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; BrafCA/wt; Trp53Δex2-10/Δex2-10 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.
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 survival1,2.
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 treatments3,4,5. Such studies require accurate tumor imaging for measurements and monitoring, which is typically performed using magnetic resonance imaging, computed tomography, or high-resolution ultrasonography6,7. 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 portable8,9. 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; BrafCA/wt; Trp53Δex2-10/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
- 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 images11,12. - 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.
- Ensure sufficient isoflurane in the vaporizer and O2 in the tank. If the supplies are inadequate, exchange the tanks for new ones.
- 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
- Switch on the isoflurane vaporizer. Transfer the mouse from the cage to the anesthetic box.
- Anesthetize the animal using a mixture of 1%-2% of isoflurane from the vaporizer and oxygen flowing at 0.8 L/min.
- 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.
- 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. - 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. - 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).
- Protect the eyes by applying ophthalmic ointment.
3. Tumor imaging
- 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. - Liberally apply ultrasound gel (see Table of Materials) to the area of bare skin.
- 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. - 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). - 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.- 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.
- 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.
- Save the recording as a cine loop, which facilitates the review of the selected images.
4. Animal recovery
- After scanning, wipe the ultrasound gel and remove the restraining tape from the animal's limbs.
- Place the mouse on the supplemental heating device (step 1.2). Lay the animal on its side (Figure 2D).
- After the mouse recovers (~5 min), transfer it back to the cage.
- Clean the ultrasonography system, probe, and platform using a soft cloth and isopropyl alcohol or glutaraldehyde wipes.
- Turn off the imaging system.
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Representative 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 on the fourth measurement had grown to almost four times larger than the size on the initial measurement. The representative measurements of four mice and the growth curves are shown in Figure 5.
High-frequency ultrasonography is the imaging modality most often used to examine the thyroid glands in humans, and the technique also seems well-suited to mice. It can visualize the entire mouse thyroid gland and details of thyroid lesion growth. This protocol of applying the method of high-frequency ultrasonography could be used to accurately scan, monitor, and measure tumors in a genetically engineered mouse model of ATC.
Figure 1: Equipment used in the present study. (A) The high-frequency ultrasonography system. (B) Laboratory supplies: (1) Electric heating blanket. (2) Paper towels. (3) Ultrasound gel. (4) Isoflurane vaporizer. (5) Depilatory cream. (6) Cotton swabs. (7) Scissors. (8) Adhesive tape. (9) Medical gloves. (10) Chamber for anesthesia induction. (11) Anesthesia system. (C) A mechanized scanning system for ultrasound imaging. The completely sedated mouse was placed on the heated platform (shown in green), and the scanning probe was attached to a precision movable arm. Please click here to view a larger version of this figure.
Figure 2: Mouse preparation and the ultrasound scan. (A) Anesthesia induction. (B) Fixing the animal on the heated platform and anesthesia maintenance. (C) Ultrasound scanning with a freehand method. (D) Recovery of the animal on the electric heating blanket. Please click here to view a larger version of this figure.
Figure 3: Ultrasound images of an orthotopic ATC tumor mouse model. The green line demarcates the trachea, the red line demarcates the ATC tumor, and the yellow line demarcates the strap muscle. Please click here to view a larger version of this figure.
Figure 4: Calculation of the tumor size. The tumor size was calculated by multiplying the anteroposterior diameter (orange line) by the left-to-right tumor diameter (white line). Please click here to view a larger version of this figure.
