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>
	<li>Jemal, A., 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=Cancer+statistics,+2006.">Cancer statistics, 2006.</a> CA Cancer J Clin. 56 (2), 106-130 (2006).</li><li>Hinerman, R. W., 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=Postoperative+irradiation+for+squamous+cell+carcinoma+of+the+oral+cavity:+35-year+experience.">Postoperative irradiation for squamous cell carcinoma of the oral cavity: 35-year experience.</a> Head Neck. 26 (11), 984-994 (2004).</li><li>Rahima, B., Shingaki, S., Nagata, M., Saito, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+perineural+invasion+in+oral+and+oropharyngeal+carcinoma.">Prognostic significance of perineural invasion in oral and oropharyngeal carcinoma.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 97 (4), 423-431 (2004).</li><li>Woolgar, J. A., Scott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+cervical+lymph+node+metastasis+in+squamous+cell+carcinoma+of+the+tongue/floor+of+mouth.">Prediction of cervical lymph node metastasis in squamous cell carcinoma of the tongue/floor of mouth.</a> Head Neck. 17 (6), 463-472 (1995).</li><li>Tai, S. K., 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=Treatment+for+T1-2+oral+squamous+cell+carcinoma+with+or+without+perineural+invasion:+neck+dissection+and+postoperative+adjuvant+therapy.">Treatment for T1-2 oral squamous cell carcinoma with or without perineural invasion: neck dissection and postoperative adjuvant therapy.</a> Ann Surg Oncol. 19 (6), 1995-2002 (2012).</li><li>George, D. 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=Nosocomial+sinusitis+in+patients+in+the+medical+intensive+care+unit:+a+prospective+epidemiological+study.">Nosocomial sinusitis in patients in the medical intensive care unit: a prospective epidemiological study.</a> Clin Infect Dis. 27 (3), 463-470 (1998).</li><li>Fagan, J. J., 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=Perineural+invasion+in+squamous+cell+carcinoma+of+the+head+and+neck.">Perineural invasion in squamous cell carcinoma of the head and neck.</a> Arch Otolaryngol Head Neck Surg. 124 (6), 637-640 (1998).</li><li>Soo, K. C., 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=Prognostic+implications+of+perineural+spread+in+squamous+carcinomas+of+the+head+and+neck.">Prognostic implications of perineural spread in squamous carcinomas of the head and neck.</a> Laryngoscope. 96 (10), 1145-1148 (1986).</li><li>Parsons, J. T., Mendenhall, W. M., Stringer, S. P., Cassisi, N. J., Million, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+factors+influencing+the+outcome+of+postoperative+irradiation+for+squamous+cell+carcinoma+of+the+oral+cavity.">An analysis of factors influencing the outcome of postoperative irradiation for squamous cell carcinoma of the oral cavity.</a> Int J Radiat Oncol Biol Phys. 39 (1), 137-148 (1997).</li><li>Liao, C. T., 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=Does+adjuvant+radiation+therapy+improve+outcomes+in+pT1-3N0+oral+cavity+cancer+with+tumor-free+margins+and+perineural+invasion.">Does adjuvant radiation therapy improve outcomes in pT1-3N0 oral cavity cancer with tumor-free margins and perineural invasion.</a> Int J Radiat Oncol Biol Phys. 71 (2), 371-376 (2008).</li><li>Fan, K. H., 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=Treatment+results+of+postoperative+radiotherapy+on+squamous+cell+carcinoma+of+the+oral+cavity:+coexistence+of+multiple+minor+risk+factors+results+in+higher+recurrence+rates.">Treatment results of postoperative radiotherapy on squamous cell carcinoma of the oral cavity: coexistence of multiple minor risk factors results in higher recurrence rates.</a> Int J Radiat Oncol Biol Phys. 77 (4), 1024-1029 (2010).</li><li>Dai, H., 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=Enhanced+survival+in+perineural+invasion+of+pancreatic+cancer:+an+in+vitro+approach.">Enhanced survival in perineural invasion of pancreatic cancer: an in vitro approach.</a> Hum Pathol. 38 (2), 299-307 (2007).</li><li>Ceyhan, G. 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. E., 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=In+vitro+dorsal+root+ganglia+and+human+prostate+cell+line+interaction:+redefining+perineural+invasion+in+prostate+cancer.">In vitro dorsal root ganglia and human prostate cell line interaction: redefining perineural invasion in prostate cancer.</a> Prostate. 49 (3), 213-223 (2001).</li><li>Na'ara, S., Gil, Z., Amit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Modeling+of+Cancerous+Neural+Invasion:+The+Dorsal+Root+Ganglion.">In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion.</a> J Vis Exp. (110), (2016).</li></ol>

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>
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			<td height="21" style="height:21px;">Gamma Irradiated 35mm glass bottom culture dishes</td>
			<td>MatTek Corporation</td>
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			<td>Ashland, MA</td>
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			<td>Carl Zeiss Microimaging</td>
			<td>495015-0021-000&#160;</td>
			<td>Thornwood, NY</td>
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			<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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			<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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			<td height="21" style="height:21px;">BD PrecisionGlide Needle 18G and 21G</td>
			<td>BD</td>
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			<td height="21" style="height:21px;">Premium Microdissecting Tweezer</td>
			<td>Harvard Apparatus</td>
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			<td>Harvard Apparatus</td>
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			<td>Holliston, MA</td>
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			<td height="21" style="height:21px;">Dressing Forceps</td>
			<td>Harvard Apparatus</td>
			<td>72-8949</td>
			<td>Holliston, MA</td>
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			<td height="21" style="height:21px;">Athymic nude mice (002019)</td>
			<td>Jackson Laboratory</td>
			<td>002019</td>
			<td>Bar Harbor, ME</td>
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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, ...

