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

Summary

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

Abstract

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's progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; Braf^CA/wt; Trp53^{Δex2-10/Δex2-10} transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of Braf^V600E 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.

Introduction

Although anaplastic thyroid carcinoma (ATC) accounts for fewer than 2% of thyroid cancers, it causes more than 50% of thyroid cancer-related deaths annually. The median survival time after diagnosis with ATC is only about 6 months, and no treatments are available that significantly improve survival^1,2.

The rarity of ATC has hampered the research studying how the disease begins and aggressively progresses. Genetically engineered mouse models that mimic the disease have recently become available, which provide insights into the disease and its responses to possible treatments^3,4,5. Such studies require accurate tumor imaging for measurements and monitoring, which is typically performed using magnetic resonance imaging, computed tomography, or high-resolution ultrasonography^6,7. Ultrasonography has been widely used in mouse organs. It has advantages over magnetic resonance imaging and computed tomography since it can be performed in real-time and does not expose the subject to radiation, and the necessary equipment is small enough to be portable^8,9. However, studies on continuously monitoring ATC growth using ultrasound are rare; therefore, this work explores the utility of ultrasound in this context.

Here, a protocol for using high-resolution ultrasonography to accurately scan, monitor, and measure tumors in a mouse model of ATC is presented.

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Protocol

The present study was performed with approval from the Animal Care and Use Committee of Sichuan University. TPO-cre/ERT2; Braf^CA/wt; Trp53^{Δex2-10/Δex2-10} transgenic mice¹⁰ were used in this study (see Table of Materials). The protocol steps can be modified for different animal species if necessary. Twelve mice, including six females and six males, with a mean age of 93 days, were used here.

1. Experimental preparation

1. Turn on the ultrasonography system (see Table of Materials) and create a new folder for capturing the images and collecting the data. Select the 40 MHz line probe and click on superficial tissue pattern to activate the superficial tissue transducer. Use the "B-mode" for thyroid imaging (Figure 1A).
   NOTE: B-mode is the basic ultrasound imaging mode. The appearance of ultrasound images relies on the physical interactions of sound with the tissues in the body. B-mode images are produced as gray images^11,12.
2. Keep the mice in specific cages with free access to food and water. Place the cage on a supplemental heating device (see Table of Materials) to ensure thermoregulation.
3. Ensure sufficient isoflurane in the vaporizer and O₂ in the tank. If the supplies are inadequate, exchange the tanks for new ones.
4. Clean the animal imaging platform with sterile saline and switch on the heating button. Verify that the temperature is 38-40 °C before placing the animal on the platform (Figure 1C).

2. Animal preparation for imaging

1. Switch on the isoflurane vaporizer. Transfer the mouse from the cage to the anesthetic box.
2. Anesthetize the animal using a mixture of 1%-2% of isoflurane from the vaporizer and oxygen flowing at 0.8 L/min.
3. Apply depilatory cream from the chest to the neck, wait 30 secs, and then wipe away the cream and fur completely. Thoroughly rinse the area and surrounding fur with warm sterile saline.
4. Place the anesthetized animal on the heated platform. Cover the snout with a nose cone connected to the anesthesia outlet (Figure 2A, B).
   NOTE: The mouse must be completely sedated within 1-2 min. If the animal is still active, perform prolonged isoflurane induction until the animal no longer shows a pedal withdrawal reflex. Ensure that the animal is breathing stably.
5. During imaging, monitor the heart rate of the mouse through the heated platform.
   NOTE: The ultrasonography imaging system is equipped with a heart rate monitor.
6. Use adhesive tape to fix the mouse's limbs to the heated platform, with the animal in a supine position. Ensure that the nose cone is stably positioned with a constant anesthetic gas flow (1.5 L/min).
7. Protect the eyes by applying ophthalmic ointment.

