The authors acknowledge the support of research grants from National Natural Science Foundation of China (31371390), the Program of the State High-Tech Development Project (2014AA020702) and the program of Guangdong Science and Technology (2016B030231001).

All animal work was performed in accordance with animal welfare act, institutional guidelines and approved by Institutional Animal Care and Use Committee, Sun Yat-sen University.
1. Preparation of TEM
<ol>
	<li>Execute mice by cervical dislocation and remove the tongues using sterile surgical scissors and tweezers.</li>
	<li>Immerse the tongues in 75% ethanol for 3 min, then put each tongue into a 1.5 mL Eppendorf (EP) tube with 1 mL of 10 mM sterile phosphate buffered solution (PBS). 
	NOTE: The concentration of PBS in all the following steps is same as the concentration in this step.</li>
	<li>Cell lysis by freeze thaw: Freeze the tongues in EP tubes at -80 &#176;C for 1 h, and then thaw the tongues at room temperature for 45 min for 3 cycles.</li>
	<li>Load each tongue onto a piece of surgical suture using a surgical needle and wrap the end of the suture with a small piece of sterile tinfoil. Perform the operation in a 3.5 cm or 6 cm culture dish containing 75% ethanol in sterile conditions. 
	NOTE: The appropriate length of each piece of surgical suture is about 20 cm, and the appropriate size of each piece of tinfoil is about 0.3 cm2 (1 cm &#215; 0.3 cm). The tongue should be loaded near the tinfoil.</li>
	<li>Rinse each tongue with 3 mL of sterile PBS in a 3.5 cm or 6 cm culture dish for 30 s. Perform this operation in sterile conditions.</li>
	<li>Wash&#160;the tongues with ultrapure water: Add ampicilin into a wide-mouth bottle with 250 mL of sterile ultrapure water to a final concentration of 90 &#181;g/mL. Put the tongues into the bottle containing a stir bar. Tighten the bottle cap with part of the suture remaining outside the bottle. Perform this operation in sterile conditions. 
	NOTE: Up to 5 tongues can be put into the same bottle in consideration of twining of the suture. The tinfoil is at the end of the suture in the bottle to prevent the tongue from slipping off. The tongues should be placed 2 cm high from the bottom of the bottle by adjusting the length of the suture remaining inside the bottle. This note is also for steps 1.8, 1.10, 1.12, and 1.16.</li>
	<li>Put the bottle on a magnetic stirrer for 12 h.</li>
	<li>Wash the&#160;tongues with NaCl: Add ampicilin into a wide-mouth bottle with 250 mL of sterile 1 M NaCl to a final concentration of 90 &#181;g/mL. Move the tongues and the stir bar into the bottle. Tighten the bottle cap with part of the suture remaining outside the bottle. Perform this operation in sterile conditions.</li>
	<li>Put the bottle on a magnetic stirrer for 24 h.</li>
	<li>Cell lysis by Triton X-100: Add ampicilin to a final concentration of 90 &#181;g/mL into a wide-mouth bottle with 250 mL of sterile 2% Triton X-100 in PBS. Move the tongues and the stir bar into the bottle. Tighten the bottle cap with part of the suture remaining outside the bottle. Perform this operation in sterile conditions.</li>
	<li>Put the bottle on a magnetic stirrer for 48 h.</li>
	<li>Wash&#160;tongues with CaCl2/MgCl2: Add ampicilin into a wide-mouth bottle with 250 mL of sterile 5 mM CaCl2/MgCl2 to a final concentration of 90 &#181;g/mL. Move the tongues and the stir bar into the bottle. Tighten the bottle cap with part of the suture remaining outside the bottle. Perform this operation in sterile conditions.</li>
	<li>Put the bottle on a magnetic stirrer for 24 h.</li>
	<li>Digestion by DNase: Add 1 mL of Hank&#39;s balanced salt solution (HBSS) to each EP tube. Add DNase into HBSS respectively to a final concentration of 300 &#181;M. Move each tongue into each EP tube, with part of the suture outside the tube. Perform this operation in sterile conditions. 
	NOTE: Make sure that the part of suture which remains inside the bottles in previous steps also remains inside the EP tube in this step, and make sure that the part of suture which remains outside the bottles in previous steps also remains outside the EP tube in this step.</li>
	<li>Incubate the tongues in EP tubes at 37 &#176;C for 24 h.</li>
	<li>Wash the&#160;tongues with PBS: Add ampicilin into a wide-mouth bottle with 250 mL of sterile PBS to a final concentration of 90 &#181;g/mL. Move the tongues and the stir bar into the bottle. Tighten the bottle cap with part of the suture remaining outside the bottle. Perform this operation in sterile conditions.</li>
	<li>Put the bottle on a magnetic stirrer for 24 h.</li>
	<li>Store the prepared TEM in sterile PBS at 4 &#176;C until use.</li>
</ol>
2. Three-dimensional (3D) Reconstitution of TSCC
<ol>
	<li>Static TSCC model construction
	<ol>
		<li>Seed 1.0 x 106 single TSCC cells (Cal27) into a 3.5 cm culture dish. Add 3 mL of Dulbecco&#39;s modified Eagle&#39;s medium/F12 (DF12) containing 10% fetal bovine serum (FBS), 90 &#181;g/mL ampicilin, and 90 &#181;g/mL kanamycin.</li>
		<li>Culture the Cal27 cells at 37 &#176;C for 2 to 3 days. Make sure that the cells cover at least 60% area of the dish bottom.</li>
		<li>Load TEM onto the Cal27 monolayer in the culture dish.</li>
		<li>Put the dish into a CO2 incubator at 37 &#176;C for 28 days.</li>
		<li>Refresh the culture medium every day during the cell culture process. The CO2 concentration in the incubator is 5%.</li>
	</ol>
	</li>
	<li>Stirred TSCC model construction
	<ol>
		<li>Preparation of a self-made stirred minibioreactor
		<ol>
			<li>Take out the plunger from a 10 mL syringe.</li>
			<li>Dig a hole (diameter of 1 cm) near the lower terminal of the rod and load a stir bar in the hole.</li>
			<li>Dig a hole (diameter of 0.5 cm) at the center of the bottle cap of a plastic wide-mouth bottle and put the piston rod through the cap.</li>
			<li>Cut half of a 50 mL centrifuge tube and weld it on the outer side of the bottle cap.</li>
			<li>Attach fishhooks to the rod by wrapping the rod with fishing lines which are tied to the fishhooks. 
			NOTE: Up to 4 fishhooks can be attached to a rod.</li>
			<li>Autoclave the self-made complex before use. 
			NOTE: Do not autoclave the plastic wide-mouth bottle. Use a new sterile plastic wide-mouth bottle while culturing cells.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Dynamic cell culture
	<ol>
		<li>Seed 1.0 x 106 single Cal27 cells in the self-made minibioreactor. Add 150 mL of DF12 medium which contains 10% FBS, 90 &#181;g/mL ampicilin, and 90 &#181;g/mL kanamycin.</li>
		<li>Load TEM onto the minibioreactor using the fishhooks attached to the rod.</li>
		<li>Tighten the bottle cap and put the minibioreactor on a magnetic stirrer. Activate the minibioreactor at 200 rpm in a CO2 incubator at 37 &#176;C for 7 to 14 days. 
		NOTE: The concentration of CO2 in the CO2 incubator is 5%.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

A well-established protocol for decellularized ECM fabrication should retain the native ECM composition while removing cellular components in tissues nearly completely21. Despite currently reported decellularization protocols which require perfusion through the vasculature to remove cellular materials by convective transport, mechanical agitation was adopted here, known as a traditional simple and cheap method22,23,24,25,26. Moreover, since the tongue is rich in lingualis and has few bulky vascular vessels, this protocol is more suitable for tongue tissues than other protocols described above.
Furthermore, this protocol for TEM production carries out an appropriate moderate-strength decellularization, avoiding the destruction or dissolution of the base membrane which may be caused by high-strength decellularization such as sodium dodecyl sulfate (SDS) treatment3. In addition, the protocol also worked effectively in the tongue of rat and pig (data not shown), suggesting that the method could be commonly used upon tongues from various species3.
It&#39;s worth noting that there are some critical steps or details in this protocol, which could directly influence the results. One important step is the digestion of tongue cells by DNase. If the digestion time is not enough or the DNase doesn&#39;t work efficiently, the decellularization of the tongue would hardly be achieved. Another thing which should be noticed is that the rotary speed for the bioreactor shouldn&#39;t be too fast, considering the damage to TEM. Moreover, a sterile environment for this operation is very important in the protocol.
In spite of the strict rules above, the protocol for TEM preparation can be adjusted to some extent. Washing the tongues with ultrapure water or PBS for a few more hours than the time which the protocol suggests would not obviously affect the fabrication of TEM. However, since the protocol needs almost one week to prepare the TEM, it cannot meet immediate demands for TEM.
The TEM shows great value in TSCC model construction. Together with a self-made minibioreactor, suspended Cal27 cells can attach in TEM and form similar infiltrative structure resembling human TSCC histopathology3. This could be an ideal model for monitoring and investigating the invasion and metastasis of TSCC in vitro. The model could also benefit the work on drug tests on TSCC. In consideration of all these, the protocol presented here may have great potential in TSCC research.

