Metastatic breast cancer

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 expend 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 [micro]L). 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 [micro]L, 9.8 vs 4.8 [micro][m.sup.3], respectively [p < 0.01]; at 100,000 cells per 100 [micro]L, 34.5 vs 17.4 [micro][m.sup.3], respectively [p < 0.10]; and at 150,000 cells per 100 [micro]L, 54.0 vs 31.5 [m.sup.3], 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 [micro]L, 8.7 vs 4.8, respectively [p < 0.001]; at 100,000 cells per 100 [micro]L, 24.3 vs 14.0, respectively [p > 0.10]; and at 150,000 cells per 100 [micro]L, 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

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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. (1-3) However, a significant amount of the variability in the natural history of the disease, including the development of metastases, remains unexplained.

Several epidemiologic studies (4-7) 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. (8-10) 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, (11,12) 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

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 elite 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-[micro]L aliquot containing 5.0 x [10.sup.4] to 1.5 x [10.sup.5] 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.

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][r.sup.3]. 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

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 [micro]L, 9.8 vs 4.8 [micro][m.sup.3], respectively [p < 0.01]; at 100,000 cells per 100 [micro]L, 34.5 vs 17.4 ++, respectively [p < 0.10]; at 150,000 (cells per 100 [micro]L, 54.0 vs 31.5 ++, 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 [micro]L, 8.7 vs 4.8, respectively [p < 0.001]; at 100,000 cells per 100 [micro]L, 24.3 vs 14.0, respectively [p > 0.10]; at 150,000 cells per 100 [micro]L, 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.

[FIGURE 1 OMITTED]

DISCUSSION