Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2026 Jun 20.
Published in final edited form as: Methods Cell Biol. 2020 Aug 24;163:153–173. doi: 10.1016/bs.mcb.2020.07.003

Chapter 11: Two-stage 3-methylcholanthrene and butylated hydroxytoluene-induced lung carcinogenesis in mice

Alison K Bauer 1,*, Lori D Dwyer-Nield 2
PMCID: PMC13282014  NIHMSID: NIHMS2162256  PMID: 33785163

Abstract

Lung cancer is one of the deadliest types of cancer and as such requires disease models that are useful for identification of novel pathways for biomarkers as well as to test therapeutic agents. Adenocarcinoma (ADC), the most prevalent type of lung cancer, is a subtype of non-small cell lung carcinoma (NSCLC) and a disease driven mainly by smoking. However, it is also the most common subtype of lung cancer found in non-smokers with environmental exposures. Chemically driven models of lung cancer, also called primary models of lung cancer, are important because they do not overexpress or delete oncogenes or tumor suppressor genes, respectively, to increase oncogenesis. Instead these models test tumor development without forcing a specific pathway (i.e., Kras). The primary focus of this chapter is to discuss a well-established 2-stage mouse model of lung adenocarcinomas. The initiator (3-methylcholanthrene, MCA) does not elicit many, if any, tumors if not followed by exposure to the tumor promoter (butylated hydroxytoluene, BHT). In sensitive strains, such as A/J, FVB, and BALB, significantly greater numbers of tumors develop following the MCA/BHT protocol compared to MCA alone. BHT does not elicit tumors on its own; it is a non-genotoxic carcinogen and promoter. In these sensitive strains, promotion is also associated with inflammation characterized by infiltrating macrophages, lymphocytes, and neutrophils, and other inflammatory cell types in addition to increases in total protein content reflective of lung hyperpermeability. This 2-stage model is a useful tool to identify unique promotion specific events to then test in future intervention studies.

Keywords: 2-stage carcinogenesis, 3-methylcholanthrene, butylated hydroxytoluene, tumor promotion, initiation, lung, inflammation, mouse, lung adenocarcinoma, carcinogen-induced

1. INTRODUCTION

Non-small cell lung carcinoma (NSCLC) is the leading cause of lung cancer deaths, with the primary subtype (adenocarcinoma (ADC)) associated with smoking and many other environmental factors (American Cancer Society, 2020; IARC, 2012, 2014, 2016; Schottenfeld, 2005; Torre et al., 2015). Approximately 40% of all lung cancers are adenocarcinomas (ADC), equating to ~94,000 and >700,000 new patients in 2018 in the US and worldwide, respectively (American Cancer Society, 2020; Torre et al., 2015). Five-year survival rates for these patients depend largely on the stage of diagnosis with stage 3 and higher <14% survival at 5 yrs (American Cancer Society, 2020). Thus, early detection is key for effectively treating lung ADC, and using models to test early stages of tumor development is valuable. Carcinogen-induced lung cancer mouse models are unique and considered primary models of lung cancer because unlike the models that overexpress or delete genes to increase oncogenes and decrease tumor suppressor genes, respectively, the tumors develop without forcing a specific pathway (i.e., oncogene or lack of tumor suppressors). As a result, the numbers of tumors that develop in these carcinogen-induced models tend to be lower and smaller than those in the models that overexpress oncogenes, for example, Kirsten rat sarcoma (Kras) (Bauer, Umer, et al., 2018; Jackson et al., 2001; Moghaddam et al., 2009). Thus, these models are ideal for studying early tumor development and chemoprevention as tumors are more diverse and grow slowly enough to evaluate chemoprevention regimens. However, carcinogen-induced models require longer periods of time which can be more costly. In this chapter, we will provide the protocol for one of our well-established carcinogen-induced cancer models below (MCA/BHT), however urethane-induced lung cancer is another well-established model that we suggest as an additional primary lung adenocarcinoma model (reviewed in Chapter 4) that is used by many laboratories (Bauer et al., 2011; Fritz et al., 2014; A.M. Malkinson, 1983, 1998; O’Donnell, Zerbe, Dwyer-Nield, Kisley, & Malkinson, 2006; Redente et al., 2010; Stathopoulos et al., 2007; Stearman et al., 2005; Zerbe et al., 2008). Lastly, another carcinogen-induced model of interest for an understudied yet prevalent type of lung cancer, squamous cell carcinoma, is the N-nitroso trischloroethylurea (NTCU) model and can be found in the following references (Ghosh et al., 2015; Hudish et al., 2012; Wang et al., 2004).

Lung adenocarcinoma development is a multi-stage process that begins with the initiation stage, where the precursor cells undergo a DNA damaging/genotoxic event, followed by the tumor promotion stage where key components lead to cellular transformation, and progression (Bauer, Malkinson, & Kleeberger, 2004; Klaunig, Kamendulis, & Xu, 2000; A.M. Malkinson, 1998). The most common lung tumor initiating mutation in both humans and mice is KRAS, although mutations can occur in several other genes such as epidermal growth factor receptor (EGFR) and serine/threonine-protein kinase B-raf (BRAF) (Fritz, Dwyer-Nield, Russell, & Malkinson, 2010; Riely et al., 2008; Sun et al., 2010). These initiating events are part of the enabling characteristic that was described for the Hallmarks of Cancer (i.e., genome instability and mutation)(Hanahan & Weinberg, 2011).Tumor promoters do not result in tumor development unless in the presence of an initiator, as we will describe below for our established model using 3-methylcholanthrene (MCA) as the initiator and butylated hydroxytoluene (BHT) as the promoter (A.M. Malkinson, Koski, Evans, & Festing, 1997; Witschi, Williamson, & Lock, 1977). However, tumor promoters are still carcinogens and often overlooked because they are non-genotoxic, meaning they do not elicit a mutation on their own. Complete carcinogens elicit tumor development (i.e., urethane, MCA) in the absence of a tumor promoter, albeit at higher doses than required for initiation (Bauer, Dwyer-Nield, Keil, Koski, & Malkinson, 2001; L. D. Dwyer-Nield et al., 2010; A.M. Malkinson, 1998). Several key components of tumor promotion are considered either hallmarks of cancer or enabling characteristics,(Hanahan & Weinberg, 2011) and have been investigated in our laboratories and others (Bauer, Velmurugan, et al., 2018; Romo, Velmurugan, Upham, Dwyer-Nield, & Bauer, 2019; Siegrist et al., 2019). Gap junctional intracellular communication (GJIC) is a component of the evasion of growth suppression hallmark, specifically during the early stages of tumor development (Cesen-Cummings, Fernstrom, Malkinson, & Ruch, 1998; Chaudhuri et al., 1993; Guan et al., 1995; Hanahan & Weinberg, 2011; Nahta et al., 2015). Tumor promoting inflammation is a critical component of cancer development and considered an enabling characteristic of cancer (Hanahan & Weinberg, 2011). We have observed numerous pro-inflammatory markers and GJIC inhibition in response to MCA/BHT in our lung models, some of which will be discussed below (Alexander et al., 2016; Bauer et al., 2005; Bauer, Dwyer-Nield, Hankin, Murphy, & Malkinson, 2001; Bauer et al., 2004; Bauer & Rondini, 2009; Bauer et al., 2017b; Keith et al., 2002).