Figure 5: Longitudinal analysis of orthotopic ATC growth in the mouse model. (A) Right thyroid lobe. (B) Left thyroid lobe. (C) Entire thyroid. Each curve corresponds to one animal measured four times. Please click here to view a larger version of this figure.
| Date | 2021.08.24 | 2021.09.16 | 2021.10.19 | 2021.11.19 | |
| Label | Location | Tumor Size (mm2) | |||
| P71 | Right | 6.39 | 6.688 | 6.327 | 8.461 |
| Left | 6.461 | 6.419 | 6.984 | 8.6 | |
| total | 12.851 | 13.107 | 13.311 | 17.062 | |
| P85 | Right | 5.962 | 7.318 | 7.057 | 7.352 |
| Left | 6.809 | 7.165 | 8.514 | 30.836 | |
| total | 12.711 | 14.483 | 15.571 | 38.188 | |
| P89 | Right | 4.423 | 5.423 | 5.988 | 8.911 |
| Left | 4.872 | 5.949 | 7.183 | 7.016 | |
| total | 9.296 | 11.372 | 13.172 | 15.928 | |
| P92 | Right | 3.593 | 3.509 | 3.769 | 6.734 |
| Left | 2.724 | 4.033 | 5.39 | 19.97 | |
| total | 6.317 | 7.542 | 9.159 | 26.704 | |
Table 1: Data on tumor size measurement. "P71", "P85", "P89", and "P92" represent the labels of the mice. Right: the tumor size of the right side. Left: the tumor size of the left side. Total: the total tumor size by adding the bilateral tumors. The first line includes the tumor size (mm2: square millimeters) and the date of the measurements.
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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; BrafCA/wt; Trp53Δex2-10/Δex2-10, was developed in our laboratory. The animals overexpress BrafV600E and lack Trp53; injecting the animals intraperitoneally with tamoxifen leads to tumor growth after approximately 1 month10. The tumors grow rapidly and reach a measurable size within 50 days. This protocol was used to monitor tumor growth for 4 months.
Ultrasonography has proven reliable in mice for imaging tissues that occupy similar body locations as human tissues, including the liver, thyroid, and fetus9. As in humans, the mouse thyroid is located on each side of the thyroid cartilage and trachea13. The presented protocol allows the analysis of ATC tumors in the thyroid, enabling the study of tumor initiation, progression, and response to treatments. The thyroid tumors in the mouse model grew quite large and occupied the space around the trachea and strap muscles. They showed solid-cystic features in ultrasound, similar to follicular structures. The non-invasiveness, short duration, and convenience of ultrasonography may make it more attractive to many research groups than magnetic resonance imaging or computed tomography8. Since lengthy sedation or anesthesia periods are unnecessary, ultrasonography's advantages could facilitate longitudinal studies.
Applying sufficient ultrasound gel during scanning is crucial to eliminate air pockets that could affect imaging and to avoid excessive compression that could lead to apnea. This protocol is routinely performed in our laboratory by experienced ultrasonography specialists who perform freehand scanning. Freehand scanning is preferred to a mechanized platform because it allows flexibility in adjusting the ultrasound probe's position according to the animal's state. When using a mechanized platform, the x- and y-coordinates must be adjusted to prevent excessive compression on the animal. The results showed that the tumors grew slowly in the early period, but from day 60, the tumors developed dramatically faster, and the maximum tumor size was 38.188 mm2. The leading cause of death was asphyxiation in the late stage. In clinical trials, due to the rarity of ATC tumors, collecting enough samples to observe the process and mechanism of development is difficult. The method of ATC lesions could be better observed in the murine model. In the future, these samples may supply more information for clinical treatments.
One limitation of ultrasound imaging is that the echogenicity of ATC tumors can resemble that of the surrounding tissues, thus obscuring tumor margins, especially in one still image. However, these margins can be identified by using dynamic contrast, so dynamic images were saved in this study for subsequent analysis. To ensure the most accurate and reliable results, the probe must be positioned in various ways to visualize the entire thyroid and tumor from different angles. In this study, only one ultrasonographer performed all the measurements, so reliability measurements between different examiners were not evaluated.
This protocol may facilitate the use of high-resolution ultrasonography for locating and measuring ATC tumors in animals, thus paving the way for detailed studies of cancer onset, progression, and treatment.
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Disclosures
The authors have no conflicts of interest to declare.
Acknowledgments
This research received no specific grant from public, commercial, or not-for-profit funding agencies.
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 |
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