Induction of Lung Tumors and Mutational Analysis in FVB/N Mice Treated with the Tobacco Carcinogen 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone

  • Natalie J. Rothenberger
  • Laura P. StabileEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2102)


Lung cancer remains the leading cause of cancer-related deaths worldwide. In order to understand lung cancer biology and evaluate novel therapeutic strategies, preclinical mouse models have been developed that mimic early and advanced-stage lung cancer. Among autochthonous models, carcinogen-induced systems are valuable preclinical tools since tobacco smoking remains the number one risk factor for lung tumor development. Among the several thousand chemicals within cigarette smoke, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen with tumorigenic effects described in both mice and humans. Herein, we describe the methodology for inducing lung tumors in mice using the tobacco carcinogen NNK and subsequent lung fixation for quantitative assessment of tumor development and analysis of oncogenic mutations in tumors.

Key words

Lung cancer Lung fixation Murine model Lung histology Tobacco carcinogen 


  1. 1.
    American Cancer Society. Cancer Facts & Figures 2019. Atlanta: American Cancer Society; 2019Google Scholar
  2. 2.
    Wakamatsu N, Devereux TR, Hong HH, Sills RC (2007) Overview of the molecular carcinogenesis of mouse lung tumor models of human lung cancer. Toxicol Pathol 35(1):75–80. Scholar
  3. 3.
    Khuder SA (2001) Effect of cigarette smoking on major histological types of lung cancer: a meta-analysis. Lung Cancer 31(2-3):139–148CrossRefGoogle Scholar
  4. 4.
    Akbay EA, Kim J (2018) Autochthonous murine models for the study of smoker and never-smoker associated lung cancers. Transl Lung Cancer Res 7(4):464–486. Scholar
  5. 5.
    Jiang Y, Zhao J, Zhang Y, Li K, Li T, Chen X, Zhao S, Zhao S, Liu K, Dong Z (2018) Establishment of lung cancer patient-derived xenograft models and primary cell lines for lung cancer study. J Transl Med 16(1):138. Scholar
  6. 6.
    Kellar A, Egan C, Morris D (2015) Preclinical murine models for lung cancer: clinical trial applications. Biomed Res Int 2015:621324. Scholar
  7. 7.
    Cancer Genome Atlas Research N (2014) Comprehensive molecular profiling of lung adenocarcinoma. Nature 511(7511):543–550. Scholar
  8. 8.
    Coggins CR (2007) An updated review of inhalation studies with cigarette smoke in laboratory animals. Int J Toxicol 26(4):331–338. Scholar
  9. 9.
    Malkinson AM (1989) The genetic basis of susceptibility to lung tumors in mice. Toxicology 54(3):241–271CrossRefGoogle Scholar
  10. 10.
    Shimkin MB, Stoner GD (1975) Lung tumors in mice: application to carcinogenesis bioassay. Adv Cancer Res 21:1–58CrossRefGoogle Scholar
  11. 11.
    Reck M, Rodriguez-Abreu D, Robinson AG, Hui R, Csoszi T, Fulop A, Gottfried M, Peled N, Tafreshi A, Cuffe S, O’Brien M, Rao S, Hotta K, Leiby MA, Lubiniecki GM, Shentu Y, Rangwala R, Brahmer JR, KEYNOTE-024 Investigators (2016) Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N Engl J Med 375(19):1823–1833. Scholar
  12. 12.
    Brahmer J, Reckamp KL, Baas P, Crino L, Eberhardt WE, Poddubskaya E, Antonia S, Pluzanski A, Vokes EE, Holgado E, Waterhouse D, Ready N, Gainor J, Aren Frontera O, Havel L, Steins M, Garassino MC, Aerts JG, Domine M, Paz-Ares L, Reck M, Baudelet C, Harbison CT, Lestini B, Spigel DR (2015) Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer. N Engl J Med 373(2):123–135. Scholar
  13. 13.
    Wang X, Bao Z, Zhang X, Li F, Lai T, Cao C, Chen Z, Li W, Shen H, Ying S (2017) Effectiveness and safety of PD-1/PD-L1 inhibitors in the treatment of solid tumors: a systematic review and meta-analysis. Oncotarget 8(35):59901–59914.
  14. 14.
    Mandel R, Samstein RM, Lee KW, Havel JJ, Wang H et al (2019) Genetic diversity of tumors with mismatch repair deficiency influences anti-PD-1 immunotherapy response. Science. 364(6439):485–91CrossRefGoogle Scholar
  15. 15.
