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.

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:
<ol>
	<li>Identify the status of the lungs at the baseline:
	<ol>
		<li>Before tumor induction, when the DT mice are 8 - 12 weeks old, perform the first micro-CT scan (see sections 2 and 3 below). This serves as the lung baseline scan, confirms the absence of spontaneously developed nodules caused by a leaky transgene, and document the absence of any existing lung pathology before tumor induction.</li>
	</ol>
	</li>
	<li>Initiate tumor induction:
	<ol>
		<li>Switch the DT mice from regular chow to doxycycline chow to induce Fibroblast Growth Factor (FGF)9 expression in the alveolar cells and to initiate tumor development. Give doxycycline chow (200 ppm) ad libitum.</li>
	</ol>
	</li>
	<li>Confirm the development of tumor nodules in mouse lungs:
	<ol>
		<li>Perform a micro-CT scan to identify the development of tumor nodules compared to the pre-induction scan.</li>
	</ol>
	</li>
	<li>Assess response to treatment:
	<ol>
		<li>To test the ability of the micro-CT to detect changes in tumor nodules in response to treatment, administer the FGF Receptor (FGFR) inhibitor AZD4547, then perform additional micro-CT scans after 5 and 10 weeks.</li>
	</ol>
	</li>
	<li>End-point evaluation:
	<ol>
		<li>Euthanize all treatment and control mice and process tissues for histological evaluation (see section 6 below).</li>
	</ol>
	</li>
</ol>
2. Preparing Mice for Micro-CT Image Acquisition:
<ol>
	<li>Turn on the micro-CT scanner and computer.</li>
	<li>Click on the software named &#34;R_m CT2&#34;, then click on &#34;warm up&#34;.</li>
	<li>Remove the sample bed upon which the mouse will be placed from the micro-CT chamber. Wrap the bed with plastic wrap.</li>
	<li>Set the anesthesia induction box by adding isoflurane to the anesthesia vaporizer up to the marked level. Set the isoflurane flow rate at 3 L/min.</li>
	<li>Open the oxygen tank to start the oxygen flow into the induction box and set the flow rate at 1 L/min.</li>
	<li>Place the mouse in the anesthesia induction box and confirm that it is deeply anesthetized by the absence of spontaneous movement and in response to a skin pinch (a gentle pinch of a small fold of skin, which does not cause tissue damage or skin break).</li>
	<li>Apply an ocular lubricant to prevent corneal dryness during anesthesia.</li>
	<li>Open the micro-CT chamber and place the mouse with the dorsal side up on the sample bed. Hold the head of the mouse and pull the body downward from the lower limbs in order to stretch and straighten the body symmetrically.</li>
	<li>Turn off the anesthesia flow to the induction box and turn it on towards the tube that connects to the micro-CT chamber.</li>
	<li>Place the anesthesia tube into the mouse&#39;s nose to administer continuous anesthetic.</li>
	<li>In order to fix the mouse in position; wrap the mouse and the sample bed with plastic wrap. 
	Note: It is critical to keep the mouse under deep anesthesia and wrapped to the sample bed because any slight movement or twist of its body during the scan will result in hazy images and difficulty in interpretation.</li>
	<li>Close the micro-CT chamber.</li>
	<li>Adjust the micro-CT system to 90 kV and 160 &#181;A and the scan time to 4.5 min. Set the image range to 24 &#215; 19 mm and the voxel size to 50 &#215; 50 &#215; 50 &#181;m. Use the synchronous mode for heartbeats.</li>
	<li>Make a new folder in the &#34;Database&#34; in order to save new images in that folder.</li>
	<li>Start the scan.</li>
	<li>After completion of the scan, move the mouse into an empty cage and observe it until it regains consciousness. Do not put it with other mice until it has fully recovered from the effects of the anesthesia.</li>
</ol>
3. Pre-induction Micro-CT Image Visualization and Analysis:
<ol>
	<li>To visualize the micro-CT images, download the free ImageJ software from the following web site:<a href="http://imagej.nih.gov/ij/"> http://imagej.nih.gov/ij/</a> . Note: Every micro-CT data file is a stack of approximately 500 TIF Files (.tif). Other image viewing software can also be used.</li>
	<li>Open the serial micro-CT images files and scroll through all of the images of each mouse from the neck to the abdomen or vice versa.</li>
	<li>Using scans of na&#239;ve wild-type mice, identify the density of different areas and normal anatomical structures in the chest based on knowledge of mouse anatomy (see Figure 1A).
	<ol>
		<li>Hold the cursor with the computer mouse and scroll up, starting from the whitish abdominal viscera and diaphragm, through the chest and up to the neck.</li>
		<li>Identify the bony chest cage landmarks (sternum in the front, vertebrae in the back and ribs on the sides).</li>
		<li>Identify the heart in the front of the chest and the major blood vessels near the heart and in the mediastinum.</li>
		<li>Observe the trachea lumen (as a small dark circle at the level of the neck and upper chest), which bifurcates into the right and left main bronchi then continue to branch into smaller and smaller bronchi. Note that each bronchus is closely associated with two or three blood vessels (Figure 1A).</li>
	</ol>
	</li>
	<li>Start examining the scans of un-induced DT mice and identify the presence of any abnormalities.
	<ol>
		<li>Exclude mice with abnormal pre-induction lung shadows (e.g., nodules, emphysematous bullae, etc.) from any further experiments. (Figure 1C-E, G, H).</li>
	</ol>
	</li>
</ol>
4. Tumor Induction:
<ol>
	<li>To induce tumor development in experimental mice that showed normal lung scans, switch their food from regular chow to doxycycline-containing chow (200 ppm).</li>
