The authors would like to thank Drs. Wenjun Li and Daniel Kreisel (Washington University School of Medicine in St. Louis) for their assistance in surgery training. R.Y.L. is supported by the National Institutes of Health Grant R01 DK068057 and the President's Research Fund of Saint Louis University.

1. Cell Preparation
Notes: 1) complete RPMI 1640 based culture media recipe (500 ml) includes 435 ml RPMI 1640, 50 ml heat inactivated FBS, 5 ml non-essential amino acids, 5 ml sodium pyruvate, 5 ml antibiotic-antimycotic; 2) place matrigel at 4 &deg;C one day prior to cell harvest.
<ol>
	<li>Human ATC cell line THJ-11T8 are grown in 6 well culture plates and harvested once 80-90% confluent.</li>
	<li>Pellet cells by centrifugation at 200 x g for 4 min at room temperature.</li>
	<li>Aspirate media and resuspend cells in RPMI 1640 media [2 ml/6 well plate].</li>
	<li>Take a small volume of the cell suspension, add it to one volume of 0.4% trypan blue dye and determine cell density using TC10 Automated Cell Counter.</li>
	<li>Calculate volume of cell suspension needed to get 5 x 105 cells/mouse times the number mice injecting.</li>
</ol>
Note: To ensure adequate cell suspension for injection, prepare cells as though you are injecting double the number of mice (e.g. if injecting 5 mice, then prepare cell suspension as though you were injecting 10 mice).
<ol start="6">
	<li>Transfer needed volume of cell suspension into a 15 ml conical tube.</li>
	<li>Pellet cells by centrifugation at 200 x g for 4 min at room temperature.</li>
	<li>Aspirate media and resuspend cells in complete RPMI 1640 media.</li>
	<li>Transfer cells to a 1.6 ml tube, add 1 volume of matrigel and mix gently by slowly pipetting in and out.</li>
</ol>
Note: Each mouse will receive 10 &mu;l of the cell suspension/matrigel cocktail. Since 5 x 105 cells suspended in a 10 &mu;l injectable volume have been successfully used in previous ATC orthotopic studies9-10, we used those parameters in this report.
<ol start="10">
	<li>Place cell suspension on ice until ready to use.</li>
</ol>
2. Mouse Preparation
<ol>
	<li>Clean the surgical zone, which includes the dissecting scope and surrounding area, by wiping all surfaces with 70% ethanol.</li>
	<li>Turn on heating lamps/pads where animals will be recovering from surgery.</li>
	<li>Administer 0.1 ml ketamine/xylazine (drug cocktail consisting of ketamine 9 mg/ml and xylazine 1mg/ml) per 10 g body weight intraperitoneally using a 1 ml, 27G &frac12;" Tuberculin syringe.</li>
	<li>Prep mouse for surgery: 1) shave neck region of mouse (from jaw line to top of sternum and out to arms), 2) scrub surgical area three times with chorhexadine soaked gauze squares, 3) final scrub done with betadine soaked gauze, 4) gently apply eye ointment to prevent eyes from drying.</li>
	<li>Check pedal reflex to ensure mouse is adequately sedated.</li>
	<li>Place animal in dorsal recumbency on a disposable sterile field barrier under the dissecting scope and secure the animal in place with cloth tape.</li>
	<li>Scrubs hands and fingernails then put on sterile surgical gloves.</li>
	<li>In a sterile fashion, open surgery pack and arrange instruments.</li>
	<li>Place sterile drape over animal, leaving only surgical site exposed.</li>
</ol>
3. Surgical Access to Thyroid and Cell Injection
<ol>
	<li>Using a sterile, disposable scalpel, make a 1-to-1.5 cm longitudinal incision along the midline of the throat.</li>
</ol>
Note: Deviation from the midline complicates deeper cutting to access the trachea. Fairly precise midline incision allows you to tease and cut through the membranous tissue holding left and right salivary glands together and reduces chance of nicking or severing large arteries.
<ol start="2">
	<li>Make a second incision into the strap muscles surrounding the trachea then pull the right side of the incised muscle to the side to expose the right thyroid gland.</li>
	<li>Hemostats can be used at this point to hold the muscle layer apart for easy access to the thyroid gland (Figure 1).</li>
</ol>
Note: Alternatively, if procedure performed by two surgical staff, the muscle layer may simply be held back with forceps while a ready injectable syringe is handed to the surgeon by the assisting staff.
<ol start="4">
	<li>Slowly inject 10 &mu;l of cell suspension into the right thyroid gland using a 31G 5/16" insulin syringe then gently remove needle.</li>
</ol>
4. Closure of Surgical Site and Mouse Recovery
<ol>
	<li>Suture muscle layer with 6.0 suturing material using either a continuous or interrupted suturing style.</li>
	<li>Stitch the skin in the same fashion.</li>
	<li>Apply a layer of triple antibiotic ointment directly over incision site.</li>
	<li>Allow animal to recover in dorsal recumbency on warm heating pad. Once animal can achieve sternal recumbency without assistance, it can be placed back with other mice.</li>
</ol>
5. Post-operative Monitoring and Tissue Collection
<ol>
	<li>Check the incision site and overall health of the animal daily for a week following surgery, then check once a week. If the sutures are still intact after 14 days, they are removed.</li>
</ol>
Note: Animals exhibiting signs of declining health (e.g. substantial weight loss, scruffy hair, labored breathing) should be euthanized and tissues collected.
<ol start="2">
	<li>Once mice have reached a predetermined post-operative time-point, administer ketamine/xylazine as describe above to sedate.</li>
	<li>Perfuse animal intracardially and harvest thyroid/trachea and other tissues of interest (not described in detail here).</li>
</ol>

