We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.
Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.
Lung cancer is the leading cause of cancer death around the world1. Research into the prevention, early detection, and treatment of lung cancer is ongoing in many research centers throughout the world2,3. Several animal models for lung cancer have been developed, and they have proven useful in studying the mechanisms of lung carcinogenesis and cell of origin, in determining the presence of cancer stem cells, and in examining various novel therapeutic strategies4. Earlier models relied on carcinogen-induced tumor initiation in sensitive strains of mice5. The development of knockout and transgenic mouse models in which lung cancer arises as a result of specifically manipulated genetic lesions has substantially improved our ability to control tumor induction and mimic several aspects of human lung cancer4. However, a major challenge in the use of lung cancer animal models is the absence of a real-time method to accurately identify and monitor the onset and development of tumors in mouse lungs and to document any later change in their sizes, such as their continued growth or reduction in response to treatments. This has forced researchers to resort to several time, effort, and resource-consuming techniques to identify the tumors and to evaluate their experimental results. The presence of inherent inter-mouse variation in response to tumor induction requires the use of large numbers of animals in each experimental group to reduce data variability. The inability to assess the tumor growth or response to treatment in real-time has forced researchers to blindly euthanize mice at multiple time-points in prolonged experimental protocols to guarantee that they will collect the right data, resulting in the waste of resources from the samples collected at time points that are either too early or too late.
In the present study, a method to exploit a small-animal micro-computed tomography (micro-CT) scanner to detect and follow-up lung tumors in living mice is introduced. We used our recently described Sftpc-rtTA and Tre-Fgf9-ires-eGfp double-transgenic (DT) mice that rapidly develop lung adenocarcinoma following induction with doxycycline6,7. The use of micro-CT enables us to (among other things) exclude mice with aberrant lung abnormalities before induction, confirm development of tumor nodules in the lung after induction, and observe changes in tumor nodules in response to experimental treatments. End-point euthanasia of mice and histological assessment confirmed the accuracy of the real-time assessment conducted with micro-CT. We believe that this technique will pave the way for conducting better-planned experiments using lung cancer animal models while saving valuable resources, shortening observation time and increasing the accuracy and understanding of results.
Animal experiments were approved by the Institutional Animal Care and Use Committee of Keio University.
Note: In this study, we used the Sftpc-rtTA and Tre-Fgf9-ires-eGfp DT mice in which lung adenocarcinoma rapidly develops after induction by feeding chow containing doxycycline6,7. However, all assessment procedures can be applied to other lung cancer mouse models.
1. Experiment Outline:
2. Preparing Mice for Micro-CT Image Acquisition:
3. Pre-induction Micro-CT Image Visualization and Analysis:
4. Tumor Induction:
5. Follow-up scans:
6. Mouse Euthanasia and Lung Collection:
7. Histological Evaluation:
Note: Although the use of a "slide scanner" for digital histological evaluation is described here, the use of regular microscopes and visual histological evaluation for assessment is also possible.
Identification of mice with lung abnormalities was performed at baseline. Before tumor induction, when the DT mice were 8 – 12 weeks of age, the lungs of all mice were scanned with micro-CT. Surprisingly, approximately 50% of mice showed abnormalities that forced us to deem them unsuitable for inclusion in the subsequent study. These abnormalities were nodule-like shadows, large single or multiple small emphysematous bullae and/or lobar atelectasis (Figure 1A, C, D-E, G-H). Then, the FGF9 transgene and tumor induction was activated.Only mice that showed normal lung scans (without abnormalities) were switched from regular chow to doxycycline chow to induce FGF9 expression in alveolar cells and subsequent tumor development. To confirm the development of tumor nodules in mouse lungs after ten weeks from the initiation of tumor induction, follow up micro-CT scans were performed. These revealed the development of multiple nodules of variable sizes in all mice (Figure 2, compare A to B and D to E). Micro-CT scans were performed to assess the response to treatment. These revealed that mice that were administered the FGFR inhibitor AZD4547 for 10 weeks indeed exhibited disappearance or reduction in the size of nodules that were visible in the pre-treatment scans (Figure 3, compare pre-, post-induction and post-treatments, A-C, E-G and I-K). On the other hand, control mice that were not given the FGFR inhibitor and were given placebo instead showed no change, increase in nodules size and appearance of new nodules (Figure 3, M-O); confirming that the decrease in size observed in the treatment group is a genuine effect of the therapeutic intervention.
