Abstract 1797: EGFR as a marker for head and neck squamous cell carcinoma-derived small extracellular vesicles
Authors: Dombroski Jenna A. , Kuhnell Damaris , Kasper Susan , Langevin Scott M. 
Published: July 2017

Abstract
Head and neck cancer will account for an estimated 61,760 new cancer cases and 13,190 cancer-related deaths in the United States in 2016, the majority of which (&gt; 90%) will be squamous cell carcinoma (HNSCC). The prognosis for HNSCC is relatively poor, with an overall 5-year survival around 60%, and the outlook worsens with increasing stage at diagnosis, which is problematic since about two-thirds of patients present with advanced stage disease. This underscores the urgent need for discovery, development and translation of novel biomarkers to facilitate early detection of HNSCC and bolster the chance for positive patient outcomes. Exosomes are nano-sized (40-150 nm) membrane-encapsulated vesicles that may offer a new avenue for discovery and development of novel HNSCC biomarkers. However, they are released by essentially all cells, both normal and malignant, into the extracellular space, resulting in a noisy environment in biofluids from the perspective of biomarker measurement. As such, a membrane-bound surface marker that allows for enrichment for cancer-derived exosomes in patient samples would provide a clear benefit. Epidermal growth factor receptor (EGFR), which belongs to the ErbB/HER family of receptor tyrosine kinases, localizes to the plasma membrane and is over-expressed in as much as 90% of HNSCC tumors. Therefore, we hypothesized that EGFR will be over-represented on membranes of HNSCC-derived exosomes and thus may offer utility as a surface marker for detection and/or enrichment. To test this, we cultured an HNSCC cell line that over-expresses EGFR (FaDu), EGFR-deficient SW620 colorectal adenocarcinoma cells, and primary human oral epithelial cells pooled from 3 healthy donors. Cells were cultivated in supplier-recommended media with 10% fetal bovine serum (FBS) that was super-depleted of exosomes via 18 hour ultracentrifugation at 100,000g and 1% penicillin/streptomycin at 37°C with 5% CO2 in 150 cm2 flasks with 25mL media. After reaching 80-90% confluence, media was harvested, and exosomes were isolated and purified via differential ultracentrifugation. Exosome isolates were whole-mounted on carbon grids, Immuno-Gold labeled with anti-EGFR, and imaged on a JEOL JEM-1230 transmission electron microscope (Immuno-EM); anti-nucleoporin 153 (NUP-153), which localizes to the nucleus, was used as a negative control. Immuno-EM images indicate abundant EGFR expression on exosomes secreted by FaDu HNSCC cells, but EGFR is not detectable on the exosomes secreted by the oral epithelial cells from healthy donors. These results indicate that EGFR may offer utility for enrichment for HNSCC-derived exosomes in human biofluid isolates using magnetic beads or flow cytometry.
Citation Format: Jenna A. Dombroski, Damaris Kuhnell, Susan Kasper, Scott M. Langevin. EGFR as a marker for head and neck squamous cell carcinoma-derived small extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1797. doi:10.1158/1538-7445.AM2017-1797