<ol>
	<li>Bibby, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+of+cancer+for+preclinical+drug+evaluation:+advantages+and+disadvantages.">Orthotopic models of cancer for preclinical drug evaluation: advantages and disadvantages.</a> Eur. J. Cancer. 40, 852-857 (2004).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+metastatic+mouse+models+for+anticancer+drug+discovery+and+evaluation:+a+bridge+to+the+clinic.">Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic.</a> Invest. New Drugs. 17, 343-359 (1999).</li><li>Killion, J. J., Radinsky, R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+are+necessary+to+predict+therapy+of+transplantable+tumors+in+mice.">Orthotopic models are necessary to predict therapy of transplantable tumors in mice.</a> Cancer Metastasis Rev. 17, 279-284 (1998).</li><li>Kim, 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+orthotopic+model+of+anaplastic+thyroid+carcinoma+in+athymic+nude+mice.">An orthotopic model of anaplastic thyroid carcinoma in athymic nude mice.</a> Clin. Cancer Res. 11, 1713-1721 (2005).</li><li>Nucera, C., 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=A+novel+orthotopic+mouse+model+of+human+anaplastic+thyroid+carcinoma.">A novel orthotopic mouse model of human anaplastic thyroid carcinoma.</a> Thyroid. 19, 1077-1084 (2009).</li><li>Todaro, M., 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=Tumorigenic+and+metastatic+activity+of+human+thyroid+cancer+stem+cells.">Tumorigenic and metastatic activity of human thyroid cancer stem cells.</a> Cancer Res. 70, 8874-8885 (2010).</li><li>Tran Cao, H. 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=Real-time+imaging+of+tumor+progression+in+a+fluorescent+orthotopic+mouse+model+of+thyroid+cancer.">Real-time imaging of tumor progression in a fluorescent orthotopic mouse model of thyroid cancer.</a> Anticancer Res. 30, 4415-4422 (2010).</li><li>Marlow, L. A., 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=Detailed+molecular+fingerprinting+of+four+new+anaplastic+thyroid+carcinoma+cell+lines+and+their+use+for+verification+of+RhoB+as+a+molecular+therapeutic+target.">Detailed molecular fingerprinting of four new anaplastic thyroid carcinoma cell lines and their use for verification of RhoB as a molecular therapeutic target.</a> J. Clin. Endocrinol. Metab. 95, 5338-5347 (2010).</li><li>Nehs, M. A., 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=Late+Intervention+with+anti-BRAF+(V600E)+therapy+induces+tumor+regression+in+an+orthotopic+mouse+model+of+human+anaplastic+thyroid+cancer.">Late Intervention with anti-BRAF (V600E) therapy induces tumor regression in an orthotopic mouse model of human anaplastic thyroid cancer.</a> Endocrinology. 153, 985-994 (2012).</li><li>Nucera, C., 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=Targeting+BRAFV600E+with+PLX4720+displays+potent+antimigratory+and+anti-invasive+activity+in+preclinical+models+of+human+thyroid+cancer.">Targeting BRAFV600E with PLX4720 displays potent antimigratory and anti-invasive activity in preclinical models of human thyroid cancer.</a> Oncologist. 16, 296-309 (2011).</li></ol>

