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
	<ol>
		<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 concentration of capillaries.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Swab the tail with alcohol. Use sterile scissors to snip the skin sample &#60;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Tamoxifen-induced TBP mice
	<ol>
		<li>Weigh 0.3 g of tamoxifen and dissolve it in 15 mL of corn oil by ultrasound (power = 20%, duration = 20 min, temperature = 4 &#176;C), at a concentration of 20 mg/mL. Store at 4 &#176;C. 
		NOTE: Tamoxifen is sensitive to light and needs to be placed in a brown container.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Dissection and imaging of mouse thyroid tumors and metastatic tumors
<ol>
	<li>Preparation
	<ol>
		<li>Prepare the dissection tools: sterile scissors and forceps, sterile blade, 75% alcohol spray bottle, 20 cm straightedge, vernier calipers, paper towels, and alcohol cotton.</li>
		<li>Mouse tissue fixing solution: prepare 4% paraformaldehyde tissue fixing solution.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Thyroid extraction
	<ol>
		<li>Euthanize the mice with carbon dioxide at a&#160;flow rate of 2.0 L/min for 5 min. Remove the mice from the cage and perform cervical dislocation.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Extraction of lung and liver
	<ol>
		<li>Disinfect the abdomen with three alternating rounds of a chlorhexidine or povidone-iodine scrub and 75% alcohol. Then,&#160;pinch just above the mouse&#39;s penis and&#160;make a small incision, cut along the midline of the abdomen to the subclavian bone, and expose the abdominal cavity.</li>
		<li>Find the liver in the upper part of the abdomen and carefully remove it. Place the liver into sterile PBS.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Tissue dehydration
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Embed the wax-impregnated tissues in the embedding machine.
		<ol>
			<li>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.</li>
			<li>Allow the wax to cool at -20 &#176;C on the freezing table. After the wax solidifies, remove the wax block from the embedding frame and trim it.</li>
		</ol>
		</li>
		<li>Place the trimmed wax block on a paraffin slicer, set at a thickness of 5 &#181;m and a size of 2 cm x 2 cm. After sectioning, allow the sections to float on a spreader with warm water at 45 &#176;C to flatten the tissue. Pick up the tissue with a slide, and bake the slices in an oven at 65 &#176;C for 2 h.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. HE staining of the primary tumor and lung
<ol>
	<li>Dewaxing
	<ol>
		<li>Put the slides with the sections in xylene for 10 min.</li>
		<li>Move into anhydrous alcohol (100%) (two bottles) for about 3 min each.</li>
		<li>Move into 95% alcohol (two bottles) for about 3 min each.</li>
		<li>Move into 80% alcohol for about 3 min.</li>
		<li>Move into 50% alcohol for about 3 min.</li>
		<li>Move into the tap water, and wash away the alcohol for about 1 min.</li>
	</ol>
	</li>
	<li>Staining
	<ol>
		<li>Move the slides into hematoxylin for 8 min.</li>
		<li>Move the slides into the water for 1 min to wash away the hematoxylin; the tissue changes from blue to red.</li>
		<li>Move the slides into 1% hydrochloric acid alcohol for about 30 s.</li>
		<li>Move the slides into the water, washing for 5 min.</li>
		<li>Move the slides into eosin for 90 s, then wash them with water for 5 min.</li>
		<li>Move the slides into 50% alcohol, 80% alcohol, 95% alcohol (two bottles), and anhydrous alcohol (100%) (two bottles), for 1 min each.</li>
		<li>Move the slides into xylene for 5 min.</li>
		<li>After the slides are dry, seal them with neutral resin.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to declare.