2. 2-STAGE MCA/BHT MODEL BACKGROUND

MCA is a model PAH carcinogen that is a polycyclic aromatic hydrocarbon and it elicits Kras codon 12 mutations (Fritz et al., 2010). Interestingly, the Kras mutations elicited by this carcinogen are strain specific. A/J mice, a highly susceptible mouse strain, develop Kras mutations similar to those observed in smokers; primarily codon 12 transversions that result in G→V and G→C changes in Kras protein. Conversely, in BALB/ByJ mice, considered an intermediate strain with respect to sensitivity, mice develop Kras mutations similar to non-smokers, primarily codon 12 transitions that result in G→D changes in Kras protein (Fritz et al., 2010). While MCA can act as a complete carcinogen, in low doses and in certain strains, it is an initiator. For example, strains that are more sensitive to lung carcinogenesis are BALB/ByJ, BALB/cJ, FVB/J, A/J, and SWR strains and can be initiated by MCA, whereas C57B/6J and 129/SVJ are relatively resistant to MCA initiation (Bauer, Dwyer-Nield, Keil, et al., 2001; A. M. Malkinson, Radcliffe, & Bauer, 2002).

BHT is a synthetic food additive (CAS: 128-37-0) and at higher doses, a well-established lung tumor promoter. Metabolism of BHT by cytochrome p4502B (Saltini et al., 1984) is required for its pneumotoxic effects to the hydroxylated form called BHTOH that is further metabolized to the BHTOH-derived quinone methide (Thompson, Bolton, & Malkinson, 1991; Witschi, Malkinson, & Thompson, 1989). P4502B is a sexually dimorphic enzyme, with P4502B9 being expressed predominantly in female mice; female mice are more sensitive to BHT-induced pneumotoxicity than male mice (Hashita et al., 2008). All mouse strains tested with a single ip. injection of BHT develop acute lung injury characterized by alveolar type 1 cell necrosis followed by alveolar type 2 cell hyperplasia (Adamson, Bowden, Cote, & Witschi, 1977; A.M. Malkinson, 1979). However, not all strains are susceptible to the more sub-chronic or chronic effects of BHT as a tumor promoter. When a strain survey was completed for differences in responsiveness to BHT (17 strains) or MCA/BHT (12 strains), we found that BHT-induced inflammation correlated with BHT-induced tumor promotion (Bauer, Dwyer-Nield, Keil, et al., 2001; A. M. alkinson et al., 2002). The inflammation observed was characterized by significant increases in infiltrating macrophages, lymphocytes, total lavage protein content, and both cyclooxygenase (COX)1 and COX2 protein content (Bauer, Dwyer-Nield, Keil, et al., 2001), however other studies demonstrated increases in neutrophils, myeloid derived suppressor cells, and specific macrophage populations (Alexander et al., 2016; Redente et al., 2010; Redente, Orlicky, Bouchard, & Malkinson, 2007; Vikis et al., 2012).

We briefly review the knockout mouse or transgenic mouse models used with the MCA/BHT model as well as chemoprevention studies performed. In Table 1, five models are listed that were used to examine the role of prostaglandin E2 receptor (Ptger2), prostacyclin (Ptgis),toll-like receptor 4 (TLR4), and epiregulin (Ereg) pathways in tumor promotion. Ptgis (PGI2) overexpression on an FVB background in the MCA/BHT model led to significantly reduced tumor development and Ptger2 deficient mice on a BALB/c background also led to reduced tumor development (Keith et al., 2006; Keith et al., 2002). PGI2 is anti-inflammatory in the lung while PGE2 (the ligand for PTGER2) is pro-inflammatory and hence pro-tumorigenic. The TLR4 pathway is important in innate immune function and TLR4 deficient mice on a BALB/cJ background were significantly more sensitive to MCA/BHT; increased tumorigenesis and inflammation were observed in these mice (Alexander et al., 2016; Bauer et al., 2005). Similarly, IFNγ an innate immune cytokine (Th1), and IFNγ deficient mice on a BALB/cJ background also demonstrated increased tumor numbers suggesting innate immunity is a protective mechanism in lung cancer (unpublished, Dr. A.K. Bauer, personal communication).

Table 1:

Inflammatory mediators tested using the MCA/BHT model.

Gene Pathway Null or transgenic Role in inflammation Response in MCA/BHT model (change in tumor multiplicity) Reference
Ereg EGFR pathway Null Augmentation ▼ (Bauer et al., 2017a)
Ptger2 Eicosanoid pathway Null Augmentation ▼ (Keith et al., 2006)
Ptgis Eicosanoid pathway Transgenic Protection ▼▼ (Keith et al., 2002)
Tlr4 Innate immune receptors Null Protection ▲▲ (Alexander et al., 2016; Bauer et al., 2005)
Open in a new tab

Ereg, epiregulin; Ptger2, Prostaglandin receptor subtype E2 (EP2); Ptgis, prostaglandin I2 (prostacyclin) synthase; Tlr4, toll-like receptor 4.

Several intervention studies also used the MCA/BHT model. Depletion of macrophages using chlorinated water significantly reduced promotion in BALB/ByJ mice supporting the importance of the macrophages in this model (Bauer, Dwyer-Nield, Keil, et al., 2001). To investigate the prostaglandin pathway in more detail, both aspirin and Celecoxib were used in the MCA/BHT model. However, neither intervention reduced tumor number and both increased tumor size (Kisley et al., 2002). These findings in the MCA/BHT model suggest that these sorts of global pathway interventions (i.e. all prostaglandins) will not be effective due to a delicate balance between the lung prostaglandins that are anti-inflammatory (PGI2) and pro-inflammatory (PGE2).

Lastly, as mentioned in section 1, another component of tumor promotion that is a known Hallmark of Cancer is the inhibition of gap junctions. Gap junctions are composed of connexin proteins and CX43 is the primary connexin protein found in lung, specifically the precursor cell types to the development of ADC, such as the alveolar type II cell. When BHT was used in vitro in type II cell lines (C10 and E10), CX43 protein was significantly reduced (Chaudhuri et al., 1993; Guan et al., 1995). We observed reductions in Cx43 mRNA expression in early tumorigenesis in the MCA/BHT model (Bauer et al., 2009) and also demonstrated that the dysregulation of gap junction activity during MCA/BHT-induced promotion is increased in TLR4-deficient animals (Hill et al., 2013). These findings were further supported by others using Cx43 heterozygous mice treated with urethane where the Cx43+/− mice were more susceptible to tumor development (Avanzo et al., 2004). Thus, both of these components, inflammation and gap junctions, are features identified in many promotion models.

Herein we provide a detailed protocol for using the MCA/BHT model in mice, including notes on troubleshooting any issues that may arise and optimization (see Section 4).

3. PROTOCOL FOR THE MCA/BHT MODEL.

3.1. Study Design for the model

3.1.1. Materials.

BHT (Sigma, St. Louis)

MCA (Sigma or Accustandard; all solid)

Corn oil* (Mazola or any brand that does not contain BHT or BHA)

5 ml glass or clear plastic tubes

Vortex

Parafilm

1 ml syringes

26 g needles

Balance to weigh mice

*The vehicle for these compounds; the oil is stored at 4° C and replaced monthly.

