Name: intramucosal inoculation of cancer cells in murine model: a procedure to establish an orthotopic head and neck cancer mouse model
Uploaded: 2023-04-30T13:39:45.000+00:00
Duration: 1 min 26 s
Description: Source: Oweida, A. J. et al. Intramucosal inoculation of squamous cell carcinoma cells in mice for tumor immune profiling and treatment response ...

intramucosal inoculation of cancer cells in murine model: a procedure to establish an orthotopic head and neck cancer mouse model

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

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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Research

JoVE Encyclopedia of Experiments

Cancer Research

Head and Neck Cancer

In vivo studies

EoE Sub Articles

eoe sub articles

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
<ol>
	<li>Prepare a 1:1 mixture of cells:basement membrane matrix (50 &#181;L each).</li>
	<li>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.</li>
	<li>Mix gently. Ensure all steps involving matrix are performed on ice. The basement membrane matrix will polymerize at room temperature.</li>
	<li>Prepare syringes for inoculation.</li>
	<li>Load 0.5 mL insulin syringes (23G) with 100 &#181;L of the cell/basement membrane matrix solution.</li>
	<li>Keep the syringes on ice to avoid basement membrane matrix polymerization.</li>
	<li>Anesthetize mice by placing them in a chamber with isoflurane and oxygen (2.5%).</li>
	<li>Ensure the mice are deeply anesthetized before performing the injection (by ensuring a lack of response to a toe pinch).</li>
	<li>Insert the needle into the right or left buccal region. This is performed through the available open space on either side of the mouth.</li>
	<li>Ensure that the mouse&#8217;s tongue is not in the way. 
	NOTE:&#160;It is easy to poke the tongue, which will result in tongue tumors. Move the tongue to the opposite side if necessary.</li>
	<li>Keep the syringe parallel to the buccal region while inside the oral cavity.</li>
	<li>When ready to inject, pull the syringe back and slowly insert the syringe at a 10&#176; angle.</li>
	<li>Inject 100 &#181;L of the cell/basement membrane matrix suspension over a period of 5 s.</li>
	<li>Hold the syringe in place for an additional 5 s to ensure all material is injected. 
	NOTE:&#160;For control nontumor-bearing mice, inject a mixture of serum-free media and matrix (as described above) without the tumor cells.</li>
	<li>Withdraw the syringe gently.</li>
	<li>Continue the above procedure with the remaining mice.</li>
	<li>Allow for 1 week until tumors begin to appear grossly (50&#8211;200 mm3&#160;for B4B8 and LY2 cells).</li>
</ol>

<table><tbody><tr><td>HBSS</td><td>ThermoFisher Scientific</td><td>14175079</td><td>No calcium, no magnesium, no pheno red</td></tr><tr><td>Helois mass cytometer</td><td>Fluidigm</td><td>NA</td><td></td></tr><tr><td>Matrigel membrane matrix</td><td>Corning&amp;nbsp;</td><td>CB-40234B</td><td></td></tr></tbody></table>

Source: Oweida, A. J. et al. <a href="https://www.jove.com/video/59195" target="_blank">Intramucosal inoculation of squamous cell carcinoma cells in mice for tumor immune profiling and treatment response assessment.</a> J. Vis. Exp. (2019)
In this video, we develop an orthotopic head and neck cancer murine model&#160;by intramucosal inoculation of the squamous cell&#160;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.

Source: Oweida, A. J. et al. Intramucosal inoculation of squamous cell carcinoma cells in mice for tumor immune profiling and treatment response ...

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. &#8203;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.

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

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