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 
ATC is a rare and highly aggressive malignancy. The clinical characteristics of mATC and ATC patients have certain similarities. Specifically, the major cause of death in mATC is asphyxiation, which is consistent with ATC patients. Therefore, during the experiment, the following should be noted: 1) gently grasp the mice and pay attention to their respiration during the operation; 2) closely monitor during the late stage to prevent sudden death and failure to obtain samples in time; 3) the thyroid and lung tissues are easily affected by the blood on the surface of the tissues for observation and photography, so the blood volume of mice can be reduced by eyeball enucleation.
Limitations of using mATC 
The occurrence and development of human ATC is complex and changeable, but the genetic background of mATC is relatively simple, which can only simulate a part of human ATC. For example, some ATC tissues harbored TERT and BRAF mutations, the NOTCH2NL copy number variant18, instead of P53 mutations, which may have different tumorigenesis mechanisms and clinical pathological characteristics7,19,20. Additionally, ATC usually has a long time-course before diagnosis, but mATC obtains a diagnosis after an induction of 2 months. Therefore, there are inherent distinctions between mATC and human thyroid cancer, animal models cannot fully imitate all characteristics of human thyroid cancer, and many therapies that are restricted to humans cannot be evaluated on mATC. Furthermore, although mATC can be used to research the therapeutic effects of various small molecule medications, several biologics (e.g., antibody or antibody-related drugs) should be designed for mice specifically. In addition, the entire process of constructing conditional knockout mice and then obtaining the target genotype of mice by crossbreeding takes a long time and requires certain mouse culture conditions and costs21,22.
Significance of using mATC in thyroid cancer research 
The heterogeneity of the human population and the rarity of human ATC hinder the exploration of potential mechanisms and therapeutic options for ATC. As a reliable mouse model, mATC facilitates the collection of ATC tissues and enriches the ATC tissue sample pool. mATC can also be used to study the effects of specific gene functions on ATC, study the cellular and molecular mechanisms of ATC development, understand the mechanisms of thyroid tumor progression and drug resistance, and ultimately improve the prognosis of ATC patients16.
Future applications of mATC 
mATC can be used for research on various aspects of radiotherapy, chemotherapy, gene therapy, immunotherapy, and targeted therapy. mATC can also be used to explore the therapeutic effects and adverse effects of regimens, such as combinations between different drugs or with radiotherapy. We subsequently utilized mATC to investigate the therapeutic effects of radiotherapy combined with immunotherapy and small molecule inhibitor drugs on ATC. In addition, mATC can be used to investigate the effects of various delivery modalities or routes on the antitumor effects of medications to develop drug delivery systems with enhanced antitumor activity. In future clinical applications, we hope to minimize the recurrence rate and mortality rate of thyroid cancer patients and thus improve the survival rate of patients.
We expect that more ATC murine models will be developed in the future, as well as more in-depth analyses of ATC in different genetic backgrounds, such as PTEN and P13K23. These will more comprehensively reveal the molecular mechanism of ATC tumorigenesis and progression, predict the outcome of ATC treatment, and could provide patients with potential therapeutic targets24,25,26,27,28. We hope that through more experimental exploration, we can improve the modeling method of the mouse model and shorten the modeling time to make more contributions to basic research.

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.