End-point histological evaluation of mouse lungs from the different time-points confirmed the accuracy of the findings detected with micro-CT scanning. Naïve mice before doxycycline induction:Mice judged to have clear lungs on micro-CT scans showed normal lung histology. Mice that showed abnormalities on the scan had various histological changes including bullae and nodules (Figure 1B, F, I). Mice after 10 – 12 weeks of induction with doxycycline: Multiple adenocarcinoma nodules with variable sizes and positions were observed in all animals (Figure 2C, F). Mice after 10 – 12 weeks of doxycycline induction followed by 10 – 12 weeks of an FGFR blocker: Nodules were much fewer in number and smaller (Figure 3D, H, L) compared to those observed post-induction. Mice after 10 – 12 weeks of doxycycline induction followed by 10 – 12 weeks of placebo: Multiple large nodules are visible (Figure 3P) similar to these seen at the post-induction time point.
Figure 1. Micro-CT Identifies Pre-induction Lung Abnormalities. All mice were examined with micro-CT before the initiation of doxycycline induction. When lung abnormalities were detected, mice were euthanized for histological confirmation. Representative micro-CT scan images for (A) a normal lung; (C) lungs with an atelectatic area; (D-E) lungs with emphysematous bullae and (G-H) lungs with an abnormal nodular shadow. Histological evaluation confirmed the accuracy of the micro-CT findings: (B) normal lung histology; (F) multiple emphysematous areas; and I) multiple tumor nodules. Scale bar: 200 µm. Please click here to view a larger version of this figure.
Figure 2. Micro-CT Identifies the Development of Tumor Nodules after Induction. Mice that showed a clean pre-induction scan were fed doxycycline chow for 10 weeks then re-evaluated with micro-CT, after which they were euthanized for histological evaluation. Representative micro-CT scan images from two different mice show the appearance of multiple nodular shadows 10 weeks after the start of doxycycline chow. (A, D) Pre-induction clean lung, (B, E) Same-position scan images showing development of multiple nodules (red arrows). Small yellow arrows point to the anatomical landmarks used to identify the same position at different time points. (C, F) Nodules seen in the micro-CT scan were confirmed in the lung histological sections. Scale bar: 200 µm. Please click here to view a larger version of this figure.
Figure 3. Micro-CT Identifies Changes In Tumor Nodules in Response to Treatment. Mice that showed clean pre-induction scans were fed doxycycline chow for 10 weeks, and then a post-induction micro-CT was performed. Next, they were administered the FGFR inhibitor AZD4547 for 10 more weeks and re-evaluated with micro-CT (post-treatment). Control mice were administered placebo instead of the FGFR inhibitor to clarify the actual effect of the therapeutic intervention. Finally, the mice were euthanized for histological evaluation.
Representative micro-CT scan images from four different mice. (A, E, I, M) Pre-induction scans confirmed that mice had no abnormalities. (B, F, J, N) Scans from the same mice after they were induced with doxycycline chow for 10 weeks; all showed multiple nodules (red arrows, the red oval encircles an area that had been completely shadowed by nodules). (C, G, K) Scans from the same mice after an additional 10 weeks of treatment with the FGFR inhibitor showing complete disappearance or marked reduction in the size of nodules. (O) Scan from a control mouse to represent the control group that was administered a placebo instead of the FGFR inhibitor showing increase in tumor nodules' size and number. Small yellow arrows point to the anatomical landmarks used to identify the same position at different time points. (D, H, L) Histological sections confirmed the marked reduction in nodule size and number (compare to Figure 2 C and F). (P) Shows the presence of multiple tumor nodules when placebo was administered. Scale bar: 200 µm. Please click here to view a larger version of this figure.
The micro-CT-based method described here for the real-time identification of lung abnormalities and monitoring of the development of tumor nodules and the response to treatment in lung cancer animal models will enable scientists who are conducting lung cancer-related experiments to plan more accurate and efficient experiments while saving time and resources. We have previously used MRI for the same purpose6. The clarity of the scan and threshold for the detection of lung nodules with MRI were inferior to those with the micro-CT scans described in this study6.