3.1.2. Treatment groups and number of animals.

Four treatment groups are essential for the MCA/BHT model. Group 1, vehicle control; group 2, MCA + vehicle; group 3, vehicle + BHT; group 4 MCA + BHT (see Figure 1; see Note 4.1). Ideally, twenty mice per treatment group are typically required to detect statistically significant differences in tumor number (i.e. 30% over control groups) given the variability observed in previous studies for BALB/c or BALB/cByJ mice (Bauer et al., 2005; Bauer, Dwyer-Nield, Keil, et al., 2001; Bauer et al., 2017b; A.M. Malkinson et al., 1997). However, this number is strain-dependent; some strains such as A/J show 15-fold differences in tumor number with BHT and therefore require fewer mice (n= ~5-10 per group). For additional endpoints, fewer animals can be used (i.e., n = 5 mice per group for molecular analysis or histopathology). As with all animal studies, it is best to repeat the entire study at least once to confirm the response. Due to differences in BHT metabolism noted above, female mice are more sensitive to BHT, and require a dose response pilot study for BHT to determine their minimum tolerated dose (MTD) (see Note 4.2).

Figure 1. Schematic of the MCA/BHT model.

Figure 1.

Open in a new tab

A. MCA (red) is injected ip. to the mouse week 1 followed by BHT (blue) or vehicle control weeks 2-7. The vehicle for both MCA and BHT is corn oil. The mice are then observed and weighed for up to 30 weeks. During that time, if a CT scanner is available, the tumors can be monitored. Numerous endpoints can be evaluated at the sacrifice time points. B. Treatment groups 1-4. Groups 2 and 4 receive MCA, groups 3 and 4 receive BHT.

3.1.3. Week 1: MCA preparation and dosing.

Weigh the mice before preparing the MCA to determine the weight range (Note 4.3). Prepare MCA in corn oil using glass or clear plastic 5 ml tubes. Weigh MCA to reach a dose of 10 μg/g in 100 μl for the number of mice being injected, add corn oil, parafilm the tube and gently heat (60° F, max. temperature) with occasional vortexing to dissolve the MCA (Note 4.4 and 4.5). For example, for 10 mice that weigh 20 g each, add 2 mg MCA in 1 ml corn oil; this is for the 10 μg/g dose. This can be made the prior day. See Note 4.4 for additional tips.

  1. Use a 1 ml syringe and 26 g needles to inject mice ip. with ~100 μl of MCA (resulting in 10 μg/g dose); volume can be adjusted to account for changes in animal weight. Corn oil (100 μl/mouse) is injected into the vehicle control mice.

3.1.4. Week 2: BHT preparation and dosing.

Weigh the mice before BHT is prepared to determine dose range (see Note 4.3). BHT is prepared in 5 ml glass or clear plastic tubes and can be made the day prior to dosing.For most inbred strains of mice, use 150 mg/kg BHT for the first dose (wk 1) (see Note 4.2 on dose response). For example, if you have 10 mice that are 20 g each, add 30 mg BHT into 1 ml of corn oil. BHT dissolves under hot water within 1 hr, but can require frequent vortexing, similar to MCA preparation. Because there is a circadian influence on BHT metabolism, mice need to be dosed in the morning, preferably before 10 am (Bauer, Dwyer-Nield, Keil, et al., 2001).

  1. Use a 1 ml syringe and 26 g needles to inject mice i.p. with ~100 μl of BHT resulting in a 150 mg/kg dose for wk 1; if the mice are not the exact weight for that dose, then adjust the amount dosed accordingly. The corn oil is used for the vehicle control animals and the MCA + vehicle group.

3.1.5. Weeks 3-7: BHT preparation and dosing.

For these weeks, the dose of BHT is increased to 200 mg/kg for most strains (see Note 4.2 on dose response). For example, for 10 mice that are 20 g each, add 40 mg BHT into 1 ml corn oil. Mice are then injected i.p. with ~100 l of BHT to reach a 200 mg/kg dose for weeks 2-7, with one dose per week on the same day and at the ~ same time each week (< 10 am).

3.1.6. Weeks 8-30.

Mice should be weighed weekly for the remainder of the study to ensure that the mice are not losing weight since caloric restriction is chemopreventative and therefore can interfere with experimental interpretation (Tannenbaum, 1940; Tannenbaum & Silverstone, 1949).

3.2. Final sacrifice for tumor endpoints

3.2.1. Materials and Equipment (vary depending on the endpoints)

All endpoints:

  • Surgical tools such as fine-tipped forceps and fine surgical scissors

  • 20-24 g plastic catheter (or metal stubber needle)

  • Dissecting microscope with fiberoptic lighting for tumor dissection

  • Digital caliper

  • Fatal Plus or similar approved euthanasia solution

Fixed tissue for tumor counts and other histology endpoints:

  • Tellyesniczky’s fixative (see note 4.7)

  • 10% Neutral Buffered Formalin (NBF)

  • 70% Ethanol

  • Sterile saline

  • 1 and 5 ml syringes

  • Olympus BX43 inverted microscope with a digital camera (or similar inverted scope)

Unfixed tissue for tumor counting and biochemical analysis:

  • 1 ml tubes with screw tops to flash freeze tissue (see 3.2.4.C)

3.2.2. Final sacrifice endpoint.

The mice are typically sacrificed between 20 - 30 wk. At 20 wk, small adenomas exist and are visible on the lung surface, while at 30 wk, the tumors are significantly larger and some adenocarcinomas are present. Earlier time points can also be used to assess smaller adenomas, hyperplasias, and the inflammatory microenvironment of the tumor bearing lungs. The manner in which the tumors are analyzed depends on the equipment available. If a micro-CT is available, then assessing tumor development from weeks 8-30, would be a valuable tool.

  1. For the final sacrifice of tumor analysis, mice are weighed and euthanized with 120 mg/kg Fatal Plus solution (MWI) or whatever accepted method of euthanasia is preferred (Note 4.6).