<table><tbody><tr><td>100x&amp;nbsp;Citrate antigen retrieval solution (PH 6.0)</td><td>MXB</td><td>Cat# MVS-0101</td><td></td></tr><tr><td>50x EDTA antigen retrieval solution(pH 9.5)</td><td>ZSGB-GIO</td><td>Cat# ZLI-9071</td><td></td></tr><tr><td>&lt;em&gt;Braf&lt;sup&gt;flox/wt&lt;/sup&gt;&lt;/em&gt; mice</td><td>Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China</td><td></td><td></td></tr><tr><td>Caspase-3</td><td>Beyotime</td><td>Cat# AC033</td><td></td></tr><tr><td>CD8</td><td>Cell Signaling Technology</td><td>Cat# 98941; RRID:AB_2756376</td><td></td></tr><tr><td>CD206</td><td>Cell Signaling Technology</td><td>Cat# 24595; RRID:AB_2892682</td><td></td></tr><tr><td>Chamber for anesthesia induction</td><td>RWDlifescience</td><td></td><td></td></tr><tr><td>Enhanced DAB chromogenic kit</td><td>MXB</td><td>Cat# DAB-2031</td><td></td></tr><tr><td>Eosin staining solution</td><td>ZSGB-GIO</td><td>Cat# ZLI-9613</td><td></td></tr><tr><td>F4/80</td><td>Abcam</td><td>Cat# 100790; RRID:AB_10675322</td><td></td></tr><tr><td>Foxp3</td><td>Cell Signaling Technology</td><td>Cat# 12653; RRID:AB_2797979</td><td></td></tr><tr><td>Fully enclosed tissue dehydrator</td><td>Leica Biosystems</td><td>ASP300S</td><td></td></tr><tr><td>Hematoxylin staining solution</td><td>ZSGB-GIO</td><td>Cat# ZLI-9610</td><td></td></tr><tr><td>HistoCore Arcadia fully automatic tissue embedding machine</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Ki67</td><td>Beyotime</td><td>Cat# AF1738</td><td></td></tr><tr><td>Rotating Slicer</td><td>RWDlifescience</td><td>&amp;nbsp;Minux S700</td><td></td></tr><tr><td>SPlink detection kits (Biotin-Streptavidin HRP Detection Systems)</td><td>ZSGB-GIO</td><td>Cat# SP-9001</td><td></td></tr><tr><td>&lt;em&gt;TPO-cre/ERT2&lt;/em&gt; mice</td><td>Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China</td><td></td><td></td></tr><tr><td>&lt;em&gt;Trp53&lt;sup&gt;flox/wt &lt;/sup&gt;&lt;/em&gt;mice</td><td>Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China</td><td></td><td></td></tr><tr><td>Ultrasonic cell crusher</td><td>Ningbo Xinyi Ultrasound Equipment Co., Ltd</td><td>JY92-IIN</td><td></td></tr><tr><td>Ultrasound gel</td><td>Keppler</td><td>KL-250</td><td></td></tr><tr><td>Ultrasound system</td><td>VisualSonics</td><td>Vevo 3100</td><td></td></tr></tbody></table>

