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. In contrast to PTC, which is a common type with mild behavior and better prognosis⁴, ATC is a rare and highly aggressive malignancy that accounts for 2% to 3% of all thyroid tumors⁵. Although ATC is rare, it is responsible for approximately 50% of thyroid cancer-related deaths, with dismal survival (6-8 months)^6,7. Over 50% of ATC cases are diagnosed as lung metastasis⁸. In addition to the aggressive nature of ATC, limited effective treatment has been developed in the clinic. Therefore, ATC patients have a bleak prognosis^9,10,11. This suggests that further in-depth studies are urgently needed on the molecular mechanisms underlying the development of ATC and treatment.

The tumorigenesis of ATC is a dynamic dedifferentiated process. The difficulty in collecting human tumor samples at each stage in clinical studies has hindered the understanding of the mechanism of development from well-differentiated to undifferentiated carcinomas. In contrast, the use of murine ATC models (mATC) favors the collection of mATC samples in the whole tumorigenesis course. Therefore, we can better understand the mechanisms of tumor formation by analyzing the dynamic dedifferentiated process. In addition, the heterogeneity of clinical ATC samples has also contributed to the difficulty in understanding the molecular mechanism. Nevertheless, mice shared the same genetic backgrounds and were maintained in similar living environments, ensuring each tumor's consistency. This facilitates exploring the generalized role of ATC development^12,13,14. Additionally, mATC is an in situ tumor model that can restore the influence of the anatomic location and tissue-specific microenvironment. As such, compared with commonly used immunodeficient mice, mATC is a spontaneous murine model with an intact immune system and immune microenvironment.

Therefore, we constructed conditionally induced mATC with the C57BL/6 strain, which is a murine model capable of reproducing the pathological features of dedifferentiated thyroid carcinoma. Based on this model, we gave a brief overview of the molecular basis, construction ideas, pathological features, and applications of mATC. In addition, we observed and reported tumor growth, survival time, metastasis, and pathological features of mATC. We believe this will be an informatic overview to assist other researchers in using this model easier.

We constructed a conditional inducible mATC model, as first reported by McFadden¹⁵; initially, we constructed mice: TPO-cre/ERT2, Braf^flox/wt, and Trp53^flox/wt. Specifically, TPO-cre/ERT2 mice included the human thyroid peroxidase (TPO) promoter (a thyroid-specific promoter), driving the expression of a cre-ERT2 fusion gene (a cre recombinase fused to a human estrogen receptor ligand binding domain). Cre-ERT2 is usually confined to the cytoplasm and enters the nucleus only when exposed to tamoxifen, which induces cre to exert recombinant enzyme activity. When the mice are crossed with mice carrying loxP-flanked sequences, after tamoxifen-induction, cre-mediated recombination deletes the floxed sequences in the thyroid cells to achieve the purpose of knocking out or knocking in specific genes.

In addition, Braf^flox/wt mice are a knock-in allele of human Braf based on the cre-loxP system. Braf^flox/wt murine transcript is encoded by endogenous exons 1-14 and loxP-flanked human exons 15-18. After cre-mediated excision of the floxed regions, the mutant exon 15 (modified with a V600E amino acid substitution linked with constitutively active Braf^V600E in human cancers) and the endogenous exons 16-18 are used to generate the transcripts. Furthermore, Trp53^flox/wt mice are knockout alleles of human Trp53 and have loxP sites flanking exons 2-10 of Trp53. When crossed with mice with a cre recombinase, cre-mediated recombination deletes the floxed sequence to knock out Trp53. Then, TPO-cre/ERT2, Braf^flox/w, and Trp53^flox/wt mice were crossed to obtain TB (TPO-cre/ERT2; Braf^flox/wt) mice and TBP (TPO-cre/ERT2; Braf^flox/wt; Trp53^flox/wt) mice, which could be used to generate PTC and ATC. After approximately 8 weeks, the mice were induced by an intraperitoneal (i.p.) administration of 150 mg/kg tamoxifen dissolved in corn oil for two administrations. Tumor growth could be monitored by high-frequency ultrasonography (the first time point of ultrasonography was recorded as Day 0). Initial ultrasonography was performed 40 days after tamoxifen introduction.

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 surface that will allow them to grip.
   3. Gently but firmly place the free hand over the shoulders, then quickly grasp the scruff of the neck between the thumb and forefinger. Hold the tail with the little finger.
   4. Swab the tail with alcohol. Use sterile scissors to snip the skin sample <5 mm and put it into a clean sample container with a label. It is not necessary to remove the fur of the mice. Compress the tail with sterile sponges to attain hemostasis.
   5. Next, lyse the murine tail and perform polymerase chain reaction (PCR) to identify the genotype.
      NOTE: After cutting one murine tail, the surface of the scissors must be wiped with alcohol cotton to avoid mutual contamination of genes between murine tails. After placing them in their cage, the mice must be watched for 5 min to look for any signs of bleeding at the wound site. Refer to Table 1 for the list of primers and the PCR settings used in this study.
2. Tamoxifen-induced TBP mice
   1. Weigh 0.3 g of tamoxifen and dissolve it in 15 mL of corn oil by ultrasound (power = 20%, duration = 20 min, temperature = 4 °C), at a concentration of 20 mg/mL. Store at 4 °C.
      NOTE: Tamoxifen is sensitive to light and needs to be placed in a brown container.
   2. At around 8 weeks, weigh the mice with electronic scales and give them an i.p. injection of tamoxifen at a dose of 150 mg/kg, administered twice with a 1 week interval.