  2. At this point, the lung tumors can be counted for in fixed tissue (see below) or unfixed tissue (section 3.2.4). There is an alternative to these two choices (see Note 4.7).
    1. For lung fixation, the mice can be fixed in several ways for surface tumor analysis. 1) Using Tellyesniczky’s fixative that turns the tumors bright white and the lung opaque (see Note 4.8) or 2) in 10% NBF.Unless the lungs are processed for other endpoints, it is best to use the entire lung for counting tumors. If one of the goals is to do a lot of immunostaining, the best fixative is NBF; Tellyesniczky’s fixative is not good for epitope retrieval for most targets. Tellyesniczky’s fixative likely turns the tumors bright white due to the acetic acid in the fixative. Acetic acid causes the color change due to the increased percentage of abnormal nuclear protein and increased number of dysplastic cells and is used in colposcopies for cervival cancer detection (https://screening.iarc.fr/colpochap.php?lang=1&chap=4).
    2. Prior to fixation, carefully perfuse the lung through the heart with sterile saline using a 5 ml syringe to remove the blood followed by careful and slow inflation of the lung with fixative via trachea (~1ml/25 g body weight). To do so, make a 1.5-2 mm small longitudinal incision in the trachea using sharp scissors on the ventral side of the neck. Insert a 20-24 g plastic catheter (or metal stubber needle) into the trachea about 0.5 cm. Ensure that the catheter is not inserted too far down into the trachea, as this can lead to damage of the lung structure. After the lung is inflated based on body weight (~1 ml/25g body weight), tie off the trachea.
    3. The lung is then carefully removed en bloc and submersed into fixative for 24 (NBF) or 48 (Tellyesniczchy’s) h. For the NBS, the lung is transferred to 70% ethanol after 24 h of fixation (to maintain epitope integrity) and for Tellyesniczchy’s, the same after 48 h.
    4. After at least 48 h, each lung lobe is carefully dissected apart from the other using a fine-tipped forceps and kept in the fixative prior to counting (see Figure 2).
  3. To evaluate the surface tumors between 20-30 wk, a dissecting microscope is used with careful analysis. Each lobe is carefully counted for tumors and each tumor is sized at the same time using a digital caliper. Tumors less than 200 μm are typically considered microadenomas if they are round and visible. However, this is subjective. Hyperplasias and dysplasias follow the normal lung architecture.
    1. All tumor counts from the 5 lobes are combined and considered tumor multiplicity for that mouse.
    2. Then, all tumor numbers per mouse are averaged to determine the tumor multiplicity differences between treatments (i.e., vehicle control, MCA, BHT, and MCA/BHT).
    3. Tumor sizes are determined using a digital caliper. For spherical lesions, the calculations for tumor volumes use (4/3 π)(r3) and for non-spherical lesions (Length × Width × Height).
    4. The lungs are then processed and sectioned for histology, typically at a histology core.
Figure 2. Lung lobes of mouse dissected apart to count tumors.

Figure 2.

Open in a new tab

Lungs were fixed in Tellyesniczky’s fixative. Red arrows indicate tumors on the surface of the lung. These are most likely adenomas.

3.2.3. Histological analysis of fixed lung tissue.

  1. To analyze the tumor numbers, tumor size, and tumor morphology by histology, the lung is serially sectioned (5 μm sections; 150 slides/mouse; H & E stained), followed by analyzing every 10th slide for a total of ~15 slides/mouse (O’Donnell et al., 2006). We then look at the location to make sure the larger tumors are not double counted. Processing the lung in this way will provide experimental consistency.

  2. Histological examination of the lung sections will allow for identification of hyperplastic lesions, microadenomas, adenomas, and adenocarcinomas using an inverted microscope. Counts are done histologically on every 10th slide and compared to the surface tumor counts. The numbers will be similar but unlikely the same; histology is more sensitive and accurate.

  3. Histological examination of tumor sizes is determined by tumor areas.

  4. Tumor morphology is evaluated by determining the number of hyperplasias, dysplasias, adenomas, adenocarcinomas, and invasive adenocarcinomas. Histopathology should be performed by two independent reviewers on blinded samples for consistency followed by 10% of the samples reviewed by a board-certified pathologist.

3.2.4. Biochemical analysis of unfixed tumors.

  1. Mice are euthanized the same way with Fatal Plus, and the lungs can be perfused prior to removal if preferred. Lungs are then removed and lobes separated, as in 3.2.2.B.4. This is an alternative to counting fixed lungs, but the tumor counting is done on the day of the sacrifice, thus less mice can be sacrificed per day.

  2. The tumors are counted the same way as described above in section 3.2.2.C (1-3).

  3. Positive features of this approach are that the tumors can be micro-dissected under the dissecting microscope and flash frozen for biochemical analysis (e.g., protein, DNA, and RNA analysis (in RNAlater)). In addition, this method allows for weighing the tumors as another measure of tumor burden. See the following references as examples (Bauer et al., 2009; L. Dwyer-Nield et al., 2017; L. D. Dwyer-Nield et al., 2010). These tumors can be used for many techniques including omics such as transcriptomics, metabolomics, and proteomics. Some of the genes and proteins that we commonly assess are Cx43 via RT-PCR and immunoblots, and numerous cytokines/chemokines using standard ELISAs.

3.3. Final sacrifice for inflammation endpoints

3.3.1. Materials and equipment required

Fatal Plus

Benchtop centrifuge for 500 x g spin

Sterile HBSS or PBS

EDTA

Scissors and forceps

20-24 g plastic catheter

1 ml syringes

5 ml plastic tubes

Hemocytometer (or any cell counter)

BioRad Protein Assay Reagent

Microscope slides*

Cytospin

Diff-quik (Baxter)

4 Coplin jars

Additional materials for specific assays (optional):

Neutrophil myeloperoxidase (Cytostore, Canada)

CD3 antibody (ab5690 Abcam)

F4/80 antibody (MF48000, Caltag)

NIMP-R14 (sc59338, Santa Cruz)

Inverted microscope with a digital camera

3.3.2. Inflammation analysis in the tumor bearing lungs.

Inflammation can be assessed in numerous manners including bronchoalveolar lavage, histology, and by flow cytometry. We will discuss the bronchoalveolar lavage method and histological analysis but will refer the readers to several papers on using flow cytometric analysis, which is beyond the scope of this methods paper (Alexander et al., 2016; Redente et al., 2010; Redente et al., 2007).

  1. Bronchoalveolar lavage (BAL) analysis.

    BAL can be done prior to lung fixation to determine the inflammatory cell content of the lungs prior to counting tumors. This is controversial but only removes a small portion of the inflammatory cell content (Meyer et al., 2012; Saltini et al., 1984). To do so, following Fatal Plus (see section 3.2.2.A) and perfusion to remove the blood (see section 3.2.2.B.2), follow these instructions.

    1. Follow 3.2.2.B.2 and make a small incision in the trachea. Insert a 20-24 g plastic catheter into the trachea, careful not to insert the catheter too far down into the trachea.

    2. Use a 1 ml syringe filled with Hanks’ balanced salt solution (HBSS) (HBSS, sterile, Sigma) with 0.6 mM EDTA and insert the fluid into the lung at ~1 ml/ 25g body weight for a whole lung lavage (or 0.5 ml/25 g BW for ½ lung lavage), thus the lavage is done based on mouse weight. Sterile PBS containing 0.6 mM EDTA can also be used instead of HBSS. Collect the first lavage into one 5 ml tube and do three more, collected into a second 5 ml tube, for a total of 4 lavages. This number can be increased if you are doing flow cytometry on the BAL cells, newer technologies such as single cell RNA sequencing, or culturing the BAL cells in vitro.

    3. Tubes should be placed on ice until lavages are completed for all mice. Samples are then centrifuged at 500 × g at 4°C for 10 min.

    4. Following centrifugation, pipette off the supernatant carefully (pellet is loose) from the first lavage return and place in a fresh tube for determination of total protein, an indicator of lung permeability. The rest of this sample can be used for cytokine/chemokine analysis.
      1. Total BALF protein concentration is measured following the method of Bradford (23) using the BioRad protein Assay reagent (BioRad, Hercules, CA). LDH can also be measured as a marker of acute lung injury, however it is a more expensive assay.
      2. Neutrophil myeloperoxidase (MPO) can be detected in BALF using a colorimetric analysis kit (Cytostore, Canada), especially if neutrophils are expected but not observed and cell lysis is suspected(Bauer et al., 2011).

    5. Following the removal of the supernatant from tube 2, the cell pellets are combined by adding 1 ml of HBSS to tube 1 and then into tube 2. The supernatant from tube two is typically too dilute and usually discarded.

