American Journal of Respiratory Cell and Molecular Biology

Lung cancer and chronic obstructive pulmonary disease are leading causes of morbidity and mortality worldwide, and cigarette smoking is a main risk factor for both. The presence of emphysema, an irreversible lung disease, further raises the risk of lung cancer in patients with chronic obstructive pulmonary disease. The mechanisms involved in smoke-induced tumorigenesis and emphysema are not fully understood, attributable to a lack of appropriate animal models. Here, we optimized a model of cigarette smoke (CS)–induced lung cancer and emphysema in A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a potent carcinogen. We investigated whether variations in CS exposure patterns with the same total amount and duration of exposure affect tumorigenesis and/or development of emphysema. Continuous CS exposure for 3 months significantly suppressed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone–induced development of adenomas and adenocarcinomas; however, emphysema independently developed during this period. Surprisingly, intermittent CS exposure increased the severity of emphysema and resulted in a higher incidence of adenocarcinomas. Furthermore, intermittent CS exposure elicited a marked increase in M2-polarized macrophages within and near the developed tumors. By employing a CS exposure protocol with repeated cycles of cessation and relapse, we provide evidence that intermittent CS exposure enhances tumorigenesis and emphysema progression more than that of continuous CS exposure.

Lung cancer is an important complication of chronic obstructive pulmonary disease (COPD), accounting for 5–38% of COPD deaths (1). We and others have recently reported that emphysema, an irreversible lung disease, is an independent risk factor of lung cancer development in COPD cohort studies (2, 3). Epidemiologically, long-term cigarette smoking is a common risk factor for both lung cancer and COPD (4, 5). Although smoking cessation reduces the incidence of lung cancer and COPD, the risks in ex-smokers remain elevated for many years after quitting (6). Mechanistic links between COPD and lung cancer have been suggested, but remain to be further elucidated (7).

Experimental mouse models, especially gene-engineered mouse models, help us to understand disease and drug response mechanisms. For lung cancer, several oncogene-engineered mouse models, such as Kirsten-ras along with tumor protein p53 or epidermal growth factor receptor models, have been established (8, 9). However, in most human lung cancers, numerous somatic genetic alterations, including synonymous and nonsynonymous, exist. In particular, lung adenocarcinomas or squamous cell carcinomas are known to be mutation-burden high, which reflects long-term exposure to carcinogens, such as that which occurs in smokers (10).

Over time, many attempts have been made to reproduce lung cancer in experimental animals exposed to chronic cigarette smoke (CS), most often with negative or only marginally positive results (11, 12). In contrast, CS-induced emphysema rodent models have been successfully established and are commonly used (13, 14). Several recent studies have confirmed the potential of two susceptible mouse strains (A/J and SWR) to develop lung tumors after 5 months of mainstream smoke inhalation and a 4-month recovery period (15, 16). The A/J mouse is known to be uniquely susceptible to lung carcinogens, and spontaneously develops tumors over time with aging (17). More recently, Takahashi and colleagues (18) reported that intermittent exposure to CS promotes tumor development in mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent carcinogen. However, the relationship between tumorigenesis and emphysema was not examined in those studies, and appropriate animal models that mimic patients with lung cancer with COPD are still missing.

In this study, we aimed to establish a mouse model of CS-induced lung tumors and emphysema. We examined whether variations in the CS exposure pattern with the same total amount and duration of exposure affect tumorigenesis and/or development of emphysema. Furthermore, we looked at differences in the inflammatory profile associated with the various CS exposure patterns, with a focus on tumor-associated macrophages (TAMs).

Some of the results of this study were presented at the American Thoracic Society International Conference 2017, and have been previously reported in the form of an abstract (19).

Details on methods are described in the supplemental materials.


Male A/J mice (7–10 wk old) were purchased from Oriental Japan (Tokyo, Japan). All experimental protocols and procedures were in accordance with the National Institutes of Health guidelines and approved by the Animal Use Committee at Keio University School of Medicine.

NNK Treatment and CS Exposure

At 1 week after 100 mg/kg intraperitoneal injection of NNK (Toronto Research Chemicals), mice were exposed to mainstream CS generated from commercially available filtered cigarettes (12 mg tar/1.0 mg nicotine, Marlboro; Phillip Morris Inc.); the mice inhaled the CS through the nose, as previously reported using a SIS-CS system (Shibata Scientific Technology) (20). To test the effects of a variety of CS exposure protocols on lung tumor and emphysema development, mice were divided into 5 groups with 5–18 mice per group. The study design and experimental protocols are described in Figure 1.

