This article describes a method for orthotopic intramucosal inoculation of tumor cells in mice. The procedure facilitates the study of tumor development and metastasis in a controlled environment.
During orthotopic intramucosal inoculation, tumor cells matching the mucosal tissue histotype get delivered into the mucosal tissue of a recipient animal.
To begin, restrain an anesthetized mouse by its neck on a surgical platform for easier access to its mouth.
Prepare a suspension of highly metastasizing cancer cells in a chilled basement membrane matrix in a syringe. The low temperature prevents the suspension from solidifying.
Carefully inject this suspension into the inner cheek or buccal region of the mouse. The buccal region is lined by the buccal mucosa, which is surrounded by a network of blood and lymphatic vessels.
Ensure complete delivery of the cancer cell suspension and gently retract the needle. Allow the matrix to solidify and embed the cells within.
Allow the mouse to recover. Within days of injection, a primary tumor develops at the site of injection. Eventually, the cells from this tumor with high metastatic potential may travel through the lymphatic system and extravasate into lymph nodes.
These cancer cells adapt to their new microenvironment and proliferate and establish metastatic colonies in lymph nodes.
Immediately before the injection, mix the cells at a 1 to 1 tumor cell suspension to basement membrane matrix ratio on ice. Load one 0.5 milliliter syringe equipped with a 23G needle with 100 microliters of cells per recipient animal. Place the syringes on ice, and confirm a lack of response to toe pinch in an anesthetized mouse.
Next, insert the needle into the right or left buccal region through the available open space on either side of the mouth, keeping the syringe parallel to the buccal region while it is inside the oral cavity, to facilitate injection of the full 100-microliter volume of the cell basement membrane matrix suspension over a period of 5 seconds. Keep the syringe in place for an additional 5 seconds to ensure that all of the material has been injected before withdrawing the syringe gently. The tumors will become grossly visible in about a week.
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