This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.
With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.
As the leading cause of cancer-related deaths worldwide, lung cancer remains refractory to treatments, mainly due to lack of relevant pre-clinical models that can recapitulate the disease in the lab1. Around 25% of lung cancer cases are due to mutations in the KRAS oncogene2. KRAS-driven lung cancer is often associated with poor prognosis and low response to therapy, highlighting the importance of further studies in this disease2.
We optimized a method that allows the relative evaluation of lung tumor growth in real time in KRAS lung cancer-induced immune-competent mice. We use Lox-Stop-Lox KRAS G12D (LSL-KRAS G12D) mice in which the KRAS G12D oncogene can be expressed by Cre lentiviral vectors3,4. These vectors are driven by carbonic anhydrase 2, allowing the viral infection to take place specifically in alveolar epithelial cells5. In addition, to accelerate the initiation and progression of lung tumors, the lentiviral construct also expresses P53 shRNA from an U6/H1 promoter (the lentiviral construct herein will be referred to as Ca2Cre-shp53)6. The biological relevance of this method lies in the natural course of lung tumor development in mice as opposed to xenografts of non-orthotopic tumors in mice. An obstacle using the orthotopic method is monitoring lung tumor growth without sacrificing the mouse. To overcome this limitation, we optimized ultrasound imaging to permit the analysis of lung tumor progression in two-dimensional (2D) mode in this mouse model. Initiating tumors at 7 weeks post-infection are reflected as B-lines in ultrasound images, which can be counted, but will not reflect the exact number of tumors present on the lung. B-lines are characterized by laser-like vertical white lines arising from the pleural line in the lung parenchyma7,8. Large tumors can be visualized after 18 weeks of infection. The relative volume of these tumors is quantified by 2D measurements done on ultrasound.
This method is optimal for researchers investigating the effect of pharmacological drugs on lung tumor growth in the LSL-KRAS G12D mouse model. In addition, lung tumor progression can be compared between mice with different genetic lineages, to examine the importance of the presence or absence of certain genes/proteins on the development of lung tumor volume.
Animal studies were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of McGill University and procedures were approved by the Animal Welfare Committee of McGill University (animal use protocol # 2009-5754).
1. Generation of CA2Cre-shp53 Lentiviral Titre
NOTE: The following protocol is the same as that described in Xia et al.6, with minor modifications.
2. Intratracheal Intubation of Lentiviruses in LSL-KRASG12D Mice
NOTE: The method of intratracheal intubation was used as described in the published protocol by Vandivort et al.9. In this protocol, mice LSL-KRASG12D mice in C57BL/6 background are used at age 6−8 weeks. A home-made working procedure board is used as described in Vandivort et al.9. The board is positioned in front of the experimenter in a convenient workspace (approximately 1 m2).
3. Ultrasound Imaging of Lung Tumors in Mice
NOTE: Ultrasound imaging was performed after 7 and 18 weeks of lentiviral intubation using the system listed in Table of Materials; however, any model can be used for the analysis.
4. 2D Analysis of Ultrasound Images
After obtaining a lentiviral infectious titer of ~2 x 106 TU/mL (Figure 1), the Ca2Cre-shp53 lentivirus was intratracheally injected when LSL-KRAS G12D mice reached an appropriate age (6−8 weeks)9. Ultrasound imaging was performed after 7 weeks of infection upon initiation of tumors (Figure 3B). Imaging was done at 7 weeks in order to include the various types of precursor lesions that occur in the LSL-KRASG12D mouse model, ranging from hyperplasia to adenoma3 as shown in Figure 4A after 8 weeks of infection. Imaging earlier than 6 weeks will not ensure the inclusion of adenoma-forming tumors in the analysis3. These precursor lesions are visualized as B-lines in ultrasound and can be counted by eye since they can be identified as narrow beams of white light vertically traversing the screen and are the result of a strong reflection of ultrasound wave (Figure 3B)10. B-lines represent small tumors that are on the surface of the lungs; tumors being initiated in deeper structures of the lung such as close to the trachea will not be reflected as B-lines. The number of tumors being initiated on the pleural surface can be quantified by counting the B-lines, since they are too small for volume measurements. We acquired 500 frames spanning the full lung area in 5 mice. B-lines were counted every 10 frames; thus, they were counted in a total of 50 images per mouse that span the full 500 frames acquired. The scatter plot in Figure 3D shows the sum of B-lines counted in every 10 images (100 frames) for 5 mice. The number of B-lines per mouse is then summed up to include B-lines present in the full lung area. The mean quantity of B-lines representing tumors in the five mice was 141.6 ± 41.52 (Figure 3E). Figure 3A shows an ultrasound image acquired of a non-infected mouse lung, for comparison.
