Name: a model for perineural invasion in head and neck squamous cell carcinoma
Uploaded: 2017-01-05T13:00:00
Description: Watch this Scientific Journal Video about A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma at JoVE.com

This work was supported in whole by funding from the NIH through the R21 grant, "Mechanisms of Perineural Invasion in Head and Neck Cancer" and the NCI T32 training grant, "Post-Doctoral Research Training in Head and Neck Oncology (2T32CA060397-21)." Thank you to Richard Steiman, MD, PhD and lab staff.

1. Preparation of Culture Medium and Dishes (10 min)
<ol>
	<li>Add 100 &#181;L of Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) to the wells of a 96-well V-bottom plate.</li>
	<li>Remove a pre-aliquoted-vial of approximately 100 &#181;L of semisolid matrix from the 20 &#176;C freezer and place it directly on ice. 
	NOTE: Do this prior to the dorsal root ganglion (DRG) harvest because the semisolid matrix takes approximately 30 min to reach liquid state on ice, which is necessary for...

<ol>
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O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+invasion+in+pancreatic+cancer:+a+mutual+tropism+between+neurons+and+cancer+cells.">Neural invasion in pancreatic cancer: a mutual tropism between neurons and cancer cells.</a> Biochem Biophys Res Commun. 374 (3), 442-447 (2008).</li><li>Gil, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+regulation+of+pancreatic+cancer+cell+invasion+by+peripheral+nerves.">Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves.</a> J Natl Cancer Inst. 102 (2), 107-118 (2010).</li><li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFRalpha1+released+by+nerves+enhances+cancer+cell+perineural+invasion+through+GDNF-RET+signaling.">GFRalpha1 released by nerves enhances cancer cell perineural invasion through GDNF-RET signaling.</a> Proc Natl Acad Sci U S A. 111 (19), 2008-2017 (2014).</li><li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+(CCL2-CCR2)+signaling+axis+mediates+perineural+invasion.">The chemokine (CCL2-CCR2) signaling axis mediates perineural invasion.</a> Mol Cancer Res. 13 (2), 380-390 (2015).</li><li>Bakst, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+impairs+perineural+invasion+by+modulating+the+nerve+microenvironment.">Radiation impairs perineural invasion by modulating the nerve microenvironment.</a> PLoS One. 7 (6), 39925 (2012).</li><li>Ayala, G. 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Critical Steps within the Protocol
The most important steps within this protocol are the precise dissection and extraction of the dorsal root ganglia. Proper transection of the spinal column and a midline-longitudinal division into two hemi-spines are critical to obtaining large numbers of DRG. During the dissection of individual DRGs, the ganglion should never be handled directly, but rather the surrounding fascia should be grasped with the microscopic forceps. Failure to do ...

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the US, with 10,000 deaths per year nationally and 300,000 deaths per year worldwide1. The overall prognosis for HNSCC has remained unchanged at 50% for the past several decades. Perineural invasion (PNI) is one of the most prominent pathological features that portend a poor prognosis in patients with HNSCC. Unfortunately, PNI is a frequent occurrence in HNSCC and can be found in up to 40% of HNSCC patients2,3. 
PNI is the process by which malignant cells track along nerves to adjacent tissues, allowing for higher rates of local and distant spread. Accordingly, PNI-positive HNSCC tumors have higher rates of locoregional recurrences and distant metastases, resulting in lower overall survival compared to HNSCC patients without PNI4-8.
Although the treatment of patients with PNI is typically maximized by employing surgery, radiation, and chemotherapy, the overall survival rates of these patients are still decreased by up to 40% compared to patients without PNI9-11. Thus, it is clear that the current treatment modalities for HNSCC are ineffective in improving the adverse prognosis associated with PNI. The approach of developing targeted therapy against PNI in HNSCC has been hindered by the poor understanding of the factors that regulate this process. This is, in part, a consequence of the lack of a consistent in vitro model for the study of PNI in HNSCC. 
In recent years, several groups have been utilizing an in vitro model for studying PNI in predominantly pancreatic and prostate cancers12-19. This model uses the neurites generated from dorsal root ganglia isolated from mice or rats as a surrogate for large-nerve invasion. The dorsal root ganglia are fixed in a factor-depleted semisolid matrix, which is a solubilized basement membrane protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. This matrix allows for the outgrowth of the neurites and the tracking of single cancer cells along these neurites. Described here is the adaption of this model for the examination of PNI in HNSCC.

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			<td height="21" style="height:21px;width:376px;">DMEM/F-12 50/50 Mix with L-glutamine &#38; 15mM HEPES</td>
			<td style="width:207px;">Corning Cellgro</td>
			<td style="width:139px;">10-090-CV</td>
			<td style="width:157px;">Manassas, VA</td>
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			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Atlanta biologicals</td>
			<td>S11150</td>
			<td>Flowery Branch, GA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA (1x)</td>
			<td>Life Technologies Corporation</td>
			<td>25200056</td>
			<td>Grand Island, NY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered Saline 1x</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td>Manassas, VA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel hESC-Qualif Mouse</td>
			<td>Corning Incorporated</td>
			<td>354277</td>
			<td>Bedford, MA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gamma Irradiated 35mm glass bottom culture dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>Ashland, MA</td>
		</tr>
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			<td height="21" style="height:21px;">SteREO Discovery.V8 Operating Microscope</td>
			<td>Carl Zeiss Microimaging</td>
			<td>495015-0021-000&#160;</td>
			<td>Thornwood, NY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Schott ACE I light source</td>
			<td>Schott</td>
			<td>A20500</td>
			<td>Germany</td>
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		<tr height="21">
			<td height="21" style="height:21px;">CellTracker&#160;</td>
			<td>Life Technologies Corporation</td>
			<td>C2925</td>
			<td>Carlsbad, CA</td>
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		<tr height="21">
			<td height="21" style="height:21px;">BD PrecisionGlide Needle 18G and 21G</td>
			<td>BD</td>
			<td>305195</td>
			<td>Franklin Lakes, NJ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Microdissecting Tweezer</td>
			<td>Harvard Apparatus</td>
			<td>60-3851</td>
			<td>Holliston, MA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Fine Operating Standard Scissors</td>
			<td>Harvard Apparatus</td>
			<td>52-2789</td>
			<td>Holliston, MA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Spring Scissors</td>
			<td>Harvard Apparatus</td>
			<td>60-3923</td>
			<td>Holliston, MA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dressing Forceps</td>
			<td>Harvard Apparatus</td>
			<td>72-8949</td>
			<td>Holliston, MA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Athymic nude mice (002019)</td>
			<td>Jackson Laboratory</td>
			<td>002019</td>
			<td>Bar Harbor, ME</td>
		</tr>
	</tbody>
</table>

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.

After the dissection of the DRG and the placement within the matrix droplet, the appearance of the assay should resemble Figure 1. Note that the DRG is not perfectly round, but it is centered within the matrix droplet. This allows for the outgrowth of neurites in 360 degrees, shown partially in Figure 2. Be aware that certain parts of the DRG send out neurites faster and in greater numbers than others, typically corresponding to where the efferent and aff...

Watch this Scientific Journal Video about A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma at JoVE.com

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

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, ...

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