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 recorded the mouse survival time, tumor features/size, and metastasized lesions.
Grossly, we observed the following: 1) the tumors were tender, and the size of the tumors on the left and right sides were inconsistent; 2) most mice (11/12) had lung metastases, but none had liver metastases. Specifically, the tumor size was monitored by animal high-frequency ultrasound and photoacoustic imaging systems (Vevo&#174;3100) throughout the whole process17. Based on the ultrasound data, the tumor growth curve (Figure 1A) was plotted to observe the dynamic alteration of the tumor size. Furthermore, the tumors grew slowly at the early stage (average tumor size varied from 9.47 mm2 to 11.75 mm2 from Day 0 to Day 60), and became dramatically faster at the late stage (average tumor size varied from 11.75 mm2 to 23.95 mm2 (from Day 60 to Day 100). Most mice were sacrificed&#160;in the late stage. In short, mATC with a certain tumor latency period needs to be closely monitored after 60 days to prevent asphyxiation-related death.
On the other hand, the survival time of the mice was recorded, and a survival curve (Figure 1B) was plotted. The median survival of mATC was 130 days, ranging from 56-166 days. In addition, lung metastasis was found in most mATC (approximately 92%) (Figure 1C). We observed that only one mouse in this cohort did not show lung metastases on gross examination, and six mice had more than one lung metastatic lesion. No liver metastasis was found. In brief, these results were consistent with the biological behavior of ATC, which are prone to lung metastasis in clinics.
Furthermore, to better observe the dynamic process of mATC, we sacrificed the mice at two time points (1 month and 2 months after induction). We performed HE staining on the primary tumors and metastatic lung tissues of mATC (Figure 1D). In 1 month inducible tissue, we observed incomplete solidified features and the coexistence of follicular structures and malignant cells. The thyroid follicular structures disappeared, and the tumor solidified completely after a 2 month induction. HE staining of the primary tumor revealed that the tumor cells were morphologically diverse, with pleomorphic giant cells (indicated by a red arrow) and spindle-shaped cells (indicated by a yellow arrow). It also showed a wide variety in nuclear size, and many cells contained multiple nuclei. Clear metastatic foci (indicated by a circle) were seen in the lung. HE staining of metastatic lung tissues showed that the normal lung tissue was a reticular structure with clear alveolar structures and airspaces. Nevertheless, lung metastases showed a loss of normal reticular structure, air cavity thickening, and lung parenchyma.
Meanwhile, IHC staining was performed to further characterize (Cd8, Foxp3, F4/80, Cd206, Ki67, and Caspase-3) mATC tumors to quantify cell proliferation and apoptosis, and investigate the infiltration of lymphocytes, T-regular (Treg) cells, and myeloid cells (Figure 2A-D). Anti-Ki67 staining was highly positive, ranging from 86.9% to 95.07%, demonstrating a high degree of cell proliferation. Anti-activated caspase-3 antibody was used to test the rate of apoptosis, ranging from 5.2% to 51.9%. Specifically, mATC presented obvious CD8+ T&#160;cell infiltration, the ratio of which ranged from 0.47% to 10.55% (mean: 5.93%). This indicated that mATC was not an immune-desert tumor, which was consistent with ATC samples. Besides, Foxp3 staining defined Treg cells, which varied from 0.45% to 25.8%. In addition to lymphocytes, F4/80 and Cd206 were used to define macrophages and M2 macrophages, respectively. We found that myeloid cells extensively infiltrated tumors (F4/80 positive cell rate from 86.6% to 94.6%; Cd206 positive cell rate from 40.4% to 67.7%), which was consistent with the previous literature16. In brief, we found highly proliferative tumor cells, lymphocyte infiltration, and extensive infiltration of myeloid cells in mATC, which was consistent with clinical samples.
In conclusion, mATC samples showed homogeneity in tumor dynamics, metastasis, and pathological features, consistent with the clinical samples. Considering the tumor formation rate, mATC was a reliable murine model.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64607/64607fig01.jpg" /> 
Figure 1:&#160;Tumor dynamics and pathological characteristics. (A) Tumor growth curve of mice (n = 5). Each line represents a mouse: the early stage ranges from Day 0 to Day 60, and the late stage ranges from Day 60 to Day 100. (B) Survival curves of mice (n = 12). The median survival of mATC was 130 days, ranging from 56 days to 166 days. (C) Lung metastasis curves of mice (n = 12). Representative dissection images of thyroid and lung metastasis. The red arrow indicates the thyroid tumor, and the white circle indicates metastasis in the lung. (D) HE staining of the primary tumor (1 month and 2 months after induction) and lung metastasis. The red arrow indicates the pleomorphic giant cell, the yellow arrow indicates the spindle-shaped cell, and the circled area indicates a metastasis in the lung. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64607/64607fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64607/64607fig02.jpg" /> 
Figure 2:&#160;Brief description of immune cell infiltration in mATC.&#160;(A) Immunohistochemical staining of ATC murine primary tumors with antibodies (Cd8, Foxp3). The red arrow indicates a CD8 positive cell, the yellow arrow indicates a Foxp3 positive cell. (B) Immunohistochemical staining of ATC murine primary tumors with antibodies (Ki67, Caspase-3). (C) Immunohistochemical staining of ATC murine primary tumors with antibodies (F4/80, Cd206). (D) The quantification of IHC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64607/64607fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: The list of primers and the PCR settings used in this study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64607/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>

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 ...

spontaneous-murine-model-of-anaplastic-thyroid-cancer

Nanyang Technological University

Research

JoVE Journal

Cancer Research

1.6K Views.  Sichuan University and Collaborative Innovation Center. We presenteren een standaard pijplijn om MATC-tumoren te verkrijgen en presenteren tumordynamiek en pathologische informatie. Deze module zal onderzoekers helpen om tumoroncogenese te begrijpen en medicijnontdekkingen te vergemakkelijken. De MATC vergemakkelijkt het verzamelen van MATC-monsters in het hele tumoroncogeneseverloop, en elk is een spontaan spiegelmodel, met het volledige immuunsysteem en de immuunmicro-omgeving.MATC kan worden gebruikt om de effecten van specifieke genfunc...