The authors have nothing to disclose.

In our model, animals demonstrated thyroid tumor metastasis and disease related cachexia and respiratory distress by 4 weeks post-injection. As with most other orthotopic models utilizing injectable delivery of a cell suspension, injection into surrounding tissues and post-injection leakage that spreads beyond the target are possible. With that being said, one recognizable potential unknown produced by this procedure is the effect of off target exposure to non-disease related metastases or complications. This is a general concern with most xenographic transplants, regardless of the disease being investigated. Nonetheless, unintentional consequences from metastasis may be significantly minimized by ensuring the entire bevel of the needle tip has penetrated the membrane snugly overlaying the thyroid gland and slowly expelling the cellular suspension. If leakage is observed during or after injection, those animals may be omitted from the study.
It is essential that the animals are perfused prior to harvesting the thyroid/trachea and other tissues. The benefits are two-fold, 1) red blood cells are purged and will not clutter histological specimens and 2) tissue morphology and integrity are preserved. Furthermore, resected thyroid/trachea that have either a tumorous mass attached or are entombed within it should be cut in a way that anatomical landmarks can be identified before being embedded for histological preparation. This will dramatically reduce specimen preparation time.
In this report we describe local growth and invasion of advanced thyroid tumor following orthotopic injection of ATC cells. ATC metastasis may be investigated by analyzing histological preparations of other tissues using standard histological stains, such as hematoxylin and eosin, or by immunohistochemistry utilizing cell or pathology relevant markers. This system can be further developed by genetically modifying thyroid cancer cells with fluorescence reporters so that live imaging may be used to monitor metastasis and the efficacy of therapeutic treatments.

<table><tbody><tr><td>RPMI 1640 cell culture media</td><td>Mediatech</td><td>10-041-CV</td><td>Media and additives used depend on cell line</td></tr><tr><td>TC10 Automated Cell Counter</td><td>Bio-Rad</td><td>145-0001</td><td></td></tr><tr><td>Matrigel</td><td>Becton, Dickinson and Company</td><td>354234</td><td></td></tr><tr><td>Graefe Forceps, Serrated; Slight Curve, 4&quot;</td><td>Roboz</td><td>RS-5135</td><td></td></tr><tr><td>Disposable scalpel, No. 15</td><td>Feather</td><td>2975#15</td><td></td></tr><tr><td>Olsen Hegar Needle Holder - 4 &amp;frac12;&quot; delicate serrated</td><td>gSource</td><td>gS 21.5400</td><td></td></tr><tr><td>6-0 nylon black monofilament suture</td><td>Surgical Specialties Corporation</td><td>1279B</td><td></td></tr><tr><td>Heating Pad, reusable, 8&quot; x 12&quot;</td><td></td><td></td><td></td></tr><tr><td>Cloth tape, 1&quot; X 10 yds</td><td>Medline</td><td>MIINON260101</td><td>For restraining anesthetized mouse</td></tr><tr><td>Peanut Clipper/Trimmer</td><td>Wahl</td><td>8655</td><td>For removing hair from surgical site</td></tr><tr><td>ChlorHex-Q SCRUB 2%</td><td>Penn Veterinary Supply</td><td>VED1222</td><td></td></tr><tr><td>Betadine</td><td>Henry Schein Company</td><td>6906727</td><td></td></tr><tr><td>Petrolatum ophthalmic ointment</td><td>Dechra Veterinary Products</td><td>17033-211-38</td><td></td></tr><tr><td>2 x 2 gauze, 12-ply</td><td>Butler Animal Health Supply</td><td>6936</td><td></td></tr><tr><td>Sterile Field, Barrier, 18&quot; x 26&quot;</td><td>Busse</td><td>696</td><td></td></tr><tr><td>Drapes (CSR Wrap)</td><td>Cardinal Health</td><td>AT 21 412</td><td></td></tr><tr><td>27G &amp;frac12;&quot; needle</td><td>Becton, Dickinson and Company</td><td>309623</td><td></td></tr><tr><td>31G 5/16&quot; needle</td><td>Becton, Dickinson and Company</td><td>328438</td><td></td></tr><tr><td>Ketamine (9 mg/ml) / xylazine (1 mg/ml) solution</td><td></td><td></td><td></td></tr><tr><td>Triple antibiotic ointment</td><td></td><td></td><td></td></tr></tbody></table>

