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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. 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>

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

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.

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

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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Research

JoVE Encyclopedia of Experiments

Cancer Research

Head and Neck Cancer

In vivo studies

1.1K Views. Źródło: Oweida, A. J. et al. Inokulacja dośluzówkowa komórek raka płaskonabłonkowego u myszy w celu profilowania immunologicznego guza i oceny odpowiedzi na leczenie. J. Vis. Exp. (2019 rok) W tym filmie rozwijamy ortotopowy model mysiego raka głowy i szyi poprzez dośluzówkowe inokulację linii komórkowej raka płaskonabłonkowego. Mysz z nowotworem wykazywałaby podobieństwo do klinicznie istotnych objawów choroby u ludzi, a zatem może być wykorzystana do badania progresji raka...

Video: Dośluzówkowa inokulacja komórek rakowych w modelu mysim: procedura mająca na celu ustalenie ortotopowego mysiego modelu raka głowy i szyi - Concept

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

JoVE Journal

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

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

Transkrypcja

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