JAMA Otol Logo Orlando 2013 AHNS Meeting Location
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American Head & Neck Society
Annual Meeting, April 10-11, 2013
JW Marriott Grande Lakes
Orlando, Florida

During the
Combined Otolaryngology Spring Meeting
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SENSORY NEURONS REGULATE HEAD AND NECK SQUAMOUS CELL CARCINOMA PROLIFERATION AND GENE EXPRESSION.

Presentation: P005
Topic: Basic Science - Molecular / Cellular Biology
Type: Poster
Date: Wednesday - Thursday, April 10 - 11, 2013
Session: Designated Poster viewing times
Authors: Scott H Troob, MD, Andrea N Flynn, PhD, Yi Ye, PhD, Chi T Viet, DDS, PhD, Brian L Schmidt, DDS, MD, PhD
Institution(s): Department of Otolaryngology - Head and Neck Surgery, New York University Langone Medical Center and Bluestone Center for Clinic Research, New York University, New York, NY

Background: Perineural invasion (PNI) is a pathologic feature of head and neck squamous cell carcinoma (HNSCC) that contributes to locoregional recurrence and decreased survival. However, the effect of neurons on SCC proliferation is not well understood.

Objective: To develop an in vitro co-culture system of HNSCC cells and primary sensory neurons. To examine the effect of dorsal root ganglion (DRG) neurons and neuron supernatant on HNSCC proliferation and gene expression.

Methods: HSC-3, a human SCC cell line, was seeded on transwell culture plates to determine optimal carcinoma seeding density. Transwell 0.4μm permeable membrane inserts were coated with laminin and poly D-lysine, alone or in combination, to determine optimal seeding conditions for primary cultured DRG neurons from nude Balb/c mice. Neurite outgrowth was examined using an antibody for βIII tubulin. DRG neuron-HSC-3 co-culture was maintained in RPMI medium. The effect of DRG neurons on HSC-3 proliferation was examined at 24, 48, and 72 hours using a MTS proliferation assay. RNA isolated from HSC-3 cultured with DRG neurons, or neuronal supernatant, was reverse transcribed, and cDNA was amplified using a gene expression assay for EGFR, KIF14, and TrkA. Human β-actin was used as endogenous control. Data were analyzed using the Student’s t-test.

Results: The optimal HSC-3 seeding density, 1.5 x 10^3 cells/well, resulted in 100% confluence at 72 hours. Laminin coating of transwell inserts resulted in optimal neuron adherence and neurite outgrowth. Proliferation of HSC-3 co-cultured with DRG neurons was significantly higher than control at 24, (p<0.001), 48 (p<0.05), and 72 hours (p<0.05). Proliferation of HSC-3 cultured in neuron supernatant was also significantly higher compared to control at 24 hours (p<0.001). HSC-3 co-cultured with either primary DRG neurons or neuron supernatant showed down regulation of KIF14 and upregulation of TrkA.

Conclusions: We established and optimized a co-culture model of HNSCC and primary DRG neurons. Sensory neuron co-culture or neuronal supernatant significantly increases HNSCC proliferation and leads to changes in expression of genes with known roles in PNI.




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