According to Jackson et al.3, 16 weeks post-intubation includes adenoma as well as adenocarcinoma, which are tumor types of interest in KRAS lung cancer. To ensure inclusion of these types of tumors when analyzing volume measurements via ultrasound in our model, we imaged the lungs of mice at 18 weeks post-infection. In order to quantify the volume of the large tumors via ultrasound, we used 2D analysis on the ultrasound software. Large tumors appear as deep clefts interrupting the pleural surface as shown in the image of Figure 3C. When a tumor is spotted, we measure the length (L) and width (W) of the tumor, using the measurement tool of the ultrasound software. Once obtained, L and W are applied in the conventional formula used for calculating tumor volume (protocol step 4.4). We analyzed the tumor volume of 10 mice, as shown in Figure 3F, where the number of tumors formed in each mouse ranged from 5 to 26 tumors. The volumes of all tumors formed per mouse are then summed up to represent the tumor volume per mouse. For this analysis, we assume that the tumors formed in the lungs of mice are epileptic in shape, thus the width of the tumor is considered as perpendicular to the imaging plane. Relative volume quantification can be done when linear size of tumors reaches 0.3 mm (either length or width). After analyzing the lung parenchyma of 10 mice, the average tumor volume obtained was 31.8 ± 6.61 mm3 (Figure 3G). Hematoxylin and Eosin (H&E) staining analysis on lung sections confirmed the formation of large tumors at 20 weeks post-infection (Figure 4B) and confirmed formation of adenoma and adenocarcinoma. It should be noted that as tumor growth progresses, different tumors might merge together to form large ones, as shown in Figure 4B. Although the ultrasound software permits 3D analysis of tumors, this type of analysis proved difficult in our model, mainly due to the high dynamic movement of the primary tumors formed that is consistent with lung sliding (movement of the pleural line with respiration) as well as cardiac beats11. We reasoned that 2D analysis would provide consistency in relative tumor volume measurements, especially when comparing lung tumor progression between different groups of mice.
Figure 1: Flow cytometry analysis of MEFs with a GFP LoxP-allele. Representative flow cytometry images showing frequency of GFP-positive MEFs that are non-infected (Fc) (A) and infected (Fi) with the Ca2Cre-shp53 lentivirus (B). Please click here to view a larger version of this figure.
Figure 2: Workspace of integrated rail system of ultrasound imaging. (A) X-axis micro-knob. (B) Y-axis micro-knob. (C) Animal platform. (D) Height control knob of scan head-probe. (E) Transducer. (F) 3D motor. (G) Heating lamp. (H) Anesthetics tube. Please click here to view a larger version of this figure.
Figure 3: Representative ultrasound images of mouse lungs before and after lung tumor induction. (A) Image of a non-infected mouse lung section. Red arrows point to ribs (R) and the pleural surface (PS) of the lung. (B) Image of a mouse lung 7 weeks after tumor induction by intratracheal intubation of lentivirus. Arrows indicate B-lines reflecting small tumors on the lung surface. (C) Image of a mouse lung section 18 weeks after tumor induction. Blue lines indicate measurements of the length (L: mm) and width (W: mm) of the tumor shown. (D) Scatter plot showing B-line quantification in 10 images spanning 500 frames (each image is separated by 10 frames). (E) Quantification of relative tumor number in lungs at 7 weeks post-induction. (F) Scatter plot showing the relative volume of the different tumors formed in the lungs of 10 mice at 18 weeks post-infection. (G) Quantification of lung tumor volume in mice (n = 10). Please click here to view a larger version of this figure.
Figure 4: Representative H&E staining of LSL-KRAS G12D lung sections. (A) Lung section at 8 weeks post-infection at 500 µm magnification. Arrows point at precursor lesions formed at this timepoint, which are reflected as B-lines in ultrasound. Black arrows point at magnified lesions at 60 µm. (B) Lung section at 20 weeks post-infection at 2 mm magnification. Arrowheads of the same color show different tumors that have merged together. Arrows show magnified tumors at 60 µm. Please click here to view a larger version of this figure.
We demonstrate a method that can assess lung tumor growth in the Cre-inducible LSL-KRAS G12D mouse model by ultrasound. This method can be used for evaluating the effect of pharmacological inhibitors on lung tumor growth. It can also be used to compare lung tumor growth between mice of different genetic backgrounds. Using this technique does not require specialized computational skills, however, it is important to be systematic in the number of frames used for analysis to allow for proper comparison if the method is used for comparing different groups of mice.