The tongue has various important functions, such as deglutition, articulation, and tasting. Thus, the impairment of tongue function has great impact on patients&#39; quality of life1. The most common malignancy in the oral cavity is tongue squamous cell carcinoma (TSCC), which usually occurs in people who drink alcohol or smoke tobacco2.
In recent years, little progress has been achieved in fundamental research on TSCC. The lack of efficient in vitro research models remains to be one of the biggest problems. Thus, the extracellular matrix (ECM) turns out to be a potential solution. Since ECM is a complex network frame composed of highly organized matrix components, scaffold materials having an ECM-like structure and composition would be competent for cancer research. Decellularized ECM can perfectly provide the niche for the cells from the same origin in vitro, which turns out to be the most significant advantage of ECM.
ECM can be retained with cellular components being removed from the tissues through the decellularization using detergents and enzymes. Various ECM components, including collagen, fibronectin, and laminin in decellularized matrix provide a native-tissue-like microenvironment for cultured cells, promoting the survival, proliferation, and differentiation of the cells3. Moreover, the immunogenicity for transplantation can be reduced to a minimal level with the absence of cellular components in ECM.
So far, fabrication methods for decellularized ECM have been tried in different tissues and organs, such as heart4,5,6,7, liver8,9,10,11, lung12,13,14,15,16,17, and kidney18,19,20. However, no relevant research has be found on similar work in the tongue to the best of our knowledge.
In this study, decellularized tongue extracellular matrix (TEM) was fabricated both efficiently and cheaply by a series of physical, chemical, and enzymatic treatment. Then the TEM was used to recapitulate TSCC in vitro, showing an appropriate simulation for TSCC behavior and development. TEM has good biocompatibility as well as the ability to guide the cells to the tissue-specific niche, which indicates that TEM may have great potential in TSCC research3. The protocol shown here provides a choice for researchers studying on either pathogenesis or clinical therapies of TSCC.

