Name: Video: Orthotopic Inoculation of Thyroid Cancer Cells into Murine Thyroid: A Procedure to Establish Orthotopic Thyroid Cancer Mouse Model - Concept
Uploaded: 2023-04-30T13:40:53.000+00:00
Duration: 1 min 24 s
Description: 1.4K Views. 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 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.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Thyroid Orthotopic Inoculation of Cells 
<ol>
	<li>Cell preparation
	<ol>
		<li>Engineer a Cal62 human thyroid cancer cell line to overexpress a transgenic construct that constitutively expresses GFP and luciferase. Select the transgenic cells by antibiotic resistance or by sorting the GFP-positive cells with flow cytometry.</li>
		<li>Grow these cells in DMEM supplemented with antibiotics (penicillin and streptomycin) and 10% FBS at 37 &#176;C and 5% CO2.</li>
		<li>Suspend 1 x 105&#160;cells in 5 &#956;L of PBS.</li>
	</ol>
	</li>
	<li>Cell inoculation into the thyroid gland of mice
	<ol>
		<li>Inject 100 &#956;L of analgesic (buprenorphine) and 100 &#956;L of antibiotic (cephalosporin) subcutaneously into 7-week-old BALB/c&#160;nu/nu&#160;mice.&#160;Disinfect the injection site with alcohol.</li>
		<li>Inject 5 &#956;L of the cell solution into the thyroid gland of the mice.
		<ol>
			<li>Anesthetize the mouse using 3% isoflurane mixed with oxygen and place it under a stereomicroscope in a sterile flow cabinet.&#160;&#160;Assess the level of anesthesia by pedal reflex (firm toe pinch) and adjust anesthetic delivery as appropriate to maintain the surgical plane.</li>
			<li>Apply ophthalmic ointment to both eyes to prevent desiccation.&#160;</li>
			<li>For each mouse, disinfect the neck with iodopovidone and make an incision of approximately 2 cm in the skin with scissors. Once open, expose the neck by displacing the salivary glands.</li>
			<li>Dissect the strap muscles using dissection forceps and/or scissors to expose the trachea and the thyroid gland. Using a 10 &#956;L syringe, inject 5 &#956;L of the cell solution into the right thyroid lobule, located at the side of the cricoid cartilage.</li>
			<li>Reposition the salivary glands and suture the incision with silk using braided, coated, non-absorbable sutures.</li>
			<li>Add iodopovidone to the wound area and place the mouse on a thermic blanket while it recovers from the anesthesia.</li>
		</ol>
		</li>
		<li>The following day post-surgery, inject subcutaneously analgesic (buprenorphine) and add 3 mL of liquid ibuprofen (40 mg/mL) into 250 mL of the drinking water for 1 week.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>Basement Membrane Matrix: Matrigel Basement Membrane Matrix High Concentration</td><td>Corning</td><td>#354248</td><td></td></tr><tr><td>In vivo&amp;nbsp;delivery reagent: Invivofectamine 3.0 Reagent</td><td>Thermo Fisher</td><td>IVF3005</td><td></td></tr><tr><td>In vivo&amp;nbsp;imaging software: IVIS-Lumina II Imaging System</td><td>Caliper Life Sciences</td><td></td><td></td></tr></tbody></table>

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.

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

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 ...

Orthotopic inoculation facilitates the delivery of cancer cells to a recipient animal at an organ or site that matches the tissue histotype. To begin, prep an anesthetized immunocompromised nude mouse in the supine position.
Next, make a surgical incision in the neck region to reveal the salivary glands. Displace the salivary glands and dissect the strap muscles to expose the underlying trachea and thyroid gland.
The thyroid gland, an endocrine gland, consists of two lobes that lie on either side of the trachea and surround the cricoid cartilage. These two lobes are connected by a thin strip of thyroid tissue called the isthmus.
Now, prepare a suspension of thyroid cancer cells with a suitable buffer in a syringe. Carefully inject the cell suspension into the mouse&#8217;s thyroid lobe.
Subsequently, reposition the salivary glands and suture the incision. Allow the mouse to recover. Monitor the mouse weekly to observe the growth of orthotopic tumors within the thyroid lobe.

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1.4K Views. 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 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. 

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

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

JoVE Journal

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

Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation

Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most common cause of the disease is accidental removal of the parathyroid glands during thyroid surgery. Novel therapies for HP are needed, but testing them requires reliable animal models of acquired HP. 
Here, we demonstrate the generation of two mouse models of acquired HP. In the GFP-PTX model, mice with green fluorescent protein (GFP) expressed specifically in the parathyroids (PTHcre-mTmG) were created by crossing PTHcre+ mice with Rosa-mTmGfl/fl mice. Green fluorescing parathyroid glands are easily identified under a fluorescence dissecting microscope and parathyroidectomy is performed in less than 20 min. After fluorescence-guided surgery, mice are profoundly hypocalcemic. Contrary to the traditional thyro-parathyroidectomy, this precise surgical approach leaves thyroid glands and thyroid function intact. The second model, which does not require surgery, is based on a diphtheria-toxin approach. PTHcre-iDTR mice, which express the diphtheria toxin (DT) receptor specifically in the parathyroids, were generated by crossing the inducible DTR mouse with the PTHcre mouse. Parathyroid cells are thus rendered sensitive to diphtheria toxin (DT) and can be selectively destroyed by systemically injecting mice with DT. The resulting hypocalcemic phenotype is stable.

Mice with acquired hypoparathyroidism would be useful for studying novel drug therapies for hypoparathyroidism. Two procedures to create such mice are demonstrated. The GFP-PTX mouse is generated by surgical parathyroidectomy guided by green fluorescing parathyroid glands. A second, non-surgical approach is based on parathyroid-specific expression of the diphtheria toxin receptor.

Hypoparathyroidism (HP) is a disorder characterized by low levels of PTH which lead to hypocalcemia, hyperphosphatemia, and low bone turnover. The most ...

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

Transcript

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

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation