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

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 ....

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 f.......

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.......

Materials

List of materials used in this article

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