Spontaneous Murine Model of Anaplastic Thyroid Cancer

Summary

Here, we present a standard pipeline to obtain murine ATC tumors by spontaneous genetically engineered mouse models. Further, we present tumor dynamics and pathological information about the primary and metastasized lesions. This model will help researchers to understand tumorigenesis and facilitate drug discoveries.

Abstract

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the carcinogenesis and development of ATC, as well as therapeutic methods, since standard treatments are essentially depleted in ATC patients. However, low prevalence has hampered thorough clinical studies and the collection of tissue samples, so little progress has been achieved in creating effective treatments. We used genetic engineering to create a conditionally inducible ATC murine model (mATC) in a C57BL/6 background. The ATC murine model was genotyped by TPO-cre/ERT2; Braf^CA/wt; Trp53^{ex2-10/ex2-10} and induced by intraperitoneal injection with tamoxifen. With the murine model, we investigated the tumor dynamics (tumor size ranged from 12.4 mm² to 32.5 mm² after 4 months of induction), survival (the median survival period was 130 days), and metastasis (lung metastases occurred in 91.6% of mice) curves and pathological features (characterized by Cd8, Foxp3, F4/80, Cd206, Ki67, and Caspase-3 immunohistochemical staining). The results indicated that spontaneous mATC possesses highly similar tumor dynamics and immunological microenvironment to human ATC tumors. In conclusion, with high similarity in pathophysiological features and unified genotypes, the mATC model resolved the shortage of clinical ATC tissue and sample heterogeneity to some extent. Therefore, it would facilitate the mechanism and translational studies of ATC and provide an approach to investigate the treatment potential of small molecular drugs and immunotherapy agents for ATC.

Introduction

Thyroid cancer is one of the most common endocrine malignancies¹, originating from the thyroid epithelium. In recent years, the incidence of thyroid cancer has increased rapidly worldwide². Thyroid cancer can be divided into distinct types according to the degree of tumor cell differentiation. On the basis of clinical behavior and histology, thyroid carcinomas are divided into well-differentiated carcinomas, including papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC), poorly differentiated carcinoma (PDTC), and undifferentiated or anaplastic carcinoma of the thyroid (ATC)³. ....

Protocol

The animal procedures described here were performed with the approval of the Animal Ethics Committee of West China Hospital, Sichuan University, Chengdu, Sichuan, China.

1. Induction of TBP mice

1. Identify mice genotype
   1. At around 3 weeks, separate the female mice from the male mice. At the same time, use ear tag clamp to fix an ear tag. Place the ear tags in the lower half and on the middle third of the ear, making sure to avoid the area with the highest concentration of capillaries.
   2. Gently but securely restrain the mice. Grasp firmly at the base of the mouse tail. Place the mice on a surfac....

Results

We induced mATC to investigate tumor growth, mouse survival time, and pathological characteristics. After induction, the mice were immediately sacrificed, and samples (thyroid, lung, and liver) were collected once one of the following conditions were found: 1) respiratory distress caused by tumor compression; 2) decreased appetite and abnormal vocalization; 3) unusually lethargy; and 4) body weight loss of over 20%. During the sampling process, we found that all mice (12/12) successfully formed tumors after induction. We.......

Discussion

Critical steps within the protocol for thyroid tumor dissection
During dissection, the anatomical location of the thyroid gland needs to be correctly understood. The thyroid gland is a butterfly-shaped gland located on the dorsal side of the submandibular gland near the thyroid cartilage and the trachea. During the procedure, severing the blood arteries on both sides of the neck was carefully avoided.

Modification and troubleshooting of the mATC breed

Materials

Name	Company	Catalog Number	Comments
100x Citrate antigen retrieval solution (PH 6.0)	MXB	Cat# MVS-0101
50x EDTA antigen retrieval solution(pH 9.5)	ZSGB-GIO	Cat# ZLI-9071
Brafflox/wt mice	Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China
Caspase-3	Beyotime	Cat# AC033
CD8	Cell Signaling Technology	Cat# 98941; RRID:AB_2756376
CD206	Cell Signaling Technology	Cat# 24595; RRID:AB_2892682
Chamber for anesthesia induction	RWDlifescience
Enhanced DAB chromogenic kit	MXB	Cat# DAB-2031
Eosin staining solution	ZSGB-GIO	Cat# ZLI-9613
F4/80	Abcam	Cat# 100790; RRID:AB_10675322
Foxp3	Cell Signaling Technology	Cat# 12653; RRID:AB_2797979
Fully enclosed tissue dehydrator	Leica Biosystems	ASP300S
Hematoxylin staining solution	ZSGB-GIO	Cat# ZLI-9610
HistoCore Arcadia fully automatic tissue embedding machine	Leica Biosystems
Ki67	Beyotime	Cat# AF1738
Rotating Slicer	RWDlifescience	Minux S700
SPlink detection kits (Biotin-Streptavidin HRP Detection Systems)	ZSGB-GIO	Cat# SP-9001
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
Ultrasonic cell crusher	Ningbo Xinyi Ultrasound Equipment Co., Ltd	JY92-IIN
Ultrasound gel	Keppler	KL-250
Ultrasound system	VisualSonics	Vevo 3100