The overall goal of this experimental protocol is to rapidly evaluate multiple treatment conditions affecting perineural invasion in head and neck squamous cancer with very high assay success rates and reproducibility. This method can help answer key questions in the field of head and neck oncology, such as what are the factors that influence head and neck cancer cell perineural invasion. The main advantage of this technique is that a large number of assays can be rapidly and reproducibly performed from one mouse in a relatively short period of time.Before beginning the dorsal root ganglion, or DRG, dissection procedure, add fresh room temperature medium to at least 40 wells of a V-bottom 96-well plate. Then, use a pair of fine scissors to make a longitudinal incision along the spinal column from the root of the tail to the head of the mouse. And transversely divide the tissue below the sacral spine, dissecting both sides of the spine, all the way up to the skull base.Transfer the specimen under the microscope, and observe the cervical end of the spinal column at low power. The white spinal cord is apparent in the middle of a bony ring, which is surrounded by variable amounts of perispinal muscles and soft tissue. Using microscopic spring scissors, split the dorsal, or superior aspect, of the first two to three vertebral bodies.Then, use the spring action of the scissors to open this initial bony cut, to ensure that the vertebral body dissection occurs at the midline. Continue in small increments toward the sacral spine, bisecting the spine with identical cuts on the ventral, or inferior, aspect of the vertebral bodies. Placing one hemispine aside, transfer the other half of the spine face down on a sterile plate with the spinal cord in place.And use two 18-gauge needles to secure both ends of the hemispine onto the polystyrene dissecting platform. Next, starting at the narrower, cervical end, gently peel back the spinal cord from about four vertebral levels. In the region where the sensory nerves insert into the DRG, gently grasp the surrounding fascia with microscopic forceps, and use microscopic spring scissors to trim this fascia and other nerve tissue to free the DRG.Under increased magnification, use the spring scissors to cut the peripheral nerve as close to the DRG as possible to release the tissue, and trim the proximal branches. Then, trim the DRG of any stray nerve fibers or fascial attachments and place the isolated tissue into one well of the 96-well V-bottom plate. To prepare the semisolid matrix droplets, transfer one glass well-bottom plate from ice onto an ice block under the operating microscope.Place the tip of a no more than 10 microliter pipette directly onto the glass at a 45-degree angle, and carefully dispense a 1.5-microliter droplet of matrix onto the glass. Slowly move the pipette tip away from the glass as the matrix becomes engaged with the plate. Deposit one droplet of matrix onto each of the four corners of the plate, and transfer the plate to a room temperature surface for about one minute.When the matrix has slightly stiffened, return the plate to the ice block, and use closed microscopic forceps to scoop the DRG gently with the left hand. Using the right hand, carefully transfer the nerve tissue to the tip of a 21-gauge needle and use the needle to gently insert the DRG into the middle of the matrix droplet. The DRG should release easily into the matrix for central positioning with the needle.After inserting a DRG piece into each of the four droplets, place the plate into a 37-degree Celsius incubator for three minutes. When all of the DRG have been embedded, hold the plates one at a time, at a slight angle and slowly add four milliliters of DMEM supplemented with 10%FBS to the plates, such that the medium only gradually comes into contact with the matrix DRG units. Return the plates to the incubator for another 48 to 72 hours.When the neurites reach at least 75%of the way to the edge of the matrix, aspirate the medium from the wells without removing the matrix DRG units. Then, using a 200-microliter pipette, with a smooth trigger, place two drops of three times 10 to the fifth cells per milliliter of medium over each matrix DRG assay. And return the plates to the incubator.After one hour, gently add four milliliters of DMEM with 10%FBS along the side wall of the glass-bottom plate, and return the plates to the incubator until their next microscopic evaluation. After their dissection and placement within the matrix droplets, the appearance of the assay should resemble the matrix-embedded DRG shown here. Note that the DRG is not perfectly round, but is centered within the matrix droplet, allowing for the outgrowth of neurites in 360 degrees.Immediately after plating, the head and neck squamous cancer cells form a circumferential ring around the matrix, expanding along the neurites over the next two to three days. Cells plated onto blank control droplets containing matrix alone actively divide around the matrices, but do not enter or extend over the top. For quantification of the perineural invasion of the cancer cells, images of the assays are divided into four quadrants via one vertical and one horizontal line.A series of points are then scored for each perineural invasion extension observed in each quadrant, allowing comparison of the cancer cell ingrowths along the neurites between different cancer cell lines. Once mastered, the dorsal root ganglia isolation technique can be complete in less than two hours if it's performed properly. While attempting this procedure, it's important to minimize excessive handling of the dorsal root ganglia as crush injury is the most common reason for assay failure.Following this procedure, the mechanisms of perineural invasion can be studied, potentially leading to the development of targeted therapies against this type of soft tissue invasion. After watching this video, you should have a good understanding of how to dissect dorsal root ganglia from a mouse and set up an assay for examining perineural invasion.

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10.4K vistas.  University of Pittsburgh Medical Center. The overall goal of this experimental protocol is to rapidly evaluate multiple treatment conditions affecting perineural invasion in head and neck squamous cancer with very high assay success rates and reproducibility. This method can help answer key questions in the field of head and neck oncology, such as what are the factors that influence head and neck cancer cell perineural invasion. The main advantage of this technique is that a large number of assays can be rapidly and reproducibly per...