OPSCCs commonly arise from the epithelium lining the lingual and palatine tonsillar crypts. The stratified epithelium at the surface gives way to complex reticulated epithelium in the crypts which contain a diverse cell population with varying cytokeratin expression patterns. How such a diverse cell population is maintained has not yet been explored. It has been observed that HPV+ HNSCC consistently arise from the tonsillar crypts. In addition, somatic stem and progenitor cells have been identified as the likely targets of tumorigenesis in many tissues. To further evaluate whether tonsillar crypts contain unique multi-potent stem cells that are targets of tumorigenesis, and similar stem cells are maintained in OPSCC tumors of tonsillar origin, we propose the following studies: Aim 1: Characterize the cellular populations of crypt epithelium and compare to oral cavity epithelium. Human tissue obtained from the IRB approved EVMS Biorepository will be evaluated. We will stain for purported markers of stem cell activity including LGR5/6, CD34, p63, Bmi-1, cytokeratins 15, 19, and hTert using tyramide signal amplification when necessary. Cytokeratins 5, 8, 14 and 17 (previously identified in subsets of crypt epithelium) will be evaluated and co-stained with the above markers. Ki67 will be used to identify proliferative regions within the crypt epithelium. Aim 2: Characterize the cellular population of oropharyngeal squamous cell carcinoma and compare to normal tonsillar crypt epithelium. Human tumor obtained from above biorepository will be evaluated. HPV+/- OPSCC cell lines will be quantified for the markers of stem cell activity listed in Aim 1. Similarly, the same markers will be evaluated in HPV+/- human OPSCC tumors and compared to normal tonsil. The cell cycle regulator, p16, which is up-regulated in HPV-induced OPSCC and has previously been identified in a subset of normal tonsillar crypt epithelial cells will also be co-stained with any identified stem cell markers to determine if it is associated with any stem/progenitor cell function. Patient data will be correlated to identify prognostic value of these markers. Aim 3: Evaluate clonal expansions of cells in tonsillar crypts and OPSCC tumors by mitochondrial lineage tracing. Acquired deficiencies in the mitochondrial gene for the respiratory chain enzyme cytochrome c oxidase (CCO) can be exploited to identify cellular lineages within human tissues and tumors. This aim will evaluate if such CCO deficient (CCO-) clones exist within normal tonsillar crypts and OPSCC tumors, which will allow for careful lineage tracing in future experiments to fully understand the cellular hierarchy underlying tonsillar crypt and OPSCC tumor morphology. Elucidating the cellular hierarchy within tonsillar crypts, and identifying whether multi-potent stem cells exist within the tissue is imperative to understanding OPSCC, including those that are HPV-induced.

Citation Format: Vivian F. Wu, Tennison Yu, Brette Harding, Robert Bruno. Unraveling the cellular hierarchywithin human tonsillar crypt epithelium and oropharyngeal squamous cellcarcinoma tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1396. doi:10.1158/1538-7445.AM2015-1396