Video: Spontaneous Murine Model of Anaplastic Thyroid Cancer

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

Medicine

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

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&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;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.

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 ...

Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its heterogeneity. Pharmacodynamic results based on PDX models are highly correlated with clinical practice. Anaplastic thyroid carcinoma (ATC) is the most malignant subtype of thyroid cancer, with strong invasiveness, poor prognosis, and limited treatment. Although the incidence rate of ATC accounts for only 2%-5% of thyroid cancer, its mortality rate is as high as 15%-50%. Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck malignancies, with over 600,000 new cases worldwide each year. Herein, detailed protocols are presented to establish PDX models of ATC and HNSCC. In this work, the key factors influencing the success rate of model construction were analyzed, and the histopathological features were compared between the PDX model and the primary tumor. Furthermore, the clinical relevance of the model was validated by evaluating the in vivo therapeutic efficacy of representative clinically used drugs in the successfully constructed PDX models.

The present protocol establishes and characterizes a patient-derived xenograft (PDX) model of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), as PDX models are rapidly becoming the standard in the field of translational oncology.

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

Immunology and Infection

Orthotopic Inoculation of Thyroid Cancer Cells into Murine Thyroid: A Procedure to Establish Orthotopic Thyroid Cancer Mouse Model

Source: Julia Ramirez-Moya, et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59322" target="_blank">In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice.</a> J. Vis. Exp. (2019)
This video describes the procedure for orthotopic inoculation of thyroid cancer cells into the thyroid gland of mouse models, which may then serve as a valuable platform to assess carcinogenesis and discover potential treatment strategies.

Source: Julia Ramirez-Moya, et al. In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice. J. Vis. Exp. (2019)
This video describes the procedure ...

JoVE Encyclopedia of Experiments

Head and Neck Cancer

In vivo studies

Modelling Intratumoral Treatment with miRNA Inhibitors: A Method to Analyze the Effect of miRNA Inhibitors on Tumor Growth in Murine Model

After establishing a Cal62 human thyroid cancer cell line in complete medium, suspend a 1 times 10 to the sixth cell aliquot in 50 microliters of PBS at 4 degrees Celsius and mix the cells with an equal volume of basement membrane matrix.
Load the cells into a 1-milliliter syringe equipped with a 27G 1/2&#34; needle and subcutaneously inject 100 microliters of the sample into the left flank of a 6-week-old immunodeficient BALB/c nude mouse. Two weeks after the injection, add the antagomiR or control treatment buffer solution to 160 microliters of room temperature in vivo delivery reagent in a 1.5-milliliter tube and immediately vortex the solution for 10 seconds to ensure complexation of the mixture.
Incubate the treatment solution for 30 minutes at 50 degrees Celsius, followed by a brief centrifugation in a microcentrifuge. Then, dilute the sedimented treatment complex six-fold with fresh PBS and thorough mixing, and inject the entire 200-microliter volume of the treatment directly into the tumor.
Inject 50 microliters of a 40 milligrams per milliliter D-luciferin substrate solution subcutaneously two times a week into each experimental animal, making sure to confirm a lack of response to toe pinch after isoflurane anesthesia. Place the animal in the chamber of an in vivo bioluminescence imaging system and image the bioluminescent signal with the in vivo imaging software according to standard protocols.

Spontaneous Murine Model of Anaplastic Thyroid Cancer

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