2. Dissection and imaging of mouse thyroid tumors and metastatic tumors

1. Preparation
   1. Prepare the dissection tools: sterile scissors and forceps, sterile blade, 75% alcohol spray bottle, 20 cm straightedge, vernier calipers, paper towels, and alcohol cotton.
   2. Mouse tissue fixing solution: prepare 4% paraformaldehyde tissue fixing solution.
   3. Clean a 10 cm dish filled with about 10 mL of sterile phosphate buffered saline (PBS) for temporary tissue storage, and wash the blood on the surface of the tissue.
2. Thyroid extraction
   1. Euthanize the mice with carbon dioxide at a flow rate of 2.0 L/min for 5 min. Remove the mice from the cage and perform cervical dislocation.
   2. Place the mouse on the dissecting board with the ventral side facing up and the head away from the experimenter, and fix the limbs on the dissecting board with sterile syringe needles. For a more convenient removal of thyroid tissue, use another sterile syringe needle to fix the head.
   3. Disinfect the neck with three alternating rounds of a chlorhexidine or povidone-iodine scrub followed by 75% alcohol. Then, make a small incision above the center of the clavicle with sterile scissors and forceps.
   4. Continue the incision midline up to the mouth. Find the submandibular gland and remove it to expose the anatomical location of the thyroid, which is placed near the thyroid cartilage and trachea.
   5. Find the thyroid, carefully dissect the thyroid from the rest of the neck region with sterile scissors, then put the removed thyroid in a 10 cm dish filled with 10 mL of sterile PBS.
      NOTE: During removal of the thyroid gland, be gentle and slow and avoid cutting the neck blood vessels. If the neck vessels are cut, the neck will be immediately filled with blood, and the blood must be promptly removed with alcohol sponges to expose the anatomical location of the thyroid. Before removing the thyroid tissue, carefully observe the characteristics of the left and right lobes of the thyroid (size, shape, etc.) to avoid being unable to distinguish between the left and right lobes of the thyroid after removal.
   6. In sterile PBS, remove the blood from the surface of the thyroid tissue with sterile scissors and carefully cut off the trachea. Then, put the thyroid tissue on a sterile cloth, and measure the size of the left and right lobes of the thyroid gland with vernier calipers.
   7. Divide the left and right lobes of the thyroid gland into two parts with a sterile blade. Put one part into 2 mL of 4% paraformaldehyde solution for fixation, and put the other part into liquid nitrogen for preservation.
3. Extraction of lung and liver
   1. Disinfect the abdomen with three alternating rounds of a chlorhexidine or povidone-iodine scrub and 75% alcohol. Then, pinch just above the mouse's penis and make a small incision, cut along the midline of the abdomen to the subclavian bone, and expose the abdominal cavity.
   2. Find the liver in the upper part of the abdomen and carefully remove it. Place the liver into sterile PBS.
   3. Carefully cut the diaphragm along the ribs to expose the thoracic cavity. Next, grab hold of the sternum and pull up to widen the space even more. Find the lung and remove it. Put the removed lung into sterile PBS.
   4. Grossly observe whether there are metastases in the lung and liver. Count the number of metastases and take a digital record. After that, use a sterile blade to divide the lung and liver tissues into two parts. Put one part into 3 mL of 4% paraformaldehyde solution for fixation and another part into liquid nitrogen for preservation.
   5. Tissue dehydration
      1. Put the tissues into the dehydration box. Put the dehydration box into the basket at gradient alcohol dehydration as follow: 70% alcohol for 1 h; 80% alcohol for 1 h; 95% alcohol for 30 min; 95% alcohol for 30 min; anhydrous ethanol I for 30 min; anhydrous ethanol II for 30 min; xylene I for 20 min; xylene II for 20 min; paraffin wax I for 30 min; paraffin wax II for 1 h; paraffin wax III for 30 min.
   6. Embed the wax-impregnated tissues in the embedding machine.
      1. Put the melted wax into the embedding frame first. Before the wax solidifies, remove the tissue from the dehydration box and put it into the embedding frame, then attach the label.
      2. Allow the wax to cool at -20 °C on the freezing table. After the wax solidifies, remove the wax block from the embedding frame and trim it.
   7. Place the trimmed wax block on a paraffin slicer, set at a thickness of 5 µm and a size of 2 cm x 2 cm. After sectioning, allow the sections to float on a spreader with warm water at 45 °C to flatten the tissue. Pick up the tissue with a slide, and bake the slices in an oven at 65 °C for 2 h.
   8. After the water dries and the wax melts, remove the slide with the tissue and use it for hematoxylin and eosin (HE) and immunohistochemical (IHC) staining¹⁶. Make sure that the sections are adhered to the slides for staining.

3. HE staining of the primary tumor and lung

1. Dewaxing
   1. Put the slides with the sections in xylene for 10 min.
   2. Move into anhydrous alcohol (100%) (two bottles) for about 3 min each.
   3. Move into 95% alcohol (two bottles) for about 3 min each.
   4. Move into 80% alcohol for about 3 min.
   5. Move into 50% alcohol for about 3 min.
   6. Move into the tap water, and wash away the alcohol for about 1 min.
2. Staining
   1. Move the slides into hematoxylin for 8 min.
   2. Move the slides into the water for 1 min to wash away the hematoxylin; the tissue changes from blue to red.
   3. Move the slides into 1% hydrochloric acid alcohol for about 30 s.
   4. Move the slides into the water, washing for 5 min.
   5. Move the slides into eosin for 90 s, then wash them with water for 5 min.
   6. Move the slides into 50% alcohol, 80% alcohol, 95% alcohol (two bottles), and anhydrous alcohol (100%) (two bottles), for 1 min each.
   7. Move the slides into xylene for 5 min.
   8. After the slides are dry, seal them with neutral resin.

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