    Dupage M, Mazumdar C, Schmidt LM, Cheung AF, Jacks T (2012) Expression of tumour-specific antigens underlies cancer immunoediting. Nature. 482(7385):405–9CrossRefGoogle Scholar
  16. 16.
    Centers for Disease Control and Prevention (US); National Center for Chronic Disease Prevention and Health Promotion (US); Office on Smoking and Health (US). (2010) How tobacco smoke causes disease: the biology and behavioral basis for smoking-attributable disease: a report of the surgeon general. Publications and Reports of the Surgeon General, Atlanta (GA)Google Scholar
  17. 17.
    Hecht SS, Hoffmann D (1988) Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis 9(6):875–884CrossRefGoogle Scholar
  18. 18.
    Hecht SS (1998) Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines. Chem Res Toxicol 11(6):559–603. Scholar
  19. 19.
    Humans IWGotEoCRt (2007) Smokeless tobacco and some tobacco-specific N-nitrosamines. IARC Monogr Eval Carcinog Risks Hum 89:1–592Google Scholar
  20. 20.
    Ge GZ, Xu TR, Chen C (2015) Tobacco carcinogen NNK-induced lung cancer animal models and associated carcinogenic mechanisms. Acta Biochim Biophys Sin Shanghai 47(7):477–487. Scholar
  21. 21.
    West KA, Brognard J, Clark AS, Linnoila IR, Yang X, Swain SM, Harris C, Belinsky S, Dennis PA (2003) Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells. J Clin Invest 111(1):81–90. Scholar
  22. 22.
    Jull BA, Plummer HK 3rd, Schuller HM (2001) Nicotinic receptor-mediated activation by the tobacco-specific nitrosamine NNK of a Raf-1/MAP kinase pathway, resulting in phosphorylation of c-myc in human small cell lung carcinoma cells and pulmonary neuroendocrine cells. J Cancer Res Clin Oncol 127(12):707–717CrossRefGoogle Scholar
  23. 23.
    Schuller HM, Cekanova M (2005) NNK-induced hamster lung adenocarcinomas over-express beta2-adrenergic and EGFR signaling pathways. Lung Cancer 49(1):35–45. Scholar
  24. 24.
    Laag E, Majidi M, Cekanova M, Masi T, Takahashi T, Schuller HM (2006) NNK activates ERK1/2 and CREB/ATF-1 via beta-1-AR and EGFR signaling in human lung adenocarcinoma and small airway epithelial cells. Int J Cancer 119(7):1547–1552. Scholar
  25. 25.
    Chen RJ, Chang LW, Lin P, Wang YJ (2011) Epigenetic effects and molecular mechanisms of tumorigenesis induced by cigarette smoke: an overview. J Oncol 2011:654931. Scholar
  26. 26.
    Siegfried JM, Farooqui M, Rothenberger NJ, Dacic S, Stabile LP (2017) Interaction between the estrogen receptor and fibroblast growth factor receptor pathways in non-small cell lung cancer. Oncotarget 8(15):24063–24076. Scholar
  27. 27.
    Stabile LP, Rothstein ME, Cunningham DE, Land SR, Dacic S, Keohavong P, Siegfried JM (2012) Prevention of tobacco carcinogen-induced lung cancer in female mice using antiestrogens. Carcinogenesis 33(11):2181–2189. Scholar
  28. 28.
    Sivakumar S, Lucas FAS, McDowell TL, Lang W, Xu L, Fujimoto J, Zhang J, Futreal PA, Fukuoka J, Yatabe Y, Dubinett SM, Spira AE, Fowler J, Hawk ET, Wistuba II, Scheet P, Kadara H (2017) Genomic landscape of atypical adenomatous hyperplasia reveals divergent modes to lung adenocarcinoma. Cancer Res 77(22):6119–6130. Scholar
  29. 29.
    Fujimoto J, Kadara H, Men T, van Pelt C, Lotan D, Lotan R (2010) Comparative functional genomics analysis of NNK tobacco-carcinogen induced lung adenocarcinoma development in Gprc5a-knockout mice. PLoS One 5(7):e11847. Scholar
  30. 30.
    Di YP (2014) Assessment of pathological and physiological changes in mouse lung through bronchoalveolar lavage. Methods Mol Biol 1105:33–42. Scholar
  31. 31.
    Keohavong P, Mady HH, Gao WM, Siegfried JM, Luketich JD, Melhem MF (2001) Topographic analysis of K-ras mutations in histologically normal lung tissues and tumours of lung cancer patients. Br J Cancer 85(2):235–241. Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Geisinger Commonwealth School of MedicineScrantonUSA
  2. 2.Department of Pharmacology & Chemical Biology, UPMC Hillman Cancer CenterUniversity of PittsburghPittsburghUSA

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