    6. Total cell counts on the BAL cells are performed using a hemocytometer or automated cell counter.

    7. Cells are cytospun onto slides by taking an aliquot (usually between 100-200 μl or ~30,000 cells) of BAL cell suspension using a cytospin such as Shandon (Thermo Scientific). Cytocentrifuge for 10 minutes at 600 rpm. (See Note 4.9).

    8. Stain slides with Wright-Giemsa stain (Diff-Quik; Baxter Scientific Products, McGaw Park, IL) for differential cell analysis. Each slide is then dipped into the solution carefully and slowly 5 times, ensuring that the entire region containing the cells is completely covered in solution. The cells are not always attached to the slides that firmly so caution must be used. There are three solutions labeled fixative, solution 1 and solution 2 and these are propriatary. The solutions should be used in that order followed by 5 dips in distilled H2O. Differential counts for macrophages, polymorphonuclear leukocytes (PMNs), lymphoctyes, monocytes, eosinophils, and epithelial cells are assessed by identifying 300 cells according to standard cytologic techniques.
      1. To calculate total cells by cell type, take total cell count per mouse and multiple by the percent for each specific cell type (e.g., 120,000 cells total x 95% macrophages = 114,000 total macrophages).

  2. Histology for analysis of inflammation.

    Histopathological analysis can be used to identify inflammatory cell types in lung sections by morphology (Pawlina, 2006). However, immunostaining can also be used to validate cell types (Note 4.10).

    1. Specific staining protocols to follow are those at the National Institute of Environmental Health Sciences histology core website (https://www.niehs.nih.gov/research/resources/protocols/protocols-immuno/index.cfm). For example, lung sections can be stained for lymphocytes using antibodies for CD3 (ab5690; Abcam, Cambridge, MA) as a pan-T-lymphocyte marker and F4/80 (MF48000; Caltag, Burlingame, CA) as a pan-macrophage marker (Bauer et al., 2009). In addition, PMNs can be stained using neutrophil-specific marker (NIMP-R14; sc-59338)(Rondini, Walters, & Bauer, 2010). Other specific markers can be used as well such as arginase 1 (Arg1) for M2 macrophages and inducible nitric oxide synthase (iNOS) for M1 macrophage markers or specific subtypes of lymphocytes (e.g., CD3 with CD4 and CD8) (Redente et al., 2010; Redente et al., 2007). Standard protocols can be found at the NIEHS website.

    2. For analysis, H scores can be determined by the percentage of staining using a 1–3 scoring method with the following formula ([1 x (% cells 1+) + 2 x (% cells 2+) + 3 x (% cells 3+)].(Hirsch et al., 2008; Hirsch et al., 2003). Immunohistochemical staining analysis should be performed by two independent reviewers on blinded samples for consistency followed by 10% of the samples reviewed by a board-certified pathologist.

5. CONCLUSIONS

The MCA-BHT 2-stage model has been used for over 20 years by numerous investigators. Understanding the different mechanisms and etiologies of lung cancer are critical to improve current therapies and identify novel targets. The MCA/BHT model allows the careful evaluation of promotion, an early stage of cancer, and depending on strain, can lead to detection of pathways that are unique to promotion. Additionally, MCA is a potent initiator at low doses and is now used for environmental or occupational tumor promoter studies and compared to the MCA/BHT model (Falcone et al., 2018; Falcone et al., 2017; Rondini et al., 2010; Sargent et al., 2014). Comparison among different promoters could identify these needed novel targets for future intervention studies in lung cancer.

ACKNOWLEDGEMENTS

This work was funded in part by the American Cancer Society RSG-10-162-01-LIB (AKB) and the R15ES024893 (AKB), R01 CA334497 (AMM, LDN), and R01 CA132552 (LDN). We also want to dedicate this chapter to our mentor, colleague, and friend, Dr. Alvin M. Malkinson, who was a pioneer in lung tumor promotion using the BHT model.

4. NOTES FOR OPTIMIZATION AND TROUBLESHOOTING

4.1.

Group 3 which is BHT only is often not run as it has been validated in numerous other studies that BHT alone is not a carcinogen. However, BHT does elicit inflammation (Bauer, Dwyer-Nield, Hankin, et al., 2001; Bauer, Dwyer-Nield, Keil, et al., 2001).

4.2.

Depending on the strain used, you may need to do a dose response for the BHT prior to performing the study. For example, most sensitive inbred strains (A/J, BALB/cJ, BALB/ByJ, FVB/J, SWR, C57BL/6J) require 75-150 mg/kg of BHT for the first dose. FVB require between 75-100 mg/kg for the first dose while BALB/ByJ receive 150 mg/kg, thus sensitivity varies by strain. If you are using a knockout or transgenic strain with BHT, it is best to do a dose response in the range of 25-150 mg/kg BHT to determine MTD. A pilot study for doses where only 1 dose of BHT is administered i.p. followed by sacrificing the animals on days 3-6 after the BHT exposure, will determine the sensitivity of that strain; this is based on several strains (GRS and CXB6) where increased sensitivity was observed up to 6 days. A two or three week study could also be performed with one dose of BHT per week and animals sacrificed between 1-6 days following BHT.

4.3.

Always dose animals based on weight and always dose them accurately for MCA and BHT dosing, i.e., do not dose all the mice the same amount if they are not the same weight. Accurate dosing will not only ensure the animals survive, it will also keep experimental variability to a minimum. In addition, keep careful track of all weights of the mice throughout the entire study, from week 1 until sacrifice.

4.4.

This can take a long time and incubation in a heat block may be necessary between 37-50°C. Every 15 minutes, vortex the tube until the MCA is completely dissolved. Sometimes there are small micro-pieces that are challenging to dissolve. Keep vortexing.

4.5.

MCA doses can be lowered if using more sensitive strains in order to decrease tumor development in the absence of BHT (e.g., A/J mice are initiated using 5-7.5 g/g dose)(L. D. Dwyer-Nield et al., 2010). It is also important to note that if your KO or transgenic mice are on a B6 or 129/Sv background, it is unlikely you will find the MCA/BHT model useful since the B6 mice are not initiated with MCA. For example, Dwyer-Nield et. al. (2010) used up to 45 g/g of MCA and were unable to initiate tumors in some of the chromosome substitution strains used, supporting the resistance of certain strains to MCA initiation (L. D. Dwyer-Nield et al., 2010).

4.6.

Because it is a lung study, it is best not to use CO asphyxiation or that may interfere with results. Additionally, decapitation is also not preferred for lung studies.

4.7.

The alternative to fixing the entire lung is to carefully clamp off the left lobe at the major left bronchus. The right lobes can be fixed for histology and counting after the left lobe is removed. If this is done, tying off the left lobe is preferred to enable proper fixation of the right lobe. The left lobe should be removed prior to fixation as all fixatives inhibit any biochemical analysis. This method is best if the endpoint is not counting tumors since counting would only be on ~½ lung. However, this approach allows the investigator to both analyze the lungs histologically and biochemically. BAL can also be done in this manner on ½ lung followed by fixation, however, this is a less well accepted method. This alternative does allow more comparisons from the same lung.

4.8.

Tellyesniczky’s fixative: 64 ml 100% EtOH, 5.5 ml PBS, 17.4 ml H2O, 8.7 ml 37% formaldehyde, 4.4 ml glacial acetic acid. Inflate lungs using the fixative and tie off trachea, remove entire lung and cardiac tissue intact, place into container with fixative for 48 h prior to counting and sizing tumors.

