Name: using micro-computed tomography for the assessment of tumor development and follow-up of response to treatment in a mouse model of lung cancer
Uploaded: 2016-05-20T15:00:00
Description: Watch this Scientific Journal Video about Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer at JoVE.com

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 t...

<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 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 border="0" cellpadding="0" cellspacing="0" width="889">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">micro-X-ray&#8211;computed tomography</td>
			<td style="width:264px;">Rigaku</td>
			<td style="width:140px;">R_mCT2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoZoomer RS Digital Pathology System</td>
			<td style="width:264px;">Hamamatsu&#160;</td>
			<td>RS C10730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NDP.view2&#160;Viewing software</td>
			<td style="width:264px;">Hamamatsu&#160;</td>
			<td>U12388-01</td>
			<td>http://www.hamamatsu.com/jp/en/U12388-01.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Isoflurane Vaporizer - Funnel-Fill</td>
			<td style="width:264px;">VETEQUIP</td>
			<td>911103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Induction chamber, 2 Liter&#160; W9.5&#215;D23&#215;H9.5</td>
			<td style="width:264px;">VETEQUIP</td>
			<td style="width:140px;">941444</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Isoflurane</td>
			<td style="width:264px;">Mylan</td>
			<td>ES2303-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AZD 4547</td>
			<td>LC Labratories</td>
			<td>A-1088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pentobarbital</td>
			<td style="width:264px;">Kyoritsu</td>
			<td style="width:140px;">SOM02-YA1312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G24 cannula&#160;</td>
			<td style="width:264px;">Terumo</td>
			<td style="width:140px;">SP-FS2419</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td style="width:264px;">Wako</td>
			<td style="width:140px;">163-20145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microtome</td>
			<td style="width:264px;">Leica</td>
			<td style="width:140px;">RM2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Doxycycline</td>
			<td style="width:264px;">SLC Japan/PMI Nutrition International</td>
			<td style="width:140px;">5TP7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ software&#160;</td>
			<td style="width:264px;">National Institute of health</td>
			<td style="width:140px;"></td>
			<td>http://imagej.nih.gov/ij/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Puralube vet ointment (Occular lubricant)</td>
			<td style="width:264px;">Dechra</td>
			<td style="width:140px;">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...

Watch this Scientific Journal Video about Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer at JoVE.com

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 ...

Research

JoVE Journal

Medicine

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...