known to increase its own endogenous production. Although we could not clearly demonstrate that TNF levels in the pulmonary vein effluent were increased, there was a strong trend toward this reaching statistical significance. It is possible that, by perfusing the pulmonary artery and collecting the pulmonary vein effluent for a longer period, the increase in endogenous TNF production could be documented. A trial of isolated limb perfusion with high-dose TNF, y-interferon, and melphalan, in combination with hyperthermia, was recently reported on for 23 patients with extremity sarcoma or melanoma. All patients experienced fever and chills and several experienced hematologic toxic reactions. There was no local toxic reaction attributed to TNF and 21 patients (91%) experienced partial or complete tumor response [7]. The striking antitumor effects of this perfusion regimen may be attributed to the combination of the four agents used. It is apparent that the combination of y-interferon and TNF enhances the antitumor effects of the latter by increasing the number of TNF receptors on tumor cells [21]. Other possible mechanisms of TNF's antitumor effects include vascular damage and direct effects on tumor cells. The vascular hypothesis explaining the tumor action of TNF was reinforced in this study by our finding of destruction of tumor neovasculature, as shown by angiograms obtained after isolated limb perfusion with TNF [7]. When administered systemically, the main toxic effect of TNF is hypotension and pulmonary toxicity resembling the adult respiratory distress syndrome [22]. We expected to see more toxicity in our in vivo experiment. Of the animals receiving systemically administered TNF, only 1
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animal died. Although we did not evaluate lung toxicity directly, it is reasonable to assume that lung toxicity would be manifested by the appearance of unilateral lung edema and adult respiratory distress syndrome, which would have caused shunting, hypoxia, and death. As no death occurred in the animals undergoing isolated lung perfusion with TNF, we believe that TNF may not have a direct pulmonary toxic effect. This is corroborated by Chang and colleagues [23], who showed that, when it is given systemically, TNF causes an increase in the lung wet-to-dry ratio (a marker of pulmonary edema) but it does not increase the lung wet-to-dry ratio after isolated lung perfusion in an ex vivo model. It is possible that lung injury is not mediated by TNF directly, but by some other cytokine or biologic response modifier. After isolated lung perfusion with TNF, a significant antitumor effect was observed. The lung perfused with TNF exhibited approximately five times less tumor than did the unperfused lung. The tumor response observed can probably be further enhanced by increasing the dose of TNF and by pretreatment with -y-interferon. The combination of TNF with -y-interferon pretreatment [21] or with hyperthermia [24] may significantly augment the antitumor effect in this model. In the present study, when we administered TNF systemically in the same doses as those used in the isolated perfusion experiments, we saw no antitumor effects, with no decrease in the pulmonary metastases in animals receiving TNF systemically. In summary, we have demonstrated that isolated lung perfusion with TNF is safe and is associated with a surprisingly low mortality. This treatment modality was highly effective in decreasing the growth of experimental sarcoma lung metastases in the rat. The systemic administration of TNF was not effective in decreasing tumor growth. Although a direct cytotoxic effect of TNF was shown, its magnitude was small and it is possible that other factors are important in the antitumor action of TNF. More experiments involving escalating doses of TNF and with the combination of TNF, interferon, and hyperthermia are needed to fully elucidate the potential role of isolated lung perfusion with TNF in the management of pulmonary metastases.
References 1. Vassalli P. The pathophysiology of tumor necrosis factors. Annu Rev Immunol 1992;10:411-52. 2. Tracey K, Beutler B, Lowry SF, et al. Shock and tissue injury induced by recombinant human cachectin. Science 1986;234: 470-4. 3. Balkwill FR, Lee A, Aldam G, et al. Human tumor xenografts treated with human tumor necrosis factor along or in combination with interferons. Cancer Res 1986;46:3990-3. 4. Schiller JH, Witt PL, Storer B, et al. Clinical and biologic effects of combination therapy with gamma-interferon and tumor necrosis factor. Cancer 1992;69:562-71. 5. Ramila P, Epstein LB. Tumor necrosis factor as immunomodu-
6. 7.
8. 9.
10. 11. 12.
13. 14. 15. 16. 17. 18.
19.
20.
21. 22. 23. 24.