4.9.

Slides used for the cytospins depend on the intent. If the slides are only for doing cell differentials, then the cheapest slides are fine with no coating. If the intention is for immunostaining of any kind, then coated slides should be used to enhance the surface for cell attachment.

4.10.

Most immunostaining will not work with Tellyesniczky’s fixative, thus if the experimental plan is to stain for any of these markers, it is best to use 10% NBF. One stain that does work with Tellyesniczky’s fixation is PCNA, used to detect proliferating cells.

REFERENCES

  1. Adamson IY, Bowden DH, Cote MG, & Witschi H (1977). Lung injury induced by butylated hydroxytoluene: cytodynamic and biochemical studies in mice. Lab Invest, 36(1), 26–32. [PubMed] [Google Scholar]
  2. Alexander CM, Xiong KN, Velmurugan K, Xiong J, Osgood RS, & Bauer AK (2016). Differential innate immune cell signatures and effects regulated by toll-like receptor 4 during murine lung tumor promotion. Exp Lung Res, 42(3), 154–173. doi: 10.3109/01902148.2016.1164263 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. American Cancer Society. (2020). Non-small cell lung cancer survival rates by stage. Retrieved from https://www.cancer.org/cancer/non-small-cell-lung-cancer/about/key-statistics.html
  4. Avanzo JL, Mesnil M, Hernandez-Blazquez FJ, Mackowiak II, Mori CM, da Silva TC, … Dagli ML (2004). Increased susceptibility to urethane-induced lung tumors in mice with decreased expression of connexin43. Carcinogenesis, 25(10), 1973–1982. [DOI] [PubMed] [Google Scholar]
  5. Bauer AK, Cho HY, Miller-Degraff L, Walker C, Helms K, Fostel J, … Kleeberger SR (2011). Targeted deletion of Nrf2 reduces urethane-induced lung tumor development in mice. PLoS One, 6(10), e26590. doi: 10.1371/journal.pone.0026590 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bauer AK, Dixon D, DeGraff LM, Cho HY, Walker CR, Malkinson AM, & Kleeberger SR (2005). Toll-like receptor 4 in butylated hydroxytoluene-induced mouse pulmonary inflammation and tumorigenesis. J Natl Cancer Inst, 97(23), 1778–1781. [DOI] [PubMed] [Google Scholar]
  7. Bauer AK, Dwyer-Nield LD, Hankin JA, Murphy RC, & Malkinson AM (2001). The lung tumor promoter, butylated hydroxytoluene (BHT), causes chronic inflammation in promotion-sensitive BALB/cByJ mice but not in promotion-resistant CXB4 mice. Toxicology, 169(1), 1–15. [DOI] [PubMed] [Google Scholar]
  8. Bauer AK, Dwyer-Nield LD, Keil K, Koski K, & Malkinson AM (2001). Butylated hydroxytoluene (BHT) induction of pulmonary inflammation: a role in tumor promotion. Exp Lung Res, 27(3), 197–216. [DOI] [PubMed] [Google Scholar]
  9. Bauer AK, Fostel J, Degraff LM, Rondini EA, Walker C, Grissom SF, … Kleeberger SR (2009). Transcriptomic analysis of pathways regulated by toll-like receptor 4 in a murine model of chronic pulmonary inflammation and carcinogenesis. Mol Cancer, 8, 107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bauer AK, Malkinson AM, & Kleeberger SR (2004). Susceptibility to neoplastic and non-neoplastic pulmonary diseases in mice: genetic similarities. Am J Physiol Lung Cell Mol Physiol, 287(4), L685–703. [DOI] [PubMed] [Google Scholar]
  11. Bauer AK, & Rondini EA (2009). Review paper: the role of inflammation in mouse pulmonary neoplasia. Vet Pathol, 46(3), 369–390. [DOI] [PubMed] [Google Scholar]
  12. Bauer AK, Umer M, Richardson VL, Cumpian AM, Harder AQ, Khosravi N, … Moghaddam SJ (2018). Requirement for MUC5AC in KRAS-dependent lung carcinogenesis. JCI Insight, 3(15). doi: 10.1172/jci.insight.120941 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Bauer AK, Velmurugan K, Plottner S, Siegrist KJ, Romo D, Welge P, … Kafferlein HU (2018). Environmentally prevalent polycyclic aromatic hydrocarbons can elicit co-carcinogenic properties in an in vitro murine lung epithelial cell model. Arch Toxicol, 92(3), 1311–1322. doi: 10.1007/s00204-017-2124-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Bauer AK, Velmurugan K, Xiong KN, Alexander CM, Xiong J, & Brooks R (2017a). Epiregulin is required for lung tumor promotion in a murine two-stage carcinogenesis model. Mol Carcinog. doi: 10.1002/mc.22475 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Bauer AK, Velmurugan K, Xiong KN, Alexander CM, Xiong J, & Brooks R (2017b). Epiregulin is required for lung tumor promotion in a murine two-stage carcinogenesis model. Mol Carcinog, 56(1), 94–105. doi: 10.1002/mc.22475 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cesen-Cummings K, Fernstrom MJ, Malkinson AM, & Ruch RJ (1998). Frequent reduction of gap junctional intercellular communication and connexin43 expression in human and mouse lung carcinoma cells. Carcinogenesis, 19(1), 61–67. [DOI] [PubMed] [Google Scholar]
  17. Chaudhuri R, Sigler K, Dupont E, Trosko JE, Malkinson AM, & Ruch RJ (1993). Gap junctional intercellular communication in mouse lung epithelial cell lines: effects of cell transformation and tumor promoters. Cancer Lett, 71(1-3), 11–18. [DOI] [PubMed] [Google Scholar]
  18. Dwyer-Nield L, Hickey GA, Friedman M, Choo K, McArthur DG, Tennis MA, … Keith RL (2017). The Second-Generation PGI2 Analogue Treprostinil Fails to Chemoprevent Tumors in a Murine Lung Adenocarcinoma Model. Cancer Prev Res (Phila), 10(11), 671–679. doi: 10.1158/1940-6207.CAPR-17-0050 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Dwyer-Nield LD, McQuillan J, Hill-Baskin A, Radcliffe RA, You M, Nadeau JH, & Malkinson AM (2010). Epistatic interactions govern chemically-induced lung tumor susceptibility and Kras mutation site in murine C57BL/6J-ChrA/J chromosome substitution strains. Int J Cancer, 126(1), 125–132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Falcone LM, Erdely A, Kodali V, Salmen R, Battelli LA, Dodd T, … Zeidler-Erdely PC (2018). Inhalation of iron-abundant gas metal arc welding-mild steel fume promotes lung tumors in mice. Toxicology, 409, 24–32. doi: 10.1016/j.tox.2018.07.