3. Tumor imaging

1. Adjust the imaging system to optimize the resolution. Set up the following parameters: two-dimensional gain, 25-30 dB; image depth, 10 mm; number of focal zones, 3; and center, 3-6 mm.
   NOTE: For the present study, a 40 MHz probe was used. The B-mode was specified for data acquisition.
2. Liberally apply ultrasound gel (see Table of Materials) to the area of bare skin.
3. Hold the probe and place it into contact with the ultrasound gel on the chest, and then scan from the chest toward the neck to locate the thyroid (Figure 2C).
   NOTE: Apply pressure gently while scanning; excessive pressure on the animal's neck may cause gasping or apnea. This protocol was developed based on handheld scanning, but mechanized scanning can also be performed using a machine to guide the probe, such as an animal imaging platform that moves along the x- and y-axes.
4. Scan up and down to identify the tumor's boundaries and assess its size and shape.
   NOTE: A healthy thyroid usually appears as a hypoechogenic, homogeneous structure in front of the trachea. Anaplastic tumors cause the thyroid to appear much larger, which can easily be identified by neck scanning (Figure 3).
5. Identify the ATC tumors from the trachea and strap muscles depending on the anatomical location and ultrasound echo.
   NOTE: The strap muscles are located in front of the thyroid and trachea, as well as behind the thyroid. The ultrasound echo of the strap muscles appears higher than that of the ATC, while attenuation exists behind the trachea¹³.
   1. Based on the impression of the total tumor, confirm the image section with the largest left-to-right tumor diameter. Press the freeze button, and measure the anteroposterior and left-to-right tumor diameters using the ultrasound caliper.
      NOTE: The anteroposterior diameter must be measured perpendicularly to the left-to-right tumor diameter (Figure 4). The tumor size of the ATC was calculated by multiplying the anteroposterior diameter by the left-to-right tumor diameter¹⁴. As the size of the tumors on the left and right sides was inconsistent, each side of the tumors was calculated separately. The total size of the tumors was obtained by adding the bilateral tumors. The sizes were recorded to observe the growth curve of the ATC.
6. Save the recording as a cine loop, which facilitates the review of the selected images.

4. Animal recovery

1. After scanning, wipe the ultrasound gel and remove the restraining tape from the animal's limbs.
2. Place the mouse on the supplemental heating device (step 1.2). Lay the animal on its side (Figure 2D).
3. After the mouse recovers (~5 min), transfer it back to the cage.
4. Clean the ultrasonography system, probe, and platform using a soft cloth and isopropyl alcohol or glutaraldehyde wipes.
5. Turn off the imaging system.

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Results

The average right ATC size at the beginning of the study was 4.867 mm², and the average left ATC size was 5.189 mm². On the fourth measurement, the average right ATC size had grown to 11.844 mm², while the tumor size of the left lobe had grown to 9.280 mm². The total ATC size increased from 10.057 mm² to 15.843 mm². In the later stage of the study, the ATC grew rapidly. In terms of the mouse labeled "P92" (Table 1), the tumor size ...

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Discussion

This protocol uses high-resolution ultrasonography to analyze orthotopic ATC tumors in a genetically engineered mouse model. The transgenic model, with a genotype of TPO-cre/ERT2; Braf^CA/wt; Trp53^{Δex2-10/Δex2-10}, was developed in our laboratory. The animals overexpress Braf^V600E and lack Trp53; injecting the animals intraperitoneally with tamoxifen leads to tumor growth after approximately 1 month¹⁰. The tumors grow rapidly and reach a m...

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Materials

Name	Company	Catalog Number	Comments
Adhesive tape	Winner
Anesthesia system	RWDlifescience
Brafflox/wt mice	Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China
Chamber for anesthesia induction	RWDlifescience
Cotton swabs	Winner
Depilatory cream	Veet
Electric heating blanket	Petbee
Isoflurane vaporizer	RWDlifescience
Medical gloves	Winner
Paper towels	Breeze	B914JY
TPO-cre/ERT2 mice	Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China
Trp53flox/wt mice	Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China
Ultrasound gel	Keppler	KL-250
Ultrasound machine	VisualSonics	Vevo 3100