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The development of animal models of lung cancer is critical to our understanding and treatment of the human disease. Conditional mouse models provide new opportunities for testing novel chemopreventatives, therapeutics and screening methods that are not possible with cultured cell lines or xenograft models. This protocol describes how to initiate tumor formation in two conditional genetic models of human non-small cell lung cancer (NSCLC) utilizing the activation of oncogenic K-ras and the loss of function of p53. We discuss methods for sporadic expression of Cre in the lungs via engineered adenovirus or lentivirus and provide a detailed protocol for the administration of the virus by intranasal inhalation or intratracheal intubation. The protocol requires 1–5 minutes per mouse with an additional 30–45 minutes to set-up and allow for the recovery of mice from anesthesia. Mice may be analyzed for tumor formation and progression starting 2–3 weeks after infection.
Lung cancer is the leading cause of cancer deaths worldwide with non-small cell lung cancer (NSCLC) being the most prevalent form of lung cancer1–3. Progress within the last decade has led to the sophisticated engineering and application of advanced preclinical models of human cancer in the mouse4,5. These models are critical to our understanding of the human disease because they shed light on events and processes that cannot be easily studied using transplantable or chemically-induced cancer models5–7. Several laboratories have constructed genetically-engineered mouse models of NSCLC that mimic the genetic and histopathological features of the human disease6,8–13. The development of Cre recombinase-controlled (Cre/LoxP) tumor models has allowed for the generation of autochthonous tumors derived from a limited number of somatic cells that become transformed in their natural location, surrounded by a normal tissue microenvironment5. By engineering LoxP DNA elements into the mouse genome that either surround (‘flox’) exons critical to a tumor suppressor gene’s function or surround a synthetic ‘stop’ element (‘LSL’) inserted in front of an oncogene, investigators can ‘turn-off’ tumor suppressors or ‘turn-on’ oncogenes with delivery of Cre recombinase to the appropriate cell types (Supp. Fig. 1 and see reference 5 for a review of Cre/LoxP-controlled genetically engineered mouse models of cancer). With this technology, investigators can not only recapitulate the genetic alterations found in the human disease, but also the timing of onset and potentially the cellular origin of the disease.
Common mutations in human NSCLC are activating mutations in K-RAS (10–30%) and loss of function point mutations in p53 (50–70%)2. Our laboratory has modeled an oncogenic mutation in K-ras by changing a glycine to aspartic acid at codon 12 in the gene’s endogenous locus. To control the expression of K-rasG12D, a lox-stop-lox (LSL) cassette was engineered into the first intron of the K-ras gene. The LSL cassette consists of transcriptional and translational stop elements flanked by LoxP sites that prevents the expression of the mutant allele until the stop elements are removed by the activity of Cre recombinase9,14. The LSL cassette thus creates a null version of the gene. It is important to note that K-ras null mice are embryonic lethal15; therefore, mice can only be heterozygous for the K-rasLSL-G12D allele. To mimic the loss of p53 function in our K-rasLSL-G12D-driven tumor model, we have utilized a conditional p53 allele from the laboratory of Anton Berns. This ‘floxed’ p53 allele (p53fl) has LoxP sites flanking exons two through ten of p53 that are deleted after Cre-mediated recombination, abolishing p53 function16 (Supp. Fig. 1). Prior to Cre-mediated recombination, the p53 locus is maintained in its wildtype state and p53 activity is normal. To more accurately recapitulate the p53 loss of function mutations commonly observed in human NSCLC, our laboratory has generated two conditional point mutant (mt) versions of p53 (R172H, R270H) that are engineered into the endogenous p53 locus, but silenced by a LSL cassette in the absence of Cre17. We use mice that are p53LSL-mt/fl or p53LSL-mt/+ to specifically express mutant p53 alone or with wildtype p53 (respectively) in tumors upon Cre-mediated recombination17,18. Importantly, as with the K-rasLSL-G12D allele, the LSL cassette in p53LSL-mt alleles blocks p53 expression in the absence of Cre, effectively creating a p53 null allele. Therefore, p53LSL-mt/fl or p53LSL-mt/+ mice are heterozygous for wildtype p53 and phenocopy p53+/− mice. To specifically express either of these mutant p53 alleles sporadically in cells of the lung, mice inhale viruses engineered to express Cre either by intranasal instillation or intratracheal intubation.