</ol>
5. Follow-up scans:
<ol>
	<li>After 10 weeks, perform a second micro-CT scan of all mice to confirm the development of tumor nodules in their lungs (see Figure 2).</li>
	<li>Split mice into two groups. Administer the FGFR blocker AZD4547 (125 &#181;g/kg/day via a gastric tube for 6 days/week for 10 weeks) to one group and a placebo to the other control group for 10 more weeks.</li>
	<li>Follow up the changes in the nodular shadows by performing a third scan 4 - 5 weeks later.</li>
	<li>At the end of 10 weeks of treatment, perform a fourth scan.</li>
	<li>Euthanize all mice with CO2 inhalation or with intraperitoneal injection of 0.1 mg/200 &#181;l of pentobarbital.</li>
	<li>To identify the dynamic changes in the tumor nodules after induction and in response to treatment, identify similar positions within the serial scan images of the same mouse at two or more different time-points then check for the appearance/disappearance of any abnormal shadows (Figure 3).</li>
	<li>To facilitate the identification of the same plane in the same mouse at different time-points, try to associate the plane of interest with anatomical landmark structures within the mouse chest.
	<ol>
		<li>Use landmarks such as the trachea, its bifurcation, the right and left main bronchi, aorta, diaphragm, and large blood vessels. 
		Note: Chest bones, including the thoracic vertebrae, ribs, and sternum are less useful as positioning landmarks because of common minor tilts in mouse body alignment on the sample bed, which increase the possibility of misinterpretation of the scan position. Similarly, the image serial number within the scan file is unreliable for identifying the same position over time because of the change in mouse body length from one time-point to another. Failure or inaccuracy in identifying the same plane at different time points may result in false positive/negative interpretation of findings.</li>
	</ol>
	</li>
</ol>
6. Mouse Euthanasia&#160;and Lung Collection:
<ol>
	<li>Euthanize mice with CO2 inhalation or with intraperitoneal injection of 0.1 mg/200 &#181;l of pentobarbital.</li>
	<li>Expose the abdominal viscera by cutting longitudinally through the abdominal wall. Bleed the mouse to reduce the volume of blood in the lungs by dissecting the abdominal aorta.</li>
	<li>Slit the diaphragm with fine scissors; this will result in loss of the negative pressure from the chest cavity, thus collapsing the lungs. Expose the lungs and heart by cutting and removing parts of the ribs of the anterior chest wall. Clean the frontal part of the neck by cutting the skin and soft tissues to expose the trachea.</li>
	<li>Cut away the heart and thymus gland. Insert forceps behind the trachea to separate it from the esophagus.</li>
	<li>Cannulate the trachea with a G24 cannula then secure it in place by tightening a thread around the inserted part.</li>
	<li>Inflate and fix the lung using ice-cold 4% paraformaldehyde (PFA) through the tracheal cannula using a 25 cm column. Detach the cannula and tighten the thread to prevent PFA leakage then cut the upper trachea off its attachment to the larynx.</li>
	<li>Pull the trachea from the suture thread and dissect it from its attachment, and continue downward to remove it with the lung en-block. Insert it into a 15 ml tube containing 5 ml of 4% PFA. Leave the lungs in PFA O/N to ensure complete tissue penetration and fixation, then process the tissue into a standard paraffin block8.</li>
	<li>Cut paraffin blocks into 6 um-thick slices on a microtome and stain with hematoxylin and eosin using standard techniques.</li>
</ol>
7. Histological Evaluation:
Note: Although the use of a &#34;slide scanner&#34; for digital histological evaluation is described here, the use of regular microscopes and visual histological evaluation for assessment is also possible.
<ol>
	<li>Turn on the slide scanner instrument and computer.</li>
	<li>Click on the &#34;NDP scan&#34; software.</li>
	<li>Select the scan mode &#34;Batch of slides&#34; and &#34;Semi-Auto Mode&#34; for scanning a series of slides. 
	Note: The robotic arm that picks up the slides during sequential scanning is very sensitive to any irregularities on the edges of the glass slides.</li>
	<li>Palpate the edges of all glass slides before loading them into the machine. If there is any protrusion of the cover glass or dried mounting medium, clean it off with a cutter or a scalpel.</li>
	<li>Load the slides into a slide cassette. Open the specimen hatch and slide the cassette into position &#34;one&#34;. Close the door.</li>
	<li>On the computer software, give short descriptive names to all slides in their corresponding position in the cassette then click the &#34;OK&#34; button.</li>
	<li>Select the profile mode: &#34;Brightfield&#34;.</li>
	<li>Click on &#34;Start Batch&#34; to start the provisional scanning. 
	Note: Once the machine has finished scanning all the slides, the software will automatically detect areas with tissues on all the slides and suggest it as a region of interest.</li>
	<li>If needed, redefine the region of interest by holding down the left mouse button and pulling the region border. 
	Note: Defining excessively large areas of the slides as the regions of interest will result in a much longer scan time.</li>
	<li>Once satisfied with the regions of interests on all of the slides, click on &#34;Scan&#34; to start scanning all slides. 
	Note: The scanned files can be observed digitally in low and high resolution, and images can be exported as JPEG files.</li>
</ol>