The intratracheal intubation of ~2 x 106 TU/mL Ca2Cre-shp53 in LSL-KRAS G12D mice led to the formation of precursor lesions after 7 weeks of infection. These appear as longitudinal white lines on sonographic images and are called B-lines (Figure 3B)12. The average number of B-lines per mouse was 141.6 ± 41.52 (Figure 3E). We chose to visualize initiating tumors at 7 weeks post-infection since this timepoint would include various types of precursor lesions; atypical adenomatous hyperplasia, epithelial hyperplasia and adenoma3. The formation of precursor lesions on the lung was verified by H&E staining after 8 weeks of lentiviral infection (Figure 4A). After 18 weeks of viral intubation, the tumors are large enough to allow 2D volume measurements in the ultrasound software (Figure 3C). Analysis of ultrasound images was done at the 18-week timepoint in order to include the quantification of well-developed adenoma and adenocarcinoma3. The formation of these types of tumors was confirmed at 20 weeks post-infection by H&E staining analysis (Figure 4B). 3D volume measurements are also possible by the ultrasound software but require high level of expertise. The titre described in this protocol is optimal for the formation of lung tumors that can be properly visualized and quantified in 2D.
Other imaging techniques can also be used to monitor lung tumor growth in mice, such as CT imaging and BLI13. However, CT imaging requires delivering a radiation dose to the tumor, which might affect tumor growth upon repeated assays13. Moreover, it requires the use of contrast agents for accurate tumor measurements13. BLI is an important imaging technique that is often used for quantifying tumor proliferation; however, its main limitation is its dependence on metabolic activities and the presence of ATP and O213. The advantage of ultrasound imaging lies in it being a non-invasive, non-irradiating, speedy and inexpensive technique for acquiring images of the lung parenchyma13. Ultrasound has been previously used to monitor growth of orthotopic human tumors xenografted in lung and pancreas of mice13,14. Raes et al. have been successful in longitudinally monitoring the growth of a single human lung tumor implanted near the posterior diaphragmic surface in mice via ultrasound13. The difference in the LSL-KRAS G12D mouse model is having spontaneous development of lung tumors that can be monitored from initiation to progression and this development can be compared between mice groups. Also, there are numerous tumors being developed on the lung of the LSL-KRAS G12D mouse model, that eventually grow, and might even merge together to form large tumors (Figure 4B). This urged the need to use a method that can monitor lung tumor initiation and progression in this mouse model. We optimized the 2D brightness (B) mode analysis to allow quantitative assessment of lung tumors developed in this mouse model.
A possible limitation with ultrasound imaging is the presence of false positives15. These are seen when a highly reflective surface is present (diaphragm or liver) that interrupts the trajectory of the beam and might create a virtual object mimicking a true object15. Such images could be false tumors but can be bypassed upon proper examination; a large tumor is usually dynamic whereas a virtual object would be static.
Ultrasound imaging for the LSL-KRAS mouse model is ideal due to the non-invasive nature of this technique. The method of analysis used in our lab to monitor lung tumor progression in the LSL-KRAS mouse model is fast and user-friendly.
The authors have nothing to disclose.
We thank Dr. I. Verma for the lentiviral Ca2Cre-shp53 vector. The work was supported by funds from the Canadian Institutes of Health Research (CIHR MOP 137113) to AEK.
0.45 μm Acrodisc Syringe Filters | Pall Corporation | PN 4614 | |
100-mm Cell Cultre Plate | CELLSTAR | 664 160 | |
6-well Cell Culture Plate | CELLSTAR | 657 160 | |
Amicon Ultra – 15 Centrifugal Filter Units | Merck Millipore Ltd. | UFC910024 | |
BD LSR-Fortessa | BD Biosciences | 649225B 3024 | |
CA2Cre-shp53 lentiviral vector | From Dr. I Verma Laboratory | ||
DMEM | Multicell | 319-005-CL | |
FBS | Multicell | 80450 | |
LSL-KRASG12D mouse | JAX Mice | 8179 | |
MX550S; Centre Transmit: 40 MHz | FUJIFILM VisualSonics | 51070 | |
OptiMEM | gibco | 11058-021 | |
Pen/strep | Multicell | 450-201-EL | |
pMD2.G | Addgene | 12259 | |
PsPAX2 | Addgene | 12260 | |
VEVO-3100 | FUJIFILM VisualSonics | 51072-50 |