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

Summary

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.

Abstract

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.

Introduction

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 patients^2,3.

PNI is the process by which malignant cells track along nerves to adjacent tissues, allowing for higher rates of local and distan....

Protocol

1. Preparation of Culture Medium and Dishes (10 min)

1. Add 100 µL of Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) to the wells of a 96-well V-bottom plate.
2. Remove a pre-aliquoted-vial of approximately 100 µL of semisolid matrix from the 20 °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 the subsequent steps. Failure to keep the semisolid matrix on ice at all times will result a poor-quality matrix droplet.
3. Label glass-botto....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

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