Intramucosal Inoculation of Cancer Cells in Murine Model: A Procedure to Establish an Orthotopic Head and Neck Cancer Mouse Model

Overview

In this video, we develop an orthotopic head and neck cancer murine model by intramucosal inoculation of the squamous cell carcinoma cell line. The tumor-bearing mouse would show resemblance to clinically relevant symptoms of human disease and can thus be used to study cancer progression and discover novel therapeutic strategies.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Cell Injection into Mice

1. Prepare a 1:1 mixture of cells:basement membrane matrix (50 µL each).
2. Add the cells first; then, gradually pipette the basement membrane matrix. Avoid introducing air bubbles. Ensure that the mixture is made immediately before the animal injection. Adding cells to the basement membrane matrix for an extended period of time can result in cells settling in the matrix mixture, which makes the mixture difficult to shake vigorously. This will cause considerable variability in tumor size between mice.
3. Mix gently. Ensure all steps involving matrix are performed on ice. The basement membrane matrix will polymerize at room temperature.
4. Prepare syringes for inoculation.
5. Load 0.5 mL insulin syringes (23G) with 100 µL of the cell/basement membrane matrix solution.
6. Keep the syringes on ice to avoid basement membrane matrix polymerization.
7. Anesthetize mice by placing them in a chamber with isoflurane and oxygen (2.5%).
8. Ensure the mice are deeply anesthetized before performing the injection (by ensuring a lack of response to a toe pinch).
9. Insert the needle into the right or left buccal region. This is performed through the available open space on either side of the mouth.
10. Ensure that the mouse’s tongue is not in the way.
    NOTE: It is easy to poke the tongue, which will result in tongue tumors. Move the tongue to the opposite side if necessary.
11. Keep the syringe parallel to the buccal region while inside the oral cavity.
12. When ready to inject, pull the syringe back and slowly insert the syringe at a 10° angle.
13. Inject 100 µL of the cell/basement membrane matrix suspension over a period of 5 s.
14. Hold the syringe in place for an additional 5 s to ensure all material is injected.
    NOTE: For control nontumor-bearing mice, inject a mixture of serum-free media and matrix (as described above) without the tumor cells.
15. Withdraw the syringe gently.
16. Continue the above procedure with the remaining mice.
17. Allow for 1 week until tumors begin to appear grossly (50–200 mm³ for B4B8 and LY2 cells).

Materials

Name	Company	Catalog Number	Comments
HBSS	ThermoFisher Scientific	14175079	No calcium, no magnesium, no pheno red
Helois mass cytometer	Fluidigm	NA
Matrigel membrane matrix	Corning	CB-40234B