Name: spontaneous murine model of anaplastic thyroid cancer
Uploaded: 2023-02-03T13:20:00
Description: Watch this Scientific Journal Video about Spontaneous Murine Model of Anaplastic Thyroid Cancer at JoVE.com

This work was supported by the National Key Research Development Program of China (2021YFA1301203); the National Natural Science Foundation of China (82103031, 82103918, 81973408, 82272933); the Clinical Research Incubation Project, West China Hospital, Sichuan University (22HXFH019); the International Cooperation Project of Chengdu Municipal Science and Technology Bureau (2020-GH02-00017-HZ); Natural Science Foundation of Sichuan, 2022NSFSC1314; the &#34;1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University&#34; (ZYJC18035, ZYJC18025, ZYYC20003, ZYJC18003); and Sichuan Science and Technology Program (2023YFS0098).

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
<ol>
	<li>Identify mice genotype
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		<li>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 ...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+genetically+engineered+mouse+models+of+papillary+and+anaplastic+thyroid+cancers:+enabling+translational+biology+for+patient+care+improvement.">Overview of genetically engineered mouse models of papillary and anaplastic thyroid cancers: enabling translational biology for patient care improvement.</a> Current Protocols in Pharmacology. 69, 1-14 (2015).</li><li>Tuttle, R. M., Haugen, B., Perrier, N. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+constrains+progression+to+anaplastic+thyroid+carcinoma+in+a+Braf-mutant+mouse+model+of+papillary+thyroid+cancer.">p53 constrains progression to anaplastic thyroid carcinoma in a Braf-mutant mouse model of papillary thyroid cancer.</a> Protocols of the National Academy of Sciences. 111 (16), 1600-1609 (2014).</li><li>Gunda, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinations+of+BRAF+inhibitor+and+anti-PD-1/PD-L1+antibody+improve+survival+and+tumour+immunity+in+an+immunocompetent+model+of+orthotopic+murine+anaplastic+thyroid+cancer.">Combinations of BRAF inhibitor and anti-PD-1/PD-L1 antibody improve survival and tumour immunity in an immunocompetent model of orthotopic murine anaplastic thyroid cancer.</a> British Journal of Cancer. 119 (10), 1223-1232 (2018).</li><li>He, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+ultrasonography+for+the+analysis+of+orthotopic+ATC+tumors+in+a+genetically+engineered+mouse+model.">High-resolution ultrasonography for the analysis of orthotopic ATC tumors in a genetically engineered mouse model.</a> Journal of Visualized Experiments. (188), e64615 (2022).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+recurrent+altered+genes+in+Chinese+patients+with+anaplastic+thyroid+cancer.">Novel recurrent altered genes in Chinese patients with anaplastic thyroid cancer.</a> The Journal of Clinical Endocrinology and Metabolism. 106 (4), 988-998 (2021).</li><li>Luo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pan-cancer+single-cell+analysis+reveals+the+heterogeneity+and+plasticity+of+cancer-associated+fibroblasts+in+the+tumor+microenvironment.">Pan-cancer single-cell analysis reveals the heterogeneity and plasticity of cancer-associated fibroblasts in the tumor microenvironment.</a> Nature Communications. 13 (1), 6619 (2022).</li><li>Luo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+dedifferentiation+of+thyroid+cancer+by+integrated+analysis.">Characterizing dedifferentiation of thyroid cancer by integrated analysis.</a> Science Advances. 7 (31), (2021).</li><li>Knostman, K. A. B., Jhiang, S. M., Capen, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+alterations+in+thyroid+cancer:+the+role+of+mouse+models.">Genetic alterations in thyroid cancer: the role of mouse models.</a> Veterinary Pathology. 44 (1), 1-14 (2007).</li><li>Kim, C. S., Zhu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+mouse+models+of+thyroid+cancer.">Lessons from mouse models of thyroid cancer.</a> Thyroid. 19 (12), 1317-1331 (2009).</li><li>Champa, D., Di Cristofano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+anaplastic+thyroid+carcinoma+in+the+mouse.">Modeling anaplastic thyroid carcinoma in the mouse.</a> Hormones and Cancer. 6 (1), 37-44 (2015).</li><li>Cabanillas, M. E., Ryder, M., Jimenez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+therapy+for+advanced+thyroid+cancer:+kinase+inhibitors+and+beyond.">Targeted therapy for advanced thyroid cancer: kinase inhibitors and beyond.</a> Endocrine Reviews. 40 (6), 1573-1604 (2019).</li><li>Ljubas, J., Ovesen, T., Rusan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+phase+II+targeted+therapy+clinical+trials+in+anaplastic+thyroid+cancer.">A systematic review of phase II targeted therapy clinical trials in anaplastic thyroid cancer.</a> Cancers. 11 (7), 943 (2019).</li><li>Huang, N. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+of+the+appropriate+treatment+strategies+in+anaplastic+thyroid+cancer:+a+population-based+study+of+735+patients.">An update of the appropriate treatment strategies in anaplastic thyroid cancer: a population-based study of 735 patients.</a> International Journal of Endocrinology. 2019, 8428547 (2019).</li><li>Subbiah, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dabrafenib+and+trametinib+treatment+in+patients+with+locally+advanced+or+metastatic+BRAF+V600-mutant+anaplastic+thyroid+cancer.">Dabrafenib and trametinib treatment in patients with locally advanced or metastatic BRAF V600-mutant anaplastic thyroid cancer.</a> Journal of Clinical Oncology. 36 (1), 7-13 (2018).</li><li>Baldini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+selective+inhibitors+of+Aurora+kinases+on+anaplastic+thyroid+carcinoma+cell+lines.">Effects of selective inhibitors of Aurora kinases on anaplastic thyroid carcinoma cell lines.</a> Endocrine-Related Cancer. 21 (5), 797-811 (2014).</li></ol>

