Published: April 17th, 2013
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 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.
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 °C one day prior to cell harvest.
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).
Note: Each mouse will receive 10 μl of the cell suspension/matrigel cocktail. Since 5 x 105 cells suspended in a 10 μl injectable volume have been successfully used in previous ATC orthotopic studies9-10, we used those parameters in this report.
2. Mouse Preparation
3. Surgical Access to Thyroid and Cell Injection
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.
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.
4. Closure of Surgical Site and Mouse Recovery
5. Post-operative Monitoring and Tissue Collection
Note: Animals exhibiting signs of declining health (e.g. substantial weight loss, scruffy hair, labored breathing) should be euthanized and tissues collected.
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&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.
Figure 1. Exposing the thyroid. Labels indicate the right and left thyroid glands flanking the trachea.
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&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.
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.
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.
The authors have nothing to disclose.
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.
|Name of Reagent/Material
|RPMI 1640 cell culture media
|Media and additives used depend on cell line
|TC10 Automated Cell Counter
|Becton, Dickinson and Company
|Graefe Forceps, Serrated; Slight Curve, 4"
|Disposable scalpel, No. 15
|Olsen Hegar Needle Holder - 4 ½" delicate serrated
|6-0 nylon black monofilament suture
|Surgical Specialties Corporation
|Heating Pad, reusable, 8" x 12"
|Cloth tape, 1" X 10 yds
|For restraining anesthetized mouse
|For removing hair from surgical site
|ChlorHex-Q SCRUB 2%
|Penn Veterinary Supply
|Henry Schein Company
|Petrolatum ophthalmic ointment
|Dechra Veterinary Products
|2 x 2 gauze, 12-ply
|Butler Animal Health Supply
|Sterile Field, Barrier, 18" x 26"
|Drapes (CSR Wrap)
|AT 21 412
|27G ½" needle
|Becton, Dickinson and Company
|31G 5/16" needle
|Becton, Dickinson and Company
|Ketamine (9 mg/ml) / xylazine (1 mg/ml) solution
|Triple antibiotic ointment
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