<ol>
	<li>Ferlay, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+incidence+and+mortality+worldwide:+sources,+methods+and+major+patterns+in+GLOBOCAN+2012.">Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.</a> Int. J. Cancer. 136, 359-386 (2015).</li><li>Mak, I. W., Evaniew, N., Ghert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation:+animal+models+and+clinical+trials+in+cancer+treatment.">Lost in translation: animal models and clinical trials in cancer treatment.</a> Am. J. Transl. Res. 15, 114-118 (2014).</li><li>Chen, Z., Fillmore, C. M., Hammerman, P. S., Kim, C. F., Wong, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-small-cell+lung+cancers:+a+heterogeneous+set+of+diseases.">Non-small-cell lung cancers: a heterogeneous set of diseases.</a> Nat. Rev. Cancer. 14, 535-546 (2014).</li><li>Kwon, M. C., Berns, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+lung+cancer.">Mouse models for lung cancer.</a> Mol. Oncol. 7, 165-177 (2013).</li><li>Malkinson, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+basis+of+susceptibility+to+lung+tumors+in+mice.">The genetic basis of susceptibility to lung tumors in mice.</a> Toxicology. 54, 241-271 (1989).</li><li>Yin, Y., Betsuyaku, T., Garbow, J. R., Miao, J., Govindan, R., Ornitz, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+induction+of+lung+adenocarcinoma+by+fibroblast+growth+factor+9+signaling+through+FGF+receptor+3.">Rapid induction of lung adenocarcinoma by fibroblast growth factor 9 signaling through FGF receptor 3.</a> Cancer Res. 73, 5730-5741 (2013).</li><li>Arai, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+cell+of+origin+and+propagation+potential+of+the+fibroblast+growth+factor+9-induced+mouse+model+of+lung+adenocarcinoma.">Characterization of the cell of origin and propagation potential of the fibroblast growth factor 9-induced mouse model of lung adenocarcinoma.</a> J. Pathol. 235, 593-605 (2015).</li><li>Curtis, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+tumor+genotype+is+an+important+determinant+in+identification+of+lung+cancer+propagating+cells.">Primary tumor genotype is an important determinant in identification of lung cancer propagating cells.</a> Cell Stem Cell. 7, 127-133 (2010).</li><li>Lau, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-propagating+cells+and+Yap/Taz+activity+contribute+to+lung+tumor+progression+and+metastasis.">Tumor-propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis.</a> EMBO J. 33, 468-481 (2014).</li><li>Santos, A. M., Jung, J., Aziz, N., Kissil, J. L., Pur&#233;, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+fibroblast+activation+protein+inhibits+tumor+stromagenesis+and+growth+in+mice.">Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice.</a> J Clin Invest. 119, 3613-3625 (2009).</li><li>Zinn, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+Bioluminescence+Imaging+in+Small+Animals.">Noninvasive Bioluminescence Imaging in Small Animals.</a> ILAR J. 49, 103-115 (2008).</li><li>Yao, R., Lecomte, R., Crawford, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-Animal+PET:+What+Is+It,+and+Why+Do+We+Need+It.">Small-Animal PET: What Is It, and Why Do We Need It.</a> J Nucl Med Technol. 40, 157-165 (2012).</li><li>Haruyama, N., Cho, A., Kulkarni, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview:+Engineering+transgenic+constructs+and+mice.">Overview: Engineering transgenic constructs and mice.</a> Curr Protoc Cell Biol. , (2009).</li></ol>

The authors declare that they have no competing financial interests.