In addition to the models of NSCLC utilized in this protocol, other conditional lung cancer models have been described which may be initiated with inhalation of viruses expressing Cre. The activation of oncogenic K-ras along with loss of p16Ink4a or Ink4a/Arf tumor suppressors, which are mutated in 20–50% of human cases, have been described11,19. In another mouse model, two other subtypes of NSCLC, squamous cell and large cell carcinoma, develop following the combined activation of oncogenic K-ras and loss of the LKB1 tumor suppressor, which is mutant in 10–30% of human cases19. NSCLC models driven by conditionally activated mutations in Braf or EGFR, mutated in 3% or 10–40% (respectively) of human lung cancers, have also been generated12,13 (and unpublished, K. Lane). A model of small cell lung cancer (SCLC) has been created in which tumors arise following the loss of both the p53 and Rb tumor suppressors20.
Altough these Cre/LoxP models provide the most sophisticated means to sporadically control genetic events at their endogenous loci, they are also limited by the difficult requirement to introduce Cre into an initiating cancer cell. In this protocol, investigators cannot restrict the genetic events exclusively to initiating cells of the disease because of the inherently non-specific nature of the viruses used to deliver Cre to cells of the lung. However, this has in fact been beneficial in our laboratory because it does not require that investigators identify and target Cre specifically to the cell of origin of the disease. Perhaps the best system to deliver Cre specifically to the cells that give rise to the disease are next generation regulatable Cre alleles, such as the tamoxifen-inducible Cre-estrogen receptor (CreERT) fusion protein5. By expressing a CreERT transgene under the control of a cell type-specific promoter, investigators can induce Cre activity specifically in the cells of a given tissue by injecting animals with tamoxifen. With proper dosing of tamoxifen, investigators can in theory control the penetrance of Cre activity and therefore the multiplicity of the disease. However, various promoter-driven CreERT alleles are not yet available and CreERT models succumb to problems encountered with many transgenic models in that the expression and activity of these alleles are often leaky and do not provide tight control of tumor initiation in the organ of interest 5. Therefore, despite the drawback of using potentially hazardous viruses, this technique is the most effective means to sporadically deliver Cre to cells of the lung to initiate lung tumor formation.
There are several alternative lung cancer models to those described here that do not require viral delivery of Cre to the lungs to generate tumors (see reference 6 for a comprehensive review of mouse models of NSCLC). Transgenic models have been created that utilize lung tissue specific promoters to drive expression of viral oncoproteins, such as E6/E7 and large T antigen, or cellular oncogenes, such as c-myc, c-Raf-1 and v-H-ras6. However, these models are limited in recapitulating the human disease because the oncogenic transgene is expressed in all of the cells of a targeted organ beginning early in the organ’s development. To overcome this limitation, our laboratory has engineered a latent oncogenic allele of K-ras that is expressed spontaneously in only a limited number of cells in the lung to initiate lung tumor formation8. However, the stochastic nature of this allele does not allow investigators to control tumor onset or multiplicity. Double transgenic models that rely on doxycycline-regulated transcriptional transactivators have allowed for the induction of oncogene expression in adult animals, making it possible to control tumor initiation6. However, these models still fail to provide sporadic oncogene expression at physiological levels due to the use of doxycyline-regulated transcription factors. Furthermore, these transgenic systems make it difficult to control tumor multiplicity and require continuous doxycycline dosing to maintain oncogene expression.
The intranasal and intratracheal infection techniques described in this protocol are not restricted to tumor-initiating studies. Investigators may theoretically probe the role of any Cre/LoxP-controlled genes in various cells of the lung using this protocol to deliver Cre. Alternatively, the intranasal or intratracheal delivery methods can be used in applications other than the viral delivery of Cre recombinase. The viral delivery systems may be adapted to express cDNAs or shRNAs in cells of the lung by infection with lentiviruses21. Viruses, such as the influenza virus, can be delivered for pathological studies22. siRNAs can be delivered as therapeutic agents for disease23. While either the intranasal or intratracheal techniques can be utilized for the aforementioned studies, only intratracheal intubation is recommended for the orthotopic transplantation of lung tumor cells or lung tumor cell lines to the alveolar space of the lungs (unpublished, C. Kim and T. Jacks). Certain chemical injurants, such as bleomycin, can be delivered intratracheally to study repair mechanisms in the lung and the effect of injury on tumor development24. Therefore, there are a number of uses of this technique for a variety of studies involving the mouse lung. Here we describe the use of the intranasal and intratracheal delivery methods to introduce viruses expressing Cre to initiate the K-rasLSL-G12D/+ (K) and the K-rasLSL-G12D/+;p53fl/fl (KP) conditional mouse models of NSCLC (Supp. Fig. 1).