Sampling of Mouse Lung Tissue and BAL

Lungs from all groups were serially sectioned in 100-μm steps for histopathological detection of tumor nodules (15). Tissue sections (6 μm) were stained with hematoxylin and eosin. In another subgroup of animals, mice were killed and underwent BAL with 0.6 ml saline three times through a tracheal cannula. Total cell counts of the BAL fluid (BALF) were determined and cell differentials in BALF were examined as previously described (15).

Morphometric Assessment of Emphysema

Mean linear intercept (Lm), a standard parameter of alveolar size, and destructive index (DI), a value representing alveolar destruction, were measured and calculated in 10 randomly selected fields per lung specimen for each mouse.

Pathological Evaluation of Lung Tumors

Bronchial-alveolar proliferative lesions were pathologically diagnosed as hyperplasia, adenoma, or adenocarcinoma on the hematoxylin and eosin–stained sections, according to published criteria (21), by a trained pathologist (M.S,). Tumor multiplicity, incidence and size were calculated as previously described (18).


The primary antibodies used included pro–surfactant protein C (SP-C) and club cell 10-kD protein (CC10; Santa Cruz Biotechnology); DyLight 488–labeled Lycopersicon esculentum (tomato) lectin (DL-1174; Vector Labs); and arginase (Arg)-1 (D4E3M; Cell Signaling Technology). All lectin or Arg-1–positive cells were visually counted from all animals with tumors. We counted cells within the tumor nodules and in the vicinity (defined as the lung tissue areas within 200 μm from the nodule).

Noninvasive Micro–computed Tomography Imaging

Mice were scanned in the prone position with inhalation anesthesia of mixed isoflurane (Pfizer Japan), and the X-ray micro–computed tomography (CT) system (R_mCT2; Rigaku) was operated as previously reported (20, 22). Maximal diameters of each lesion were determined for the axial direction.

Statistical Analysis

Data are expressed as means (±SE). Continuous data were analyzed using the Student’s t test, paired t test, and ANOVA, followed by the Tukey-Kramer test. Categorical data were analyzed using the χ2 test. All data were analyzed using JMP version 11.0 (SAS Institute).

Inhibitory Effects of CS on Increasing Body Weight

The mice were exposed to CS for 60 min/d, 5 d/wk. The study design and experimental protocols are described in Figure 1. Mice that were continuously exposed to CS (3 M CS group) failed to gain weight (Figure 2A). However, the continuously CS-exposed mice rapidly resumed weight gain, nearly reaching that of the air-exposed mice (5 M air group) after a 2-month withdrawal (3 M CS 2 M air group) (Figure 2B). In the intermittent CS exposure group (5 M CS at 1 M intervals group), the mice showed a reduction in body weight during the second and third exposure periods (Figure 2B). These findings indicate that our CS exposure model replicates a hallmark feature of human smokers as a systemic response in a corresponding time frame (23).

Differences in CS-induced Inflammation by Changes in CS Exposure Patterns

We then examined whether varying the CS exposure patterns with the same amount and duration affects lung inflammation. Total cell number in BALF was significantly higher in the 3 M CS and 5 M CS at 1 M intervals groups than that in the 3 M and 5 M air groups, respectively (Figure 3A). Although the total cell number in BALF did not differ between the 3 M CS and 5 M CS at 1 M intervals groups, differential cell counts revealed significant differences in the cell type profile between these two groups. The 5 M CS at 1 M intervals group showed significantly more macrophages and fewer neutrophils than that of the 3 M CS group (Figures 3B and 3C). Furthermore, a lower trend in the number of lymphocytes was observed in the 5 M CS at 1 M intervals group; however, the difference was not statistically significant (Figure 3D).