Previous studies that used similar lung adenocarcinoma mice models (with different genetic backgrounds and tumor induction methods) obviously had several disadvantages that could have been avoided or improved had they used micro-CT scanning9,10. They had no means to identify any pre-induction lung abnormalities or nodules, thus risking confounding their results by the inclusion of unsuitable animals. They also had no means to confirm the development of tumors after induction or after propagating cancer cells into wild-type mice (to induce secondary tumors), so they had to wait for 4-8 months then blindly euthanize the mice for the histological identification of tumors. When these models were used to assess the therapeutic potential of a drug, researchers had to allocate several groups to be euthanized in a chronological sequence to detect the effect of treatments on tumor nodules with histology and to be able to identify the time point of maximal effect11.
One limitation of our method, however, is the appearance of hazy shadows that obscure the detection of nodules in the lungs when some foreign substances are injected intratracheally7 (data not shown). Some mice seemed to develop a prolonged inflammatory reaction to the injected substance and the shadow of this reaction and accompanying interstitial edema masked the existing nodules. In such cases, MRI can be tried as an alternative.
Recently, other imaging modalities have been introduced to assess various pathologies in small animals; like bioluminescence imaging12 and positron emission tomography (PET) scan13. To our knowledge, these imaging systems have not been used yet to evaluate lung tumors in mice models so comparison of detection efficiency and image clarity with small animal MRI and micro-CT images is not yet possible. The availability and cost of these machines are a limiting factor to their widespread use.
We detected lung abnormalities in approximately 50% of mice at the pre-induction screening and accordingly these mice were excluded from further experiments. This high incidence of abnormalities might be a result of a "leaky" FGF9 transgene in some animals resulting in the development of tumor nodules even before they are induced14. Indeed, roughly half of the excluded animals when examined histologically showed the presence of multiple small nodules. These could also be caused by an aberrant effect of the inserted transgene on the neighboring genes in the mouse genome14. The development of these sort of abnormalities are by no means specific to the Sftpc-rtTA and Tre-Fgf9-ires-eGfp double-transgenic mice and thus other lung cancer models might be suffering from similar situations. This high incidence of abnormality highlights even-more the importance of pre-inclusion screening of mice using micro-CT. Naïve wild type mice never showed any abnormal shadows on micro-CT (Figure 1A).
In conclusion, we have described a method that exploits a small animal micro-CT machine for the accurate detection and monitoring of the development of tumor nodules in the lungs of an adenocarcinoma mouse model. By using it to monitor changes in nodules over time, more accurate data can be collected, and a cost and time-efficient time course can be adapted.
The authors have nothing to disclose.
This work was supported by a Grant-in-Aid from JSPS KAKENHI for A.E.H. (Grant Number 25461196) and T.B. (Grant Numbers 23390218 and 15H04833) and National Institutes of Health grant HL111190 (D.M.O.). The authors would like to acknowledge Miyuki Yamamoto for her efforts in helping with animal genotyping and the preparation of histological sections. We are grateful to the Collaborative Research Resources, School of Medicine, Keio University for technical support and reagents.
micro-X-ray–computed tomography | Rigaku | R_mCT2 | |
NanoZoomer RS Digital Pathology System | Hamamatsu | RS C10730 | |
NDP.view2 Viewing software | Hamamatsu | U12388-01 | http://www.hamamatsu.com/jp/en/U12388-01.html |
Isoflurane Vaporizer – Funnel-Fill | VETEQUIP | 911103 | |
Induction chamber, 2 Liter W9.5×D23×H9.5 | VETEQUIP | 941444 | |
Isoflurane | Mylan | ES2303-01 | |
AZD 4547 | LC Labratories | A-1088 | |
Pentobarbital | Kyoritsu | SOM02-YA1312 | |
G24 cannula | Terumo | SP-FS2419 | |
Paraformaldehyde | Wako | 163-20145 | |
Microtome | Leica | RM2265 | |
Doxycycline | SLC Japan/PMI Nutrition International | 5TP7 | |
ImageJ software | National Institute of health | http://imagej.nih.gov/ij/ | |
Puralube vet ointment (Occular lubricant) | Dechra | NDC 17033-211-38 |