After deciding to utilize autochthonous tumor models, investigators must consider the genetic tumor model, the viral system to express Cre recombinase, and the viral delivery method when initiating an experiment. Each option has certain advantages and limitations that are highlighted in the text and the Tables.
Although both the K-rasLSL-G12D/+ (K) and the K-rasLSL-G12D/+;p53fl/fl (KP) NSCLC models induce tumors that resemble the human disease histopathologically, they have distinct features that make them more suitable for different applications (Table 1). In our laboratory’s mouse models of NSCLC, the activation of an oncogenic allele of K-ras is sufficient to initiate the tumorigenesis process, while the additional deletion or point mutation of p53 significantly enhances tumor progression, leading to a more rapid development of adenocarcinomas that have features of a more advanced disease. K-ras and p53 mutant tumors exhibit a greater incidence of cellular and nuclear pleomorphism, desmoplasia, and a high frequency of metastases to the mediastinal lymph nodes and the pleural spaces of the thoracic cavity, and less frequently to the liver and kidneys.
To generate tumors sporadically in the lungs of K-rasLSL-G12D/+ mice, our laboratory initially used replication-deficient adenoviruses expressing Cre (Ad-Cre) to deliver transient Cre expression to infected cells of the lung9,10. Prior to administration, Ad-Cre is precipitated with calcium phosphate (see procedure) to improve the delivery of Cre by increasing the efficiency of viral infection of the lung epithelium25. Recently, we have used lentiviruses to deliver Cre to the lung (Lenti-Cre)26. Lentiviruses are beneficial because they integrate into the genome of infected cells27, allowing for further modification of the tumors by simultaneously introducing Cre recombinase and stable expression of cDNAs to overexpress, or short-hairpin RNAs to silence, genes of interest (Table 2).
In order to control for the number of tumors generated, viruses are titered prior to use in experiments. Ad-Cre is titered at the University of Iowa, while Lenti-Cre is titered in our laboratory by assaying for Cre activity after infection of the 3TZ reporter cell line, a mouse fibroblast cell line modified to express β-galactosidase after Cre-mediated recombination28. We infect mice with 2.5×107 infectious particles of Ad-Cre (titered at Univ. Iowa) or approximately 104–105 infectious particles of Lenti-Cre (3TZ titered in-house). It is important to note that Ad-Cre and Lenti-Cre are titered differently, and as a result, the titers cannot be directly compared.
While we initially delivered Cre to the lungs of anesthetized mice using intranasal (IN) instillation of the virus, we now prefer to deliver the virus to the lungs by intratracheal (IT) intubation. IT delivery provides the most direct and consistent method for the virus to reach the lungs. Reproducible delivery of the virus is critical because it directly affects the number of tumors generated in the mice. However, intratracheal intubation requires additional equipment and practice to perform it correctly and in a timely manner (Table 3). Therefore, it may be easier to begin with the IN delivery method to assess the tumor model and practice the IT method while continuing to breed animals for future experiments.
We recommend using avertin (2-2-2 Tribromoethanol) to anesthetize the mice. The amount of avertin administered to the mice is crucial to the success of the procedure. Mice administered too much avertin are more likely to stop breathing during the infection procedure, and recover poorly from the anesthesia. Conversely, mice administered too little avertin may struggle to inhale the virus and should be given more avertin before continuing. Therefore, we recommend using the smallest volume of avertin required to keep mice anesthetized during the procedure. Following the procedure, mice will recover better if they are kept warm to maintain their normal body temperature after anesthesia.
Our laboratory has utilized mice between 6 and 12 weeks of age for tumor initiation by IN or IT delivery of viruses expressing Cre. Mice of this age are old enough to recover from the anesthetic, the volume of virus administered to the lung, and the intubation of the trachea with the catheter.