331
lator and mediator of monocyte cytotoxicity induced by itself, gamma interferon and interleukin-1. Nature 1986;323:86-9. Watanabe N, Yoshiro N, Umeno H, et al. Toxic effect of tumor necrosis factor on tumor vasculature in mice. Cancer Res 1988;48:2179-83. Lienard 0, Ewalenko P, Delmotte Renard N, Lejeune FJ. High-dose recombinant tumor necrosis factor alpha in combination with interferon gamma and melphalan in isolation perfusion of the limbs for melanoma and sarcoma. J Clin Oncol 1992;10:52-60. Potter DA, Glenn J, Kinsella T. Patterns of recurrence in patients with high grade soft-tissue sarcomas. J Clin Oncol 1985;3:353-66. [ablons 0, Steinberg SM, Roth J, Pittaluga S, Rosenberg SA, Pass HI. Metastasectomy for soft tissue sarcoma. Further evidence for efficacy and prognostic indicators. J Thorac Cardiovasc Surg 1989;97:695-705. Weksler B, Schneider A, Ng B, Burt ME. Isolated single lung perfusion in the rat: an experimental model. J Appl Physiol 1993;74:2736-9. Weksler B, Ng B, Lenert J, Burt M. Isolated single lung perfusion with doxorubicin is pharmacokinetically superior to systemic administration. Ann Thorac Surg 1993;56:209-14. Weksler B, Lenert J, Ng B, Burt M. Isolated single lung perfusion with doxorubicin is effective in eradicating softtissue sarcoma lung metastases in the rat. J Thorac Cardiovasc Surg 1994;107:50-4. Burt ME, Lowry SF, Gorschboth CM, Brennan MF. Metabolic alteration in a noncachectic animal tumor system. Cancer 1981;47:2138-46. Weksler B, Ng B, Lenert J, Burt M. A simple method for endotracheal intubation in the rat. J Appl Physiol 1994;76: 1823-5. Wexler H. Accurate identification of experimental pulmonary metastasis. J Nat! Cancer Inst 1966;36:641-5. Debs RJ, Fuchs HJ, Philip R, et al. Immunomodulatory and toxic effects of free and liposome-encapsulated tumor necrosis factor alpha in rats. Cancer Res 1990;50:375-80. Tracey KJ, Fong Y, Hesse DG, et al. Anti-cachectin/TNF antibodies prevent septic shock during lethal bacteremia. Nature 1987;330:662-4. Abbruzzese JL, Levin B, Ajani JA, et al. Phase I trial of recombinant human gamma-interferon and recombinant human tumor necrosis factor in patients with advanced gastrointestinal cancer. Cancer Res 1989;49:4057-61. Stolfi VM, Milson JW, Finke JH, Fazio VW, Fiocchi C. Tumor necrosis factor alpha selectively enhances growth and cytotoxic activity of tumor infiltrating lymphocytes from human colorectal cancer. J Surg Res 1992;52:39-45. Fong Y, Lowry SF. Cytokines and the cellular response to injury and infection. In: Wilmore OW, Brennan MF, Harkin 0, Holcroft JW, Meakins JL, eds. American College of Surgeons: care of the surgical patient. Baltimore: Scientific America, 1992:1-17. Aggarwal BB, Eessalu TE, Hass PE. Characterization of receptors for human tumor necrosis factor and their regulation by interferon-gamma. Nature 1985;318:665-7. Ferrari-Baliviera E, Mealy K, Smith RJ, Wilmore OW. Tumor necrosis factor induces adult respiratory distress syndrome in rats. Arch Surg 1989;124:1400-5. Chang SW, Ohara N, Kuo G, Voelkel NF. Tumor necrosis factor-induced lung injury is not mediated by plateletactivating factor. Am J Physiol 1989;257:L232-9. Niitsu Y, Watanabe N, Umeno H, et al. Synergistic effects of recombinant human tumor necrosis factor and hyperthermia on in vitro cytotoxicity and artificial metastasis. Cancer Res 1988;48:654-7.
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DISCUSSION DR HARVEY I. PASS (Baltimore, MD): For 1 year, we have been conducting an ongoing phase I dose-escalating trial of tumor necrosis factor (TNF), interferon, and hyperthermia in human patients using isolated lung perfusion. We treat rather large nodules in human patients using TNF, and the TNF may actually be working on the microvasculature, but the nodules in your animals are small, and still you are seeing very good effects. Do you think that the TNF mechanism of action is going to be critically important as far as the vasculature? In other words, do you think that large nodules are going to be more responsive than small nodules in the human patient? DR WEKSLER: The mechanism of TNF's antitumor effects is definitely complex. The study by Lienard and colleagues using isolated limb perfusion revealed the remarkable vascular toxicity of TNF to thrombose vessels to the tumor, and this has also been shown in other animal experimental models. But, the other mechanism of TNF, which I think will playa larger role in our model, is the induction of the cytokine cascade, complement, stimulation of lymphocytes, and injury to the cells brought about by oxygen free radicals. lt has been shown that cells that are very sensitive to TNF have less protection against this action of TNF, and this is actually independent of the number of TNF receptors on the cell surface. So, I think that, in terms of larger nodules, as in your experiment and in the experiment of Lienard's group, the vascular effect will be the predominant one. I suspect that all other antineoplastic effects of TNF in the smaller nodules will be more important,
although I am sure that this will have to be sorted out in carefully designed experiments. DR FRANK A. BACIEWICZ (Detroit, MI): We have been conducting clinical studies in a small number of patients with isolated lung perfusion using Adriamycin (doxorubicin). One of my concerns with the cannula position, and the fact that the lung is being inflated and deflated during the operation, is whether there are equal levels of drug in all lobes. In this one lobe model, is the tumor eradicated in all the different areas of the lobe or are there certain parts that are preferentially perfused using your technique? Second, and this is similar to the question asked by Dr Pass, when you operate on the animal 7 days after administering the tumor cells, are the larger lesions affected the same as the smaller ones? DR WEKSLER: In regard to your first question, when we set up this model of isolated lung perfusion, we tested those lungs with methylene blue, and we perfused at more than one rate using methylene blue in the perfusate. We found there was an even distribution of the blue dye in the lung. We did not measure the TNF lung tissue levels. When we did our doxorubicin experiments, we also did not measure the doxorubicin levels in several areas of the lung. But, in our feasibility experiments, we saw an even distribution of tumor eradication. Regarding your second question, if we inject sarcoma cells on day 0, both lungs are massively infiltrated with tumor by day 14. On day 7, tumors are present, but barely visible.