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.

We detected 19 invasive thyroid tumors from a total of 20 mice injected with ATC cells after 4 weeks. In the example shown in Figure 2A, mice receiving an orthotopic injection of 5X105 ATC cells grown under attached conditions, exhibit significant infiltration of cancer cells into the thyroid by 4 weeks post-injection. The nature of the invading ATC cells with a characteristic spindled cells and medium- to giant-sized cells with eosinophilic cytoplasm and large nuclei was verified by hematoxylin and eosin (H&amp;E) staining. For comparison, the thyroid and trachea from a non-injected control animal is shown in Figure 2B. Follicles of the uninjected animal display a close, yet loose, association and have essentially a round morphology.
Due to the small size of the thyroid and use of injectable delivery, unintentional outcomes may arise. Figure 3A illustrates the most commonly observed, which is development of a tumor mass outside of, but not encompassing, the thyroid. This is more than likely due to needle penetration through and beyond the thyroid when cells are expelled. Occasionally, mice presenting with no tumor growth, both grossly and histologically, are detected as shown in Figure 3B. Absence of detectable tumor development can be caused by expulsion of the cell suspension from the injection site and dissemination into neighboring tissues and cavities.
<img src="/files/ftp_upload/50097/50097fig1.jpg" alt="Figure 1"/> 
 Figure 1. Exposing the thyroid. Labels indicate the right and left thyroid glands flanking the trachea.
<img src="/files/ftp_upload/50097/50097fig2.jpg" alt="Figure 2"/> 
 Figure 2. Histological examination of tumor growth and invasion after successful thyroid injection. Tissue specimens were sectioned along the coronal plane. A) Image taken at 200X of mouse exhibiting substantial tumor growth with characteristic spindled cells and medium- to giant- sized cells with eosinophilic cytoplasm and large nuclei in hematoxylin and eosin (H&amp;E) stain. Tu indicates the primary lesion from which tumor cells invade the thyroid. B) Trachea and thyroid from control animal (image taken at 100X). Tu ,Tumors; S, smooth muscle; Tr, trachea; Thy, thyroid gland.
<img src="/files/ftp_upload/50097/50097fig3.jpg" alt="Figure 3"/> 
 Figure 3. Unintentional outcomes of orthotopic injection. Tissue specimens were sectioned along the coronal plane. A) Peripheral tumor growth with no growth evident in the thyroid (black arrow, image taken at 100X). B) Lack of tumor detection upon gross (not shown) or histological examination (image taken at 50X). Thy, thyroid gland.

Watch this Scientific Journal Video about An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma at JoVE.com

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

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 ...

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21.0K Views.  Saint Louis University School of Medicine. 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.

Video: An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

Biology

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

Cancer Research

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 ...

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.

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 ...

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 ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

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/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

Intracranial Orthotopic Model: A Procedure to Implant Cancer Cells in Mouse Brain

Source: Geisler, J. A. et al. <a href="https://www.jove.com/v/61272/modeling-brain-metastases-through-intracranial-injection-magnetic">Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging.</a> J. Vis. Exp. (2020)
This video demonstrates implantation of tumor cells in the mouse brain by performing intracranial injection.

Source: Geisler, J. A. et al. Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging. J. Vis. Exp. (2020)
This video ...