Amplifying Effects of Intermittent CS on Emphysema Development

Alveolar size was evaluated by quantifying Lm, and alveolar damage was evaluated by quantifying DI. The 3 M CS group exhibited significant enlargement of the alveoli and more destruction than that of the 3 M air group (Lm = 61.2 ± 0.9 vs. 55.3 ± 1.7 μm, P = 0.009; DI = 28.8 ± 1.3 vs. 22.4 ± 1.8, P = 0.024; n = 18 and 5, respectively; Figures 4A–4C). Interestingly, after the 5-month exposure period, Lm and DI in the 5 M CS at 1 M intervals group were significantly higher than those in the 3 M CS 2 M air and 5 M air groups (Lm = 82. 4 ± 1.8 vs. 61.8 ± 2.0 or 63.5 ± 2.9 μm; P < 0.0001; DI = 39.4 ± 1.2 vs. 30.7 ± 1.3 or 26.6 ± 1.9, P = 0.0001; n = 13, 11, and 5, respectively; Figures 4A, 4D, and 4E). Although we did see an increase in the air-exposed group over time (3 M air vs. 5 M air, Lm = 55.3 ± 1.7 vs. 63.5 ± 2.9 μm, P = 0.02; DI = 22.4 ± 1.9 vs. 26.6 ± 1.8, P = 0.16), the Lm and DI were consistent, with no further increase in the 3 M CS 2 M air group after smoking cessation (3 M CS vs. 3 M CS 2 M air, Lm = 61.2 ± 0.9 vs. 61.8 ± 2.0 μm, P = 0.79; DI = 28.8 ± 1.3 vs. 30.7 ± 1.3, P = 0.28). These data suggest that intermittent CS exposure increases the severity of emphysema more than that of continuous exposure, whereas continuous cessation appears to stop further progression of emphysema.

Promoting Effects of Intermittent CS Exposure on Lung Tumorigenesis

NNK-treated and CS-exposed mice developed lung adenomas and adenocarcinomas (Figures 5A and 5B). Adenocarcinoma criteria for diagnosis included a high nuclear/cytoplasm ratio, dyskaryosis, cell aggregation, and destruction of the alveolar structure (Figure 5B). Histological examination of adenoma and adenocarcinoma was similar regardless of which smoking protocol was used. We analyzed lung tumor multiplicity, incidence, and size by microscopic examination (Figures 6A–6F). The 3 M CS group showed the lowest tumor multiplicity and incidence, followed by the 3 M air group (multiplicity = 0.1 ± 0.1 vs. 0.8 ± 0.4 per mouse, P = 0.008; incidence = 11.1 vs. 60.0%, P = 0.03; Figures 6A and 6B), implying that continuous CS exposure suppressed NNK-induced tumor multiplicity and incidence by 86.3% and 81.5%, respectively. The suppressive effects of CS exposure on tumorigenesis were lost after CS withdrawal in the 3 M CS 2 M air group (Figures 6D and 6E). However, alternating exposure to CS and CS-free air in the 5 M CS at 1 M intervals group resulted in significantly higher tumor multiplicity and incidence than those in the 5 M air group and 3 M CS 2 M air group (multiplicity = 4.1 ± 0.6 vs. 1.0 ± 0.5 or 0.6 ± 0.3 per mouse, P = 0.002 and P < 0.0001, respectively; incidence = 100.0 vs. 60.0 or 45.5%, P = 0.02 and P = 0.002, respectively). The 5 M CS at 1 M intervals group showed higher lung adenocarcinoma multiplicity and incidence compared with the 5 M air group and the 3M CS 2M air group (multiplicity = 1.08 ± 0.26 vs. 0.0 ± 0.0 or 0.18 ± 0.12 per mouse, P = 0.02 and P = 0.01, respectively; incidence = 69.2 vs. 0.0 or 18.2%, P = 0.009 and P = 0.01, respectively; Figures 6D and 6E). Importantly, these results suggest that intermittent CS exposure caused an acceleration in the progression of adenoma to adenocarcinoma. However, there were no statistical differences in tumor size between the two groups at 3 months (0.31 ± 0.07 vs. 0.34 ± 0.10 mm2, P = 0.85), or among the three groups at 5 months (0.38 ± 0.05 vs. 0.37 ± 0.10 vs. 0.46 ± 0.07 mm2, P = 0.82) (Figures 6C and 6F)

Molecular Characterization of CS-induced Adenocarcinoma

To assist in determining the phenotype of epithelial cells in adenomas and adenocarcinomas from 5 M CS at 1 M intervals group, the tissues were immunostained for SP-C and CC10, distinct lung epithelial cell markers that indicate alveolar type II and club cells, respectively (Figures 5C and 5D). Most tumor cells were uniformly positive for SP-C and exclusively negative for CC10, although airway epithelial cells in normal bronchus from the same sections did stain positive for CC10. All tumors in different smoking protocols exhibited no differences in the SP-C and CC10 status.