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...

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&#160;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&#160;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.

<table><tbody><tr><td>micro-X-ray&amp;ndash;computed tomography</td><td>Rigaku</td><td>R_mCT2</td><td></td></tr><tr><td>NanoZoomer RS Digital Pathology System</td><td>Hamamatsu&amp;nbsp;</td><td>RS C10730</td><td></td></tr><tr><td>NDP.view2&amp;nbsp;Viewing software</td><td>Hamamatsu&amp;nbsp;</td><td>U12388-01</td><td>http://www.hamamatsu.com/jp/en/U12388-01.html</td></tr><tr><td>Isoflurane Vaporizer - Funnel-Fill</td><td>VETEQUIP</td><td>911103</td><td></td></tr><tr><td>Induction chamber, 2 L&amp;nbsp; W9.5 &amp;times; D23 &amp;times; H9.5</td><td>VETEQUIP</td><td>941444</td><td></td></tr><tr><td>Isoflurane</td><td>Mylan</td><td>ES2303-01</td><td></td></tr><tr><td>AZD 4547</td><td>LC Labratories</td><td>A-1088</td><td></td></tr><tr><td>Pentobarbital</td><td>Kyoritsu</td><td>SOM02-YA1312</td><td></td></tr><tr><td>G24 cannula&amp;nbsp;</td><td>Terumo</td><td>SP-FS2419</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Wako</td><td>163-20145</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2265</td><td></td></tr><tr><td>Doxycycline</td><td>SLC Japan/PMI Nutrition International</td><td>5TP7</td><td></td></tr><tr><td>ImageJ software&amp;nbsp;</td><td>National Institute of health</td><td></td><td>http://imagej.nih.gov/ij/</td></tr><tr><td>Puralube vet ointment (Occular lubricant)</td><td>Dechra</td><td>NDC 17033-211-38</td><td></td></tr></tbody></table>

using micro-computed tomography for the assessment of tumor development and follow-up of response to treatment in a mouse model of lung cancer

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.

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...

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Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

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 ...

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10.6K Views.  Keio University School of Medicine. 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.

Video: Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Cancer Research

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

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.

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 ...

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...

Intrapulmonary Inoculation of Multicellular Spheroids to Model Orthotopic NSCLC

Construction of An Orthotopic Xenograft Model of Non-Small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model.
Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease&#39;s progression and facilitate the development of potential anticancer drugs.

Author Spotlight: Establishing a Murine Non-Small Cell Lung Cancer Model for Developing Nanoformulations of Anticancer Drugs

This study presents an orthotopic non-small cell lung cancer (NSCLC) model based on intrapulmonary inoculation of multicellular spheroids of fluorescent A549-iRFP cells. The model recapitulates clinical NSCLC stages and responds to cisplatin, according to dynamic in vivo monitoring of long-wavelength fluorescence.

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Immunology and Infection

In vivo Evaluation of Mucociliary Clearance in Mice

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a metachronal, synchronal fashion, these multiple, motile, actin-based organelles generate a cephalad fluid flow clearing the respiratory tract of inhaled pollutants and pathogens. With increasing environmental pollution, novel viral pathogens and emerging multi-drug resistant bacteria, cilia generated mucociliary clearance (MCC) is essential for maintaining lung health. MCC is also depressed in multiple congenital disorders like primary ciliary dyskinesia, cystic fibrosis as well as acquired disorders like chronic obstructive pulmonary disease. All these disorders have established, in some case multiple, mouse models. In this publication, we detail a method using a small amount of radioactivity and dual-modality SPECT/CT imaging to accurately and reproducibly measure MCC in mice in vivo. The method allows for recovery of mice after imaging, making serial measurements possible, and testing potential therapeutics longitudinally over time. The data in wild-type mice demonstrates the reproducibility of the MCC measurement as long as adequate attention to detail is paid, and the protocol strictly adhered to.

In this publication, we describe protocols for assessing airway mucociliary clearance (MCC) in mice in vivo utilizing dual-modality radionuclide imaging. This protocol is designed for a single photon emission computed tomography (SPECT) and computed tomography (CT) acquisition protocol using mouse whole body (MWB) collimators in a dual SPECT/CT system.

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

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Chapters in this video

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Utilizing ¹⁸F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Construction of An Orthotopic Xenograft Model of Non-Small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

In vivo Evaluation of Mucociliary Clearance in Mice