Mice can be infected with a volume ranging from 50–125 µl per mouse, but we recommend using a total volume of 75 µl per mouse. Although a volume of 125 µl can be administered, it is not recommended for very young mice (6 weeks of age or younger). If 125 µl is administered, then the mice should receive two doses of 62.5 µl each, with a 1–5 minute break in between the doses, to allow the mice to recover a normal breathing pattern before receiving the second dose.
CRITICAL The following list of reagents represents our laboratory’s preference. All reagents, with the exception of Ad-Cre, can be modified according to investigator preference.
Add 15.5 ml tert-amyl alcohol to 25 g of avertin. Stir overnight to dissolve. Stable at room temperature (18–25 °C) for approximately 1 year.
CRITICAL Seal bottle tightly and protect from light. Discard the solution if it yellows.
Dilute avertin stock in PBS, stir overnight, and protect from light. Sterilize by passing solution through a 0.22 µm filter. Aliquots may be safely stored at 4 °C in the dark for approximately 4 months.
Dissolve in distilled water and store at 4 °C for up to 5 years.
Ad-Cre should be prepared fresh each time and used within one hour of preparation. For extended storage times, Lenti-Cre should be stored at −80 °C or alternatively, at 4 °C for periods of a few days. Keep viruses on ice prior to infection.
To set-up for either IN or IT administrations, arrange the virus, a heat lamp (or gloves filled with warm water), and a beaker filled with 50% bleach (to disinfect catheters and pipette tips that have contacted the virus) in a biosafety hood.
For IT administration, set up the platform and light source on a flat surface near the biosafety hood (Fig. 1a). Insert the catheter into the trachea outside of the hood, and then move the mouse into the biosafety hood to inhale the virus. A sharps waste container is also required for the proper disposal of the needles from the Exel Safelet IV Catheter.
We have reported that recombination of K-rasLSL-G12D and p53fl alleles in tumors can be examined by PCR for the presence of a “1 lox”, or recombined, product (see Reagents in Materials section for the protocols)9. Although it is possible to perform PCR on DNA isolated from whole lung after infection to assess infection efficiency, this is not recommended. Typically very few cells in the lung have undergone recombination of these alleles, making it difficult to detect recombination. Instead, it is more informative to examine Cre expression after infection by using conditional reporter strains such as Rosa26LSL-LacZ or Rosa26LSL-eGFP and examining reporter expression by immunohistochemistry, immunofluorescence, or fluorescent activated cell sorting33. Polyclonal antibodies that can specifically detect the oncogenic K-rasG12D protein are no longer available; however, increased Ras-GTP levels in tumors or cells can be assessed using a Raf-GST pulldown assay14.
The time to tumor development and progression will vary depending upon the model chosen (K v. KP), while survival time will also depend on the amount and type of virus administered to the mice. The survival of mice is reduced approximately twofold in KP mice compared to K mice (median survival with Ad-Cre: K, 185 days; KP, 76 days, with Lenti-Cre: K, 266 days; KP, 170 days) (Fig. 3a, b). Decreased survival is due to a greater growth rate of tumors lacking p53, leading to the more rapid development of a tumor burden that disrupts the normal function of the lungs. Reduced survival after p53 loss is not due to an increased number of tumors or metastatic disease. Survival of mice is also reduced after infection with Ad-Cre as compared to Lenti-Cre (Fig. 3a, b). This is due to the higher titer of virus typically administered to mice with Ad-Cre but may also reflect the viral tropism or the efficiency of the virus to infect the cell of origin of the disease. Mice infected with 2.5×107 infectious particles of Ad-Cre (Univ. Iowa, Gene Transfer Vector Core) can generate greater than 200 tumors per mouse, whereas mice infected with roughly 104–105 infectious particles of Lenti-Cre (3TZ titered) can generate 10–100 tumors per mouse. Our laboratory has had success titrating the viruses to lower levels (Ad-Cre: 5×106 infectious particles/mouse, Lenti-Cre: 5×103 infectious particles/mouse) which reduce the number of primary tumors and increase the survival time of mice, allowing for a greater frequency of metastatic disease in the KP model.