Accumulation of Macrophages with M2 Polarization in CS-induced Adenocarcinoma

Significantly more macrophages were observed in BALF from the 5 M CS at 1 M intervals group than in the 3 M CS group. Therefore, we examined whether this increase in macrophages represents a general change in the overall lung inflammatory response or a tumor-associated phenomenon (e.g., TAMs) (24). As such, we examined the distribution of macrophages in all lung tissue sections bearing adenocarcinomas in the 5 M CS at 1 M intervals group (n = 14) and in the 3 M CS 2 M air group (n = 2). To identify macrophage polarization toward the M2 phenotype, we co-stained for pan-macrophage and M2 macrophage markers (tomato lectin and arginase-1, respectively) (Figures 7A, 7B, 7E, and 7F). Macrophage density was significantly higher in the vicinity and within the adenocarcinoma than that in the emphysematous area in the 5 M CS at 1 M intervals group (Figure 7C). Arg-1–positive (M2) macrophages predominantly accumulated inside and around the adenocarcinoma, but were rarely observed in the emphysematous area in the 5 M CS at 1 M intervals group (Figure 7D). There seemed no difference in the number and localization of lectin-positive cells and Arg-1–positive cells among the different smoking protocols (Figures 7A–7H). Representative adenomas in all groups were examined. Arg-1–positive (M2) macrophages were very few or absent in adenomas. These data suggest that M2-polarized macrophages are associated with CS-induced adenocarcinoma, but not with alveolar destruction.

Monitoring the Development and Growth of Lung Tumors via CT Scans

In this model, many tumors from the 5 M CS at 1 M intervals group were detectable via micro-CT, appearing as nodular lesions clearly distinguishable from the surrounding tissue without the use of a contrast agent (Figure 6G). We thus monitored mice from another set of the 5 M CS at 1 M intervals group (n = 7) to assess further changes in tumor size after an additional 4 months of smoking cessation using micro-CT. Our results show that tumors detectable on micro-CT at the end of 5 months grew further in size after smoking cessation in the absence of treatment (Figure 6H). These data imply that the tumors developing in mice exposed to intermittent CS were aggressive in nature.

This is a novel study showing that modification of the CS exposure protocol enhances both tumorigenesis and progression of emphysema without altering the total dose and duration of exposure, implying that repeated cycles of CS exposure and cessation enhance the adverse effects more than those observed after continuous CS exposure.

Adenomas developed in response to treatment with a combination of NNK and CS exposure, corresponding to a relatively early stage of lung adenocarcinoma as reported previously (8). Tumor cells displayed markers for alveolar type II cells, but not for club cells. However, this phenomenon does not necessarily dictate the alveolar origin of tumor cells. We recognize that tumors may derive from bronchoalveolar stem cells (25); one possibility is that SP-C– and CC10–double-positive cells progressively lose CC10 expression. It has also been reported that loss of CC10 expression is associated with increased tumorigenicity in human non–small-cell lung cancer (NSCLC) (26) and in mouse models of lung cancer (27).

It is well known that macrophages, neutrophils, and lymphocytes are increased in the airways of patients with COPD (28, 29), as well as in the BALF from experimental models of CS-induced emphysema (30). However, it is now evident that inflammation is involved in all stages of tumorigenesis, and macrophages make up the majority of the immune infiltrate in tumors and are a key cell type linking inflammation and cancer (31). Although a large spectrum of macrophage phenotypes exists, two major phenotypes have been recognized in tumors, namely the classically activated (M1) and alternatively activated (M2) macrophage phenotypes (32). TAMs from established tumors are generally skewed toward the M2 end of the spectrum, promoting tumor survival, progression, and dissemination through cellular processes, such as enhanced angiogenesis, epithelial-to-mesenchymal transition, and immune suppression (33, 34). Indeed, macrophages have been correlated with negative outcomes and poor prognoses in most cancers (34). In the group of mice intermittently exposed to CS, macrophages preferentially accumulated within and around the tumors, and most of those macrophages were positive for Arg-1, indicating M2-polarized macrophages. This finding agrees with reports using a lung urethane-induced carcinoma model that showed an important role of M2 macrophages in lung tumor promotion (35, 36) and M2 macrophages only within the tumor and peritumor areas, but not in normal appearing alveoli (36) and FGF9-induced lung adenocarcinoma model (our unpublished data), and thus supports the association between M2 macrophages and more aggressive tumorigenesis in this model.

