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

Summary

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.

Abstract

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.

Introduction

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 world^2,3. 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 ca....

Protocol

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 doxycycline^6,7. However, all assessment procedures can be applied to other lung cancer mouse models.

1. Experiment Outline:

1. Identify the status of the lungs at the baseline:
   1. Before tumor induction, when the DT mice are 8 - 12 weeks old, perform the first micro-CT scan (see sections 2 and 3 below). ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 L  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