Abstract PO066: Effect of radiation on oral cancer cell viability and anti-tumor T-cell responses
Authors: Pero Travis , Ryan Nathan , Anderson Kelvin , Oghumu Steve 
Published: February 2021

Abstract
Background: Oral cancer is a malignant, neoplastic disorder most commonly presenting as squamous cell carcinoma (HNSCC). It is responsible for nearly 300,000 new diagnoses and 145,000 deaths globally each year, and in the United States alone, approximately one person will die from oral cancer every hour every day in 2020. Although primary surgical resection remains the standard of care for these patients, tumor recurrence is common (40%) and late stage five year survival rate is low (&lt;50%). As a result, ionizing radiation has gained traction as an adjunctive therapy to improve oral cancer clinical outcomes. However, the effects of radiation on oral cancer cell viability and subsequent anti-tumor T cell responses to oral cancer are not fully understood. Objective: To examine the effects of ionizing radiation on HNSCC cell viability and determine the ability of irradiated HNSCC cells to elicit antigen specific T-cell immune responses.
Methods: A metastatic mouse HNSCC cell line, LY2, was seeded and exposed to increasing doses of ionizing radiation (0, 20, 50, 100 200 Gy) generated via an RS2000 Irradiator, a ten minute exposure to ultraviolet (UV) light, freeze/thaw cycling, or a combination of these treatments. Following exposure, cells were cultured for 96 hours. Cell viability was assessed by microscopic imaging using a Leica microscope, and analysis was performed in ImageJ. Proteins were extracted from the cells, and protein concentration was quantified using a BCA assay. Next, single cell suspensions from the lymph nodes of LY2 tumor bearing, immunocompetent BALB/c mice were obtained and stimulated using various concentrations (0, 1.6, 8, and 40 µg) of proteins obtained from the LY2 cells. Antigen specific T cell proliferative responses, as determined by Alