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(Chest. 2004;125:1467-1471.)
© 2004 American College of Chest Physicians

The Effect of Cigarette Smoke Exposure on Pulmonary Metastatic Disease in a Murine Model of Metastatic Breast Cancer*

Susan Murin, MD, MSc, FCCP; Kent E. Pinkerton, PhD; Neil E. Hubbard, PhD and Kent Erickson, PhD

* From the Division of Pulmonary and Critical Care Medicine (Dr. Murin), Department of Internal Medicine, Department of Cell Biology and Human Anatomy (Drs. Hubbard and Erickson), University of California Davis School of Medicine, Davis, CA; and Center for Health and the Environment (Dr. Pinkerton), University of California, Davis, CA.

Correspondence to: Susan Murin, MD, MSc, FCCP, 4150 V St, Suite 3400, Sacramento, CA 95817; e-mail: sxmurin{at}ucdavis.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Introduction: Women who smoke have a higher rate of fatal breast cancer than nonsmoking women. An association between smoking and pulmonary metastases from breast cancer has been suggested by epidemiologic studies.

Study objectives: To examine the relationship between exposure to cigarette smoke and pulmonary metastasis in a murine model of metastatic mammary cancer.

Study design: Prospective, randomized study.

Setting: Animal research laboratory.

Experimental subjects: Female sexually mature BALB/cAnN mice.

Interventions: Mice were randomly divided into experimental and control groups. Experimental animals were exposed to cigarette smoke in specialized exposure chambers, at concentrations chosen to approximate active cigarette smoking. Control animals were exposed to filtered air. One week after the initiation of exposures, mouse mammary tumor cells (tumor cell line 4526) were injected into the tail veins of experimental animals at one of three concentrations (50,000, 100,000, or 150,000 cells per 100 µL). Three weeks later, the mice were killed, and pulmonary metastases were counted and measured.

Results: The mean metastatic burden in the lungs was consistently greater for smoke-exposed animals at each concentration of cells injected (at 50,000 cells per 100 µL, 9.8 vs 4.8 µm3, respectively [p < 0.01]; at 100,000 cells per 100 µL, 34.5 vs 17.4 µm3, respectively [p < 0.10]; and at 150,000 cells per 100 µL, 54.0 vs 31.5 µm3, respectively [p < 0.05]). This was largely attributable to a significant increase in the number of metastatic nodules per animal (at 50,000 cells per 100 µL, 8.7 vs 4.8, respectively [p < 0.001]; at 100,000 cells per 100 µL, 24.3 vs 14.0, respectively [p > 0.10]; and at 150,000 cells per 100 µL, 42.0 vs 20.1, respectively [p < 0.02]) rather than to a change in nodule size.

Conclusions: Cigarette smoke exposure is associated with an increase in the total pulmonary metastatic burden in this murine model of metastatic mammary cell cancer. This study provides experimental support for an adverse effect of smoking on the metastatic process and suggests a possible mechanism for smokers’ increased breast cancer mortality.

Key Words: breast cancer • metastasis • smoking


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Carcinoma of the breast is the most common cancer among women. The development of metastases, for this and most other tumors, usually signifies incurable disease. A number of factors, including disease stage, degree of tumor differentiation, and tumor genetic profile, as assessed by DNA microarray, predict the risk of metastatic disease.123 However, a significant amount of the variability in the natural history of the disease, including the development of metastases, remains unexplained.

Several epidemiologic studies4567 have shown that smokers have an increased rate of breast cancer death compared with nonsmokers, although smoking is not associated with an increased incidence of breast cancer.8910 This suggests that smoking might adversely affect the biology and natural history of breast cancer. Smoking causes a variety of pulmonary and systemic effects that could contribute to an increased propensity to metastasis from breast and other cancers. Two epidemiologic studies,1112 including one from our group, have demonstrated an association between smoking and pulmonary metastases among women with breast cancer. Smoking also has been associated with worse outcomes from a variety of other malignancies, suggesting that a possible adverse effect of smoking on cancer biology may not be unique to breast cancer. This study directly examined the relationship between exposure to cigarette smoke and pulmonary metastasis in a murine model of metastatic mammary cancer.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Female BALB/cAnN mice, 5 weeks old (sexually mature, young adult) were used for the study (Charles River Laboratories; Wilmington, MA). Animals were handled in accordance with the standards set forth by the National Institutes of Health and the University of California, Davis, Animal Care and Use Committee. Animals were fed a standard rodent chow. Mice were randomly divided into experimental and control groups (36 mice per group). Experimental animals were exposed to a mixture of sidestream and mainstream cigarette smoke in a smoking apparatus built in our laboratory, as previously described.13 The cigarettes were 1R4F research cigarettes (Tobacco Health Research Institute; Lexington, KY), were conditioned prior to use to the proper temperature and humidity to ensure uniform burning during combustion. An automated, metered puffer was used to smoke the cigarettes under Federal Trade Commission conditions (35 mL per puff, 2-s duration, and one puff per minute). The smoke was collected in a chimney, diluted with filtered air, and delivered to whole-body exposure chambers. The exposures were characterized for the three major constituents of cigarette smoke, including nicotine, carbon monoxide, and total suspended particulates. Smoke concentrations were selected to replicate concentrations associated with active smoking. Animals in the smoke-exposed group were exposed continuously for 6 h/d, 5 d/wk. Carbon monoxide was measured every 30 min, total suspended particulates were measured every 2 h, and nicotine was measured once daily. Average exposure conditions are summarized in Table 1 . Control animals were exposed to filtered room air. Cells of the mouse mammary tumor cell line 4526 (a cloned subpopulation derived from a spontaneously arising mammary adenocarcinoma of a BABL/cfC3H mouse)14 were maintained and prepared as previously described.15 A 100-µL aliquot containing 5.0 x 104 to 1.5 x 105 tumor cells was injected into the tail vein of each animal 1 week after cigarette smoke or filtered air exposures began (12 mice for each concentration). The tumor cell dose range was chosen based on prior experiments with a similar model. Injection was facilitated by briefly warming the mice using a heat lamp. Animals were not exposed to cigarette smoke on the day of injection as logistical considerations precluded such exposure on the day of injection.


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  		Table 1.. Mean Concentrations of Cigarette Smoke Constitutents in Exposure Chambers

 
The mice were killed 3 weeks after tumor cell injection. This time point was selected based on the results of prior experiments in a similar model. Three weeks is a sufficient period to allow the establishment and growth of metastasis, without posing a risk of respiratory insufficiency and death from progressive metastasis. The quantification of pulmonary metastases was facilitated by insufflation of the lungs with India ink/Karnovsky fixative (2% formaldehyde, 2.5% glutaraldehyde, in 0.06 M phosphate buffer, pH 7.2), which allowed the nodules to appear white against a black background, as described previously.15 Nodules were counted by direct visualization using a stereoscopic microscope. The volume of the nodules was calculated from diameters estimated using the stereoscopic microscope equipped with an intraocular reticule. Nodules were observed to be spherical, and thus the volume of the nodules was calculated from diameters according to the formula v = 4/3{pi}r3. Total metastatic burden, the primary outcome variable of our study as determined a priori, was calculated by multiplying the number of lung nodules by the average volume/nodule. No metastases were observed in other organs on gross inspection. Comparisons between experimental groups were performed using nonparametric (Wilcoxon) tests. Comparisons were considered to be significant if a p value of < 0.05 was observed. Statistical analysis was performed with (Medcalc; Medcalc Software; Brussels, Belgium).


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
The experimental results are summarized in Figure 1 . The total metastatic burden in the lungs was consistently greater for smoke-exposed animals than for control animals (at 50,000 cells per 100 µL, 9.8 vs 4.8 µm3, respectively [p < 0.01]; at 100,000 cells per 100 µL, 34.5 vs 17.4 µm3, respectively [p < 0.10]; at 150,000 cells per 100 µL, 54.0 vs 31.5 µm3, respectively [p < 0.05]). The number of metastatic nodules increased with an increasing concentration of tumor cells injected, and at each concentration the number of tumors in the lungs of smoke-exposed animals was greater than in those of control animals (at 50,000 cells per 100 µL, 8.7 vs 4.8, respectively [p < 0.001]; at 100,000 cells per 100 µL, 24.3 vs 14.0, respectively [p > 0.10]; at 150,000 cells per 100 µL, 42.0 vs 20.1, respectively [p < 0.02]). Metastatic nodules were slightly larger for smoke-exposed vs control animals, but the difference was not significant.



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  		Figure 1.. Top: total tumor load. Mean pulmonary tumor load per animal for smoke-exposed and filtered air-exposed animals, at three different doses of injected tumor cells (50,000 to 150,000 cells per 100 µL). Twelve mice were used for each experimental condition. Bottom: number of pulmonary metastases. The mean number of pulmonary metastases per animal for smoke-exposed and filtered air-exposed animals, at three different doses of injected tumor cells (50,000 to 150,000 cells per 100 µL). Twelve mice were used for each experimental condition.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
Prior epidemiologic investigations have suggested that breast cancer is a more lethal disease among smokers.4567 Smokers have been found to have more frequent axillary metastases, and larger metastases, than nonsmokers, after controlling for tumor size and other variables.1617 Two studies1112 have found an association between cigarette smoking and the development of pulmonary metastases from breast cancer. Such epidemiologic studies have shortcomings, such as potential bias and unrecognized confounding factors, that necessitate caution in their interpretation. This study was designed to directly examine the effects of exposure to cigarette smoke on the development of pulmonary metastasis, using a simple animal model.

In this murine, tail-vein injection model of metastatic mammary cancer, cigarette smoke exposure at concentrations akin to those in active cigarette smoking was associated with an increased burden of metastatic disease in the lung, which manifested as an increased number of metastatic nodules. This provides direct evidence of an effect of smoke-exposure on the metastatic process. It supports the previously observed relationship between cigarette smoking and pulmonary metastatic disease among women with breast cancer, and suggests that the facilitation of metastasis by cigarette smoke exposure may contribute to the increased rate of fatal breast cancer seen among women who smoke.

The specific mechanisms underlying smoke exposure’s effect on the metastatic burden in the lungs are presently unknown. Smoking is known to cause a host of pulmonary and systemic effects that could contribute to a change in the number or behavior of pulmonary metastases.18 These include increased vascular and epithelial permeability,1920 changes in local immune function,21222324252627 changes in systemic antitumor defenses,28 coagulation status,29 platelet adhesiveness,30 and cellular adhesion molecules.31 In addition, exposure to smoke constituents has been shown to directly affect the metastatic potential of tumor cells via effects on signal transduction.32 In this model, animals were preexposed to cigarette smoke before the injection of tumor cells in order to best replicate the human condition of breast cancer metastasizing in a human who is an active smoker. We cannot determine from the current experiments whether the effects of smoking that lead to the potentiation of metastasis are limited to the state of active exposure or would be sustained in a past smoker.

The tail vein injection model of metastasis was used in our investigation. With this model, tumor cells are placed directly into the circulation, without a period of growth in the experimental animal under conditions of exposure, making a direct, smoke-induced change in the metastatic potential of the tumor cells extremely unlikely. The increased tumor burden seen in the lungs of smoke-exposed animals in these experiments was due to an increase in the number of metastatic nodules per animal. The increased number of individual metastases demonstrates that a greater number of the injected tumor cells was able to establish metastatic foci. Research33 involving the direct visualization of the steps in early hematogenous metastasis has shown that pulmonary metastasis is initiated by the proliferation of intravascular tumor cells that have adhered to the vascular endothelium with subsequent extravasation. This suggests that the potentiation of metastasis seen in our model is due to increased adherence, proliferation, or extravasation of the injected tumor cells. Which of these steps in the metastatic process was affected by smoke exposure is currently unclear. The lack of difference in the size of metastatic nodules between smoke-exposed and unexposed experimental animals suggests that smoke exposure does not affect the growth rate of metastases once they are established.

Several potential shortcomings of this study warrant mention. The cigarette smoke exposure utilized in this and other similar studies differs somewhat from that of active cigarette smoking. Mice and other experimental animals do not actively smoke but are exposed via intermittent residence in a smoke- containing environment, and the cigarette smoke to which the animals are exposed is a mixture of both mainstream and sidestream smoke. It is not possible to precisely correlate the exposure levels in mice to those of humans, but serum cotinine levels in mice exposed at the levels used in our experiments approximate those in human smokers. It is plausible that sidestream smoke and mainstream smoke have different effects on the metastatic process that are not separable in our experimental model, or that the conditions of exposure (6 h of continuous exposure per day) result in effects that would not be seen under the condition of active cigarette smoking throughout the day. Future experiments are necessary to assess whether lower dose exposures, such as those experienced by second-hand smokers, also affect the metastatic process.

In addition, while murine models are commonly used in the study of mammary and other cancers, such animal models have obvious limitations in their replication of the metastatic process in humans. In particular, the tail-vein injection model of hematogenous metastasis, a popular model for the study of metastasis because of its simplicity, differs from spontaneous metastasis in several important ways. Because tumor cells are injected directly into the circulation, this model does not allow study of the effects of exposure on the early steps in the metastatic process, such as acquisition of a metastatic phenotype and detachment from the primary tumor. Given evidence that in vitro exposure of tumor cells to constituents of cigarette smoke leads to molecular events that may facilitate metastasis,32 it is very unlikely that this limitation in the model would lead to an overestimation of the adverse effects of smoke exposure on the metastatic process. In addition, the tail-vein injection model differs from spontaneous metastasis in that all tumor cells enter the circulation simultaneously. It is possible that the effects of smoking on tumor-lung interactions differ in the face of this larger intravascular tumor load. Subsequent experiments in a related, spontaneous metastasis model should address this potential limitation. Similarly, further study is necessary to ascertain whether a similar effect of smoking on metastasis occurs with other tumor types and in organs other than the lung.


   Conclusion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
In this murine model of metastatic mammary cancer, the exposure of experimental animals to cigarette smoke under controlled conditions was associated with a significant increase in the total pulmonary metastatic burden, chiefly due to an increase in the number of metastatic foci developing within the lungs. These data complement epidemiologic studies showing an association between smoking and pulmonary metastasis among women with breast cancer, and they suggest that smoking-associated changes in metastatic behavior might play a role in the increased mortality seen among smokers with breast and other cancers. Further studies to explore the potential mechanisms for the potentiating effect of cigarette smoke exposure on pulmonary metastasis are planned.


   Footnotes
 
Supported by the American Lung Association, California Breast Cancer Research Program, and California Tobacco Related Diseases Research Program.

Received for publication July 7, 2003. Accepted for publication September 12, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

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