NNK-induced tumorigenesis with morphological characteristics of adenomas and adenocarcinomas was significantly suppressed by continuous CS exposure for 3 months as previously reported (37); however, emphysema independently developed during this period. This finding suggests that there is a mechanism by which continuous CS exposure induces emphysema, but suppresses tumorigenesis in the lungs. Our data also show that complete withdrawal stopped progression of emphysema as well as the development of new tumors.

A surprising finding of this study was that intermittent CS exposure increases the number of tumors, especially adenocarcinoma, along with further progression of emphysema, even though the total amount of CS and duration were the same. The question then arises as to how repeated CS exposure and withdrawal further the severity of tumorigenesis and emphysema. The molecular pathways associated with enhanced lung tumorigenesis during the progression of smoking-associated emphysema are complex (7). CS exposure induces a wide range of systemic alterations in mechanistic pathways, including oxidative stress, metabolic alterations, autonomic nervous system alterations, and hormonal changes (38). Nicotine, the addictive constituent of cigarettes, and its derived carcinogenic nitrosamines contribute to cancer promotion and progression through the activation of nicotinic acetylcholine receptors (nAChRs) (39). It is also reported that nAChR α7 modulates the response to chronic CS exposure in animal experiments (40), and human genome-wide association studies revealed that genetic variants in the nAchR on chromosome 15q24/25 was a risk for lung cancer (41) and emphysema (42). On the other hand, long-term nicotine treatment down-regulates nAChR expression and function (43). Down-regulation of nAChR might be the mechanism of attenuation in tumorigenesis and emphysema progression in continuous CS exposure. An extensive effort will be required to elucidate the pathways mediating the coordinated repertoire of such responses in both diseases. This model would provide an approach to investigate pathogenetic links between these two life-threatening diseases.

Iskandar and colleagues (44) recently established an animal model promoting lung cancer in NNK-treated A/J mice that simultaneously develop emphysema via repeated intraperitoneal administration of nicotine. An advantage of our model is that it mimics the behavior of human smokers by applying a daily exposure of main stream CS through nose inhalation (20). There are some issues regarding differences in the respiratory anatomy and physiology between mice and humans that need to be considered, such as the reduced number of bronchial branches in mice. This fact might explain the lack of squamous and small-cell carcinomas in our model, both of which are commonly observed in human smokers.

Comprehensive analyses in this study revealed the effects of various patterns of CS exposure on inflammatory cell profiles in alveolar spaces, progression of emphysema, and lung tumorigenesis. We included a unique experimental dimension by comparing groups exposed to continuous and intermittent CS. There are minimal specific pathway mechanistic data in this study; however, the impact between the exposure treatment groups outweighs the descriptive nature of the results.

This model is also suitable for screening putative chemopreventive agents, and serves as a therapeutic intervention that mimics progressive human lung cancer with emphysema in smokers.

The authors thank Naomi Ishikawa and Miyuki Yamamoto for exposing the mice to smoke and for technical assistance, and the Collaborative Research Resources, Keio University School of Medicine, for technical support and reagents.

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Correspondence and requests for reprints should be addressed to Shotaro Chubachi, M.D., Ph.D., Division of Pulmonary Medicine, Department of Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: .

This work was supported by Japan Society for the Promotion of Science Grant-in-Aid for Young Scientists 17K16063 (S.C.), Grant-in-Aid for Scientific Research B 05-045-0215 (T.B.), and GlaxoSmithKline research grant 2016 A-34 (S.C.).

Author Contributions: N.K., S.C., and T.B. developed the concept and designed the research; N.K., S.C., A.E.H., H.Y., S.K., A.T., and M.S. performed the experiments and analyzed data for the work; N.K., S.C., A.E.H., H.Y., S.K., A.T., K.F., M.S., Y.K., K.S., and T.B. interpreted the data and reviewed the manuscript critically for important intellectual content.

This article has a data supplement, which is accessible from this issue’s table of contents at

Originally Published in Press as DOI: 10.1165/rcmb.2017-0375OC on February 14, 2018

Author disclosures are available with the text of this article at

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