Monitoring tumor development and progression histologically is an important, though difficult, way to follow the disease, and it is especially useful in experiments where genetic events or modifications in addition to K-ras activation and/or p53 loss may be expected to impact tumor progression. We stress that the NSCLC model generates a multi-focal disease and therefore, investigators should expect some tumor heterogeneity; tumors do not always progress in exactly the same way or at the same time. To follow these changes objectively, we have employed a 4-stage grading system for tumor progression in our NSCLC models (adapted from ref. 18). The earliest lesions, designated as grade 1, are atypical adenomatous hyperpasias (AAH) or small adenomas that feature uniform nuclei and can appear as early as 2–3 weeks post-infection (Fig. 4a). These earliest lesions can only be identified through careful histological analysis. To see visible lesions on the surface of the lung, investigators should wait until 6–8 weeks after tumor initiation or later. Grade 2 tumors are larger adenomas that have slightly enlarged nuclei with prominent nucleoli and are observed 6–8 weeks post-infection (Fig. 4b, c). Adenocarcinomas are classified as grade 3; they have a great degree of cellular pleomorphism and nuclear atypia and can develop as early as 16 weeks post-infection (Fig. 4d, e). Grade 4 tumors are invasive adenocarcinomas (Fig. 4f) that harbor all the cellular characteristics of Grade 3 tumors but with a higher mitotic index - including irregular mitoses, a distinctive highly invasive stromal reaction (desmoplasia) (Fig. 4g), and invasive edges bordering lymphatic vessels, blood vessels, or the pleura (Fig. 4h). Grade 4 tumors may develop as early as 18 weeks post-infection in KP animals, but are not observed in K animals. Finally, locally metastatic disease to the mediastinal lymph nodes (Fig. 4i) or the pleural cavity develops in approximately 50% of KP mice as early as 18–20 weeks post-infection. In some mice, distant metastases can be found seeding the liver or the kidneys as early as 20 weeks post-infection (Fig. 4j). Although the time to progression in K and KP mice is described here to be similar up to Grade 3 lesions, mice with p53 deficient tumors often harbor cells that exhibit nuclear atypia at very early time points after tumor initiation. In addition, a greater proportion of tumors that lack p53 progress to higher grades during tumor development18. For example, at 6 weeks after infection the distribution of tumor grades in the K model is ~90% grade 1 and ~10 grade 2, whereas in the KP model it is ~40% grade 1, ~40% grade 2 and ~20% grade 3 18. At 19 weeks after infection in the KP model, the tumor distribution is ~5% grade 1, ~20% grade 2, ~70% grade 3 and ~5% grade 4 18. At 26 weeks after infection in the K model, ~30% of tumors are grade 1, ~40% are grade 2 and ~30% are grade 3 18. However, as with most autochthonous mouse tumor models, there is some variation in the results, such as tumor number and approximate time to progression, depending on the strain/ background of mice as well as other factors that vary between institutions.
Engineering of LoxP elements and a stop element allow for the controlled expression of oncogenic K-ras and the loss of p53 function after Cre expression.
Upon opening the Exel Safelet IV catheter, the needle is exposed (a). Slide the catheter over the end of the needle to completely cover the tip (b) and the Exel Safelet IV catheter is now ready to use.
Mice can be placed under a heat lamp (a) or on a glove filled with warm water (b) to recover following anesthesia in the biosafety hood.
CAUTION All experiments should be done in accordance with protocols approved by the Institutional Animal Care and Use Committee.
CAUTION All experiments should be done in accordance with protocols approved by the Institutional Animal Care and Use Committee.
We would like to thank Carla Kim and Amber Woolfenden for originally training the authors to perform the intratracheal intubation technique. The technique was implemented in our lab with help from Kwok-Kin Wong and Samanthi Perera. We would like to thank Trudy Oliver and Peter Sandy for providing data to compile the Kaplan-Meier survival curves as well as Denise Crowley and Roderick Bronson for providing key histological and pathological advice. We also thank Keara Lane, Etienne Meylan, Eric Snyder, and Anne Deconinck for reviewing this protocol. This work was supported by funding from the Howard Hughes Medical Institute, the NCI (including a Cancer Center Support grant), and the Ludwig Center for Molecular Oncology at MIT. T.J. is the David H. Koch Professor of Biology and a Daniel K. Ludwig Scholar. Research was conducted in compliance with the Animal Welfare Act Regulations and other Federal statutes relating to animals and experiments involving animals and adheres to the principles set forth in the Guide for Care and Use of Laboratory Animals, National Research Council, 1996.
Competing financial interests
The authors declare that they have no competing financial interests.