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Falcone LM, Erdely A, Meighan TG, Battelli LA, Salmen R, McKinney W, … Zeidler-Erdely PC (2017). Inhalation of gas metal arc-stainless steel welding fume promotes lung tumorigenesis in A/J mice. Arch Toxicol, 91(8), 2953–2962. doi: 10.1007/s00204-016-1909-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fritz JM, Dwyer-Nield LD, Russell BM, & Malkinson AM (2010). The Kras mutational spectra of chemically induced lung tumors in different inbred mice mimics the spectra of KRAS mutations in adenocarcinomas in smokers versus nonsmokers. J Thorac Oncol, 5(2), 254–257. doi: 10.1097/JTO.0b013e3181c8ce04 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fritz JM, Tennis MA, Orlicky DJ, Lin H, Ju C, Redente EF, … Dwyer-Nield LD (2014). Depletion of tumor-associated macrophages slows the growth of chemically induced mouse lung adenocarcinomas. Front Immunol, 5, 587. doi: 10.3389/fimmu.2014.00587 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ghosh M, Dwyer-Nield LD, Kwon JB, Barthel L, Janssen WJ, Merrick DT, & Keith RL (2015). Tracheal dysplasia precedes bronchial dysplasia in mouse model of N-nitroso trischloroethylurea induced squamous cell lung cancer. PLoS One, 10(4), e0122823. doi: 10.1371/journal.pone.0122823 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Guan X, Hardenbrook J, Fernstrom MJ, Chaudhuri R, Malkinson AM, & Ruch RJ (1995). Down-regulation by butylated hydroxytoluene of the number and function of gap junctions in epithelial cell lines derived from mouse lung and rat liver. Carcinogenesis, 16(10), 2575–2582. [DOI] [PubMed] [Google Scholar]
  26. Hanahan D, & Weinberg RA (2011). Hallmarks of cancer: the next generation. Cell, 144(5), 646–674. doi: 10.1016/j.cell.2011.02.013 [DOI] [PubMed] [Google Scholar]
  27. Hashita T, Sakuma T, Akada M, Nakajima A, Yamahara H, Ito S, … Nemoto N (2008). Forkhead box A2-mediated regulation of female-predominant expression of the mouse Cyp2b9 gene. Drug Metab Dispos, 36(6), 1080–1087. doi: 10.1124/dmd.107.019729 [DOI] [PubMed] [Google Scholar]
  28. Hill T 3rd, Osgood RS, Velmurugan K, Alexander CM, Upham BL, & Bauer AK (2013). Bronchoalveolar Lavage Fluid Utilized Ex Vivo to Validate In Vivo Findings: Inhibition of Gap Junction Activity in Lung Tumor Promotion is Toll-Like Receptor 4-Dependent. J Mol Biomark Diagn, 5(1). doi: 10.4172/2155-9929.1000160 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hirsch FR, Dziadziuszko R, Thatcher N, Mann H, Watkins C, Parums DV, … Franklin WA (2008). Epidermal growth factor receptor immunohistochemistry: comparison of antibodies and cutoff points to predict benefit from gefitinib in a phase 3 placebo-controlled study in advanced nonsmall-cell lung cancer. Cancer, 112(5), 1114–1121. doi: 10.1002/cncr.23282 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hirsch FR, Varella-Garcia M, Bunn PA Jr., Di Maria MV, Veve R, Bremmes RM, … Franklin WA (2003). Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol, 21(20), 3798–3807. doi: 10.1200/JCO.2003.11.069 [DOI] [PubMed] [Google Scholar]
  31. Hudish TM, Opincariu LI, Mozer AB, Johnson MS, Cleaver TG, Malkoski SP, … Keith RL (2012). N-nitroso-tris-chloroethylurea induces premalignant squamous dysplasia in mice. Cancer Prev Res (Phila), 5(2), 283–289. doi: 10.1158/1940-6207.CAPR-11-0257 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. International Agency for Research on Cancer (IARC). (2012). Arsenic, Metals, Fibres and Dusts. Retrieved from Lyon, France: [Google Scholar]
  33. International Agency for Research on Cancer (IARC). (2014). Diesel and Gasoline Engine Exhausts and Some Nitroarenes. Retrieved from Lyon, France: [Google Scholar]
  34. International Agency for Research on Cancer (IARC). (2016). Air pollution and cancer. Retrieved from Lyon, France: [Google Scholar]
  35. Jackson EL, Willis N, Mercer K, Bronson RT, Crowley D, Montoya R, … Tuveson DA (2001). Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev, 15(24), 3243–3248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Keith RL, Geraci MW, Nana-Sinkam SP, Breyer RM, Hudish TM, Meyer AM, … Dwyer-Nield LD (2006). Prostaglandin E2 receptor subtype 2 (EP2) null mice are protected against murine lung tumorigenesis. Anticancer Res, 26(4B), 2857–2861. [PubMed] [Google Scholar]
  37. Keith RL, Miller YE, Hoshikawa Y, Moore MD, Gesell TL, Gao B, … Geraci MW (2002). Manipulation of pulmonary prostacyclin synthase expression prevents murine lung cancer. Cancer Res, 62(3), 734–740. [PubMed] [Google Scholar]
  38. Kisley LR, Barrett BS, Dwyer-Nield LD, Bauer AK, Thompson DC, & Malkinson AM (2002). Celecoxib reduces pulmonary inflammation but not lung tumorigenesis in mice. Carcinogenesis, 23(10), 1653–1660. [DOI] [PubMed] [Google Scholar]
  39. Klaunig JE, Kamendulis LM, & Xu Y (2000). Epigenetic mechanisms of chemical carcinogenesis. Hum Exp Toxicol, 19(10), 543–555. [DOI] [PubMed] [Google Scholar]
  40. Malkinson AM (1979). Prevention of butylated hydroxytoluene-induced lung damage in mice by cedar terpene administration. Toxicol.Appl.Pharmacol, 49(3), 551–560. [DOI] [PubMed] [Google Scholar]
  41. Malkinson AM (1983). Modulation of the incidence of urethane induced mouse lung adenomas by butylated hydroxytoluene (BHT). Proc.West Pharmacol.Soc, 26, 417–419. [PubMed] [Google Scholar]
  42. Malkinson AM (1998). Molecular comparison of human and mouse pulmonary adenocarcinomas. Exp.Lung Res, 24(4), 541–555. [DOI] [PubMed] [Google Scholar]
  43. Malkinson AM, Koski KM, Evans WA, & Festing MF (1997). Butylated hydroxytoluene exposure is necessary to induce lung tumors in BALB mice treated with 3-methylcholanthrene. Cancer Res., 57(14), 2832–2834. [PubMed] [Google Scholar]
  44. Malkinson AM, Radcliffe RA, & Bauer AK (2002). Quantitative trait locus mapping of susceptibilities to butylated hydroxytoluene-induced lung tumor promotion and pulmonary inflammation in CXB mice. Carcinogenesis, 23(3), 411–417. [DOI] [PubMed] [Google Scholar]
  45. Meyer KC, Raghu G, Baughman RP, Brown KK, Costabel U, du Bois RM, … American Thoracic Society Committee on, B. A. L. i. I. L. D. (2012). An official American Thoracic Society clinical practice guideline: the clinical utility of bronchoalveolar lavage cellular analysis in interstitial lung disease. Am J Respir Crit Care Med, 185(9), 1004–1014. doi: 10.1164/rccm.201202-0320ST [DOI] [PubMed] [Google Scholar]
