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 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.
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.
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
2. Animal preparation for imaging
3. Tumor imaging
4. Animal recovery
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.
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.
The authors have nothing to disclose.
This research received no specific grant from public, commercial, or not-for-profit funding agencies.
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 |