<table border="0" cellpadding="0" cellspacing="0" style="width:891px;" width="891">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C57-BL/6J mice</td>
			<td style="width:292px;">Sun Yat-sen University Laboratory Animal Center</td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:292px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">HB15-GR-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A501218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium chloride</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A100395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dibasic Sodium Phosphate</td>
			<td style="width:292px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">BE14-GR-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Phosphate Monobasic&#160;</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A501211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL EP tube</td>
			<td style="width:292px;">Axygen</td>
			<td style="width:119px;">MCT-150-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-low temperature freezer&#160;</td>
			<td style="width:292px;">Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3.5 cm cell culture dish</td>
			<td style="width:292px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">153066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 cm cell culture dish</td>
			<td style="width:292px;">Greiner</td>
			<td style="width:119px;">628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">V900502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">449164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium sulphate</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A601988</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">158968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kanamycin</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">PHR1487</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical suture</td>
			<td style="width:292px;">Shanghai Jinhuan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">250 mL wide-mouth bottle</td>
			<td style="width:292px;">SHUNIU</td>
			<td style="width:119px;">1407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic stirrer</td>
			<td style="width:292px;">AS ONE</td>
			<td style="width:119px;">1-4602-32</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">CO2 incubator</td>
			<td style="width:292px;">SHEL LAB</td>
			<td style="width:119px;">SCO5A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL syringe</td>
			<td style="width:292px;">Hunan Pingan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL centrifuge tube</td>
			<td style="width:292px;">Greiner</td>
			<td style="width:119px;">227270</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cal27 cell</td>
			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
			<td style="width:119px;"></td>
			<td>Tongue squamous cell carcinoma cell line</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">U2OS cell</td>
			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
			<td style="width:119px;"></td>
			<td>Human osteosarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes free acid</td>
			<td style="width:292px;">BBI</td>
			<td style="width:119px;">A600264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:292px;">Hyclone</td>
			<td style="width:119px;">SH30084.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4 &#176;C fridge</td>
			<td style="width:292px;">Haier</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water purifier</td>
			<td style="width:292px;">ELGA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:292px;">BLAU</td>
			<td style="width:119px;">717805</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

This protocol for the preparation of TEM turns out to be efficient and appropriate. The TEM showed perfect decellularization compared with native tongue tissues. The efficacy of decellularization was confirmed by hematoxylin-eosin (HE) staining (Figure 1A-B). The HE staining results revealed complete disappearance of nuclear staining in TEM (Figure 1B). Moreover, DNA content quantification from previous work showed that DNA was almost completely removed from TEM3. This protocol also showed rare damage to the tissue integrity while removing cell components (Figure 1B).
3D reconstitution of TSCC using TEM and a self-made minibioreactor (Figure 2A-B) achieved satisfying results. HE staining showed that Cal27 cells in the TEM presented typical TSCC pathological characteristics (Figure 2C-D). The cells in stirred culture conditions presented single-cell migration (Figure 2C) or collective migration (Figure 2D) in different lesion areas. In static culture conditions, Cal27 cells also formed invasive structures in the TEM, but it took a longer time (Figure 2E). Furthermore, a human osteosarcoma cell line U2OS was also introduced to the same stirred culture system. Though U2OS cells could live in the culture medium, they were not found in the TEM (Figure 2F). The TEM showed different biocompatibility for different types of cancer cells, suggesting that different tumor cells may need different microenvironments to flourish.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57235/57235fig1.jpg" /> 
Figure 1: Preparation of TEM. (A) HE staining of native tongues from mice. Scale bar = 100 &#181;m. (B) HE staining of decellularized TEM from mice. Scale bar = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57235/57235fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57235/57235fig2.jpg" /> 
Figure 2: Reconstitution of TSCC by TEM. (A) The overview of a self-made minibioreactor. (B) The view of the TEM-loading position of a self-made minibioreactor. (C) HE staining of 14-day stirred cultured TSCC with TEM. Single cell invasion phenomena are indicated by black arrows. Scale bar = 50 &#181;m. (D) HE staining of 14-day stirred cultured TSCC with TEM. Collective invasion phenomena are indicated by black arrows. Scale bar = 50 &#181;m. (E) HE staining of 28-day static cultured TSCC with TEM. Single cell invasion phenomena are indicated by black arrows. Scale bar = 100 &#181;m. (F) HE staining of 14-day stirred cultured U2OS cells with TEM. Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57235/57235fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

fabrication-tongue-extracellular-matrix-reconstitution-tongue

Nanyang Technological University

Research

JoVE Journal

Cancer Research

6.2K Views.  Sun Yat-sen University. A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

Video: Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

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 ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...