  46. Moghaddam SJ, Li H, Cho SN, Dishop MK, Wistuba II, Ji L, … Demayo FJ (2009). Promotion of lung carcinogenesis by chronic obstructive pulmonary disease-like airway inflammation in a K-ras-induced mouse model. Am J Respir Cell Mol Biol, 40(4), 443–453. doi: 10.1165/rcmb.2008-0198OC [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Nahta R, Al-Mulla F, Al-Temaimi R, Amedei A, Andrade-Vieira R, Bay SN, … Bisson WH (2015). Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression. Carcinogenesis, 36 Suppl 1, S2–18. doi: 10.1093/carcin/bgv028 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. O’Donnell EP, Zerbe LK, Dwyer-Nield LD, Kisley LR, & Malkinson AM (2006). Quantitative analysis of early chemically-induced pulmonary lesions in mice of varying susceptibilities to lung tumorigenesis. Cancer Lett, 241(2), 197–202. doi: 10.1016/j.canlet.2005.10.012 [DOI] [PubMed] [Google Scholar]
  49. Pawlina M. H. R. a. W.(2006). Respiratory System. In Histology: A text and atlas with correlated cell and molecular biology (pp. 612–645). Baltimore: Lippincott Williams and Wilkins. [Google Scholar]
  50. Redente EF, Dwyer-Nield LD, Merrick DT, Raina K, Agarwal R, Pao W, … Malkinson AM (2010). Tumor progression stage and anatomical site regulate tumor-associated macrophage and bone marrow-derived monocyte polarization. Am J Pathol, 176(6), 2972–2985. doi: 10.2353/ajpath.2010.090879 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Redente EF, Orlicky DJ, Bouchard RJ, & Malkinson AM (2007). Tumor signaling to the bone marrow changes the phenotype of monocytes and pulmonary macrophages during urethane-induced primary lung tumorigenesis in A/J mice. Am J Pathol, 170(2), 693–708. doi: 10.2353/ajpath.2007.060566 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Riely GJ, Kris MG, Rosenbaum D, Marks J, Li A, Chitale DA, … Ladanyi M (2008). Frequency and distinctive spectrum of KRAS mutations in never smokers with lung adenocarcinoma. Clin Cancer Res, 14(18), 5731–5734. doi: 10.1158/1078-0432.CCR-08-0646 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Romo D, Velmurugan K, Upham BL, Dwyer-Nield LD, & Bauer AK (2019). Dysregulation of Gap Junction Function and Cytokine Production in Response to Non-Genotoxic Polycyclic Aromatic Hydrocarbons in an In Vitro Lung Cell Model. Cancers (Basel), 11(4). doi: 10.3390/cancers11040572 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Rondini EA, Walters DM, & Bauer AK (2010). Vanadium pentoxide induces pulmonary inflammation and tumor promotion in a strain-dependent manner. Part Fibre Toxicol, 7, 9. doi: 10.1186/1743-8977-7-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Saltini C, Hance AJ, Ferrans VJ, Basset F, Bitterman PB, & Crystal RG (1984). Accurate quantification of cells recovered by bronchoalveolar lavage. Am Rev Respir Dis, 130(4), 650–658. doi: 10.1164/arrd.1984.130.4.650 [DOI] [PubMed] [Google Scholar]
  56. Sargent LM, Porter DW, Staska LM, Hubbs AF, Lowry DT, Battelli L, … Reynolds SH (2014). Promotion of lung adenocarcinoma following inhalation exposure to multi-walled carbon nanotubes. Part Fibre Toxicol, 11, 3. doi: 10.1186/1743-8977-11-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Schottenfeld D (2005). Etiology and Epidemiology of Lung Cancer. In Pass HI, Mitchell JB, Johnson DH, Turrisi AT, & Minna JD (Eds.), Lung Cancer-Principles and Practice (3rd ed., pp. 3–24). Philadelphia: Lippincott Williams and Wilkins. [Google Scholar]
  58. Siegrist KJ, Romo D, Upham BL, Armstrong M, Quinn K, Vanderlinden L, … Bauer AK (2019). Early Mechanistic Events Induced by Low Molecular Weight Polycyclic Aromatic Hydrocarbons in Mouse Lung Epithelial Cells: A Role for Eicosanoid Signaling. Toxicol Sci. doi: 10.1093/toxsci/kfz030 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Stathopoulos GT, Sherrill TP, Cheng DS, Scoggins RM, Han W, Polosukhin VV, … Blackwell TS (2007). Epithelial NF-kappaB activation promotes urethane-induced lung carcinogenesis. Proc Natl Acad Sci U S A, 104(47), 18514–18519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Stearman RS, Dwyer-Nield L, Zerbe L, Blaine SA, Chan Z, Bunn PA Jr., … Geraci MW (2005). Analysis of orthologous gene expression between human pulmonary adenocarcinoma and a carcinogen-induced murine model. Am J Pathol, 167(6), 1763–1775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Sun Y, Ren Y, Fang Z, Li C, Fang R, Gao B, … Ji H (2010). Lung adenocarcinoma from East Asian never-smokers is a disease largely defined by targetable oncogenic mutant kinases. J Clin Oncol, 28(30), 4616–4620. doi: 10.1200/JCO.2010.29.6038 [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Tannenbaum A (1940). The initiation and growth of tumors. Introduction. I. Effect of feeding. Am.J.Cancer, 38, 335. [Google Scholar]
  63. Tannenbaum A, & Silverstone H (1949). The influence of the degree of caloric restriction on the formation of skin tumors and hepatomas in mice. Cancer Res, 9(12), 724–727. [PubMed] [Google Scholar]
  64. Thompson JA, Bolton JL, & Malkinson AM (1991). Relationship between the metabolism of butylated hydroxytoluene (BHT) and lung tumor promotion in mice. Exp.Lung Res, 17(2), 439–453. [DOI] [PubMed] [Google Scholar]
  65. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, & Jemal A (2015). Global cancer statistics, 2012. CA Cancer J Clin, 65(2), 87–108. doi: 10.3322/caac.21262 [DOI] [PubMed] [Google Scholar]
  66. Vikis HG, Gelman AE, Franklin A, Stein L, Rymaszewski A, Zhu J, … You M (2012). Neutrophils are required for 3-methylcholanthrene-initiated, butylated hydroxytoluene-promoted lung carcinogenesis. Mol Carcinog, 51(12), 993–1002. doi: 10.1002/mc.20870 [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Wang Y, Zhang Z, Yan Y, Lemon WJ, LaRegina M, Morrison C, … You M (2004). A chemically induced model for squamous cell carcinoma of the lung in mice: histopathology and strain susceptibility. Cancer Res, 64(5), 1647–1654. [DOI] [PubMed] [Google Scholar]
  68. Witschi H, Malkinson AM, & Thompson JA (1989). Metabolism and pulmonary toxicity of butylated hydroxytoluene (BHT). Pharmacol.Ther, 42(1), 89–113. [DOI] [PubMed] [Google Scholar]
  69. Witschi H, Williamson D, & Lock S (1977). Enhancement of urethan tumorigenesis in mouse lung by butylated hydroxytoluene. J.Natl.Cancer Inst, 58(2), 301–305. [DOI] [PubMed] [Google Scholar]
  70. Zerbe LK, Dwyer-Nield LD, Fritz JM, Redente EF, Shroyer RJ, Conklin E, … Malkinson AM (2008). Inhibition by erlotinib of primary lung adenocarcinoma at an early stage in male mice. Cancer Chemother Pharmacol, 62(4), 605–620. [DOI] [PubMed] [Google Scholar]

RESOURCES