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<br...

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 prognosis9,10,11. 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&#39;s consistency. This facilitates exploring the generalized role of ATC development12,13,14. 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, Brafflox/wt, and Trp53flox/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, Brafflox/wt mice are a knock-in allele of human Braf based on the cre-loxP system. Brafflox/wt&#160;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 BrafV600E in human cancers) and the endogenous exons 16-18 are used to generate the transcripts. Furthermore, Trp53flox/wt&#160;mice are knockout alleles of human Trp53 and&#160;have loxP sites flanking exons 2-10 of Trp53.&#160;When crossed with mice with a cre recombinase, cre-mediated recombination deletes the floxed sequence to knock out Trp53. Then, TPO-cre/ERT2, Brafflox/w, and Trp53flox/wt mice were crossed to obtain TB (TPO-cre/ERT2; Brafflox/wt) mice and TBP (TPO-cre/ERT2; Brafflox/wt; Trp53flox/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.

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			<td>100x&#160;Citrate antigen retrieval solution (PH 6.0)</td>
			<td>MXB</td>
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			<td>50x EDTA antigen retrieval solution(pH 9.5)</td>
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			<td>Brafflox/wt mice</td>
			<td>Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China</td>
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			<td>Caspase-3</td>
			<td>Beyotime</td>
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			<td>CD206</td>
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			<td>Chamber for anesthesia induction</td>
			<td>RWDlifescience</td>
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			<td>Enhanced DAB chromogenic kit</td>
			<td>MXB</td>
			<td>Cat# DAB-2031</td>
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			<td>Eosin staining solution</td>
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			<td>F4/80</td>
			<td>Abcam</td>
			<td>Cat# 100790; RRID:AB_10675322</td>
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			<td>Foxp3</td>
			<td>Cell Signaling Technology</td>
			<td>Cat# 12653; RRID:AB_2797979</td>
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			<td>Fully enclosed tissue dehydrator</td>
			<td>Leica Biosystems</td>
			<td>ASP300S</td>
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			<td>Hematoxylin staining solution</td>
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			<td>Cat# ZLI-9610</td>
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			<td>HistoCore Arcadia fully automatic tissue embedding machine</td>
			<td>Leica Biosystems</td>
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			<td>Ki67</td>
			<td>Beyotime</td>
			<td>Cat# AF1738</td>
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			<td>Rotating Slicer</td>
			<td>RWDlifescience</td>
			<td>&#160;Minux S700</td>
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			<td>SPlink detection kits (Biotin-Streptavidin HRP Detection Systems)</td>
			<td>ZSGB-GIO</td>
			<td>Cat# SP-9001</td>
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			<td>TPO-cre/ERT2 mice</td>
			<td>Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China</td>
			<td></td>
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			<td>Trp53flox/wt mice</td>
			<td>Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China</td>
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			<td>Ultrasonic cell crusher</td>
			<td>Ningbo Xinyi Ultrasound Equipment Co., Ltd</td>
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			<td>Keppler</td>
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			<td>Ultrasound system</td>
			<td>VisualSonics</td>
			<td>Vevo 3100</td>
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spontaneous murine model of anaplastic thyroid cancer

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; BrafCA/wt; Trp53ex2-10/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.

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...

Watch this Scientific Journal Video about Spontaneous Murine Model of Anaplastic Thyroid Cancer at JoVE.com

Spontaneous Murine Model of Anaplastic Thyroid Cancer

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.

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 ...

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An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Performing Data Mining And Integrative Analysis Of Biomarker in Breast Cancer Using Multiple Publicly Accessible Databases

In recent years, emerging databases were designed to lower the barriers for approaching the intricate cancer genomic datasets, thereby, facilitating ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...