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

Abstract

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.

Protocol

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

1. Cell preparation
   1. 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.
   2. Grow these cells in DMEM supplemented with antibiotics (penicillin and streptomycin) and 10% FBS at 37 °C and 5% CO₂.
   3. Suspend 1 x 10⁵ cells in 5 μL of PBS.
2. Cell inoculation into the thyroid gland of mice
   1. Inject 100 μL of analgesic (buprenorphine) and 100 μL of antibiotic (cephalosporin) subcutaneously into 7-week-old BALB/c nu/nu mice. Disinfect the injection site with alcohol.
   2. Inject 5 μL of the cell solution into the thyroid gland of the mice.
      1. Anesthetize the mouse using 3% isoflurane mixed with oxygen and place it under a stereomicroscope in a sterile flow cabinet.  Assess the level of anesthesia by pedal reflex (firm toe pinch) and adjust anesthetic delivery as appropriate to maintain the surgical plane.
      2. Apply ophthalmic ointment to both eyes to prevent desiccation. 
      3. 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.
      4. Dissect the strap muscles using dissection forceps and/or scissors to expose the trachea and the thyroid gland. Using a 10 μL syringe, inject 5 μL of the cell solution into the right thyroid lobule, located at the side of the cricoid cartilage.
      5. Reposition the salivary glands and suture the incision with silk using braided, coated, non-absorbable sutures.
      6. Add iodopovidone to the wound area and place the mouse on a thermic blanket while it recovers from the anesthesia.
   3. 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.

Materials

Basement Membrane Matrix: Matrigel Basement Membrane Matrix High Concentration	Corning	#354248
In vivo delivery reagent: Invivofectamine 3.0 Reagent	Thermo Fisher	IVF3005
In vivo imaging software: IVIS-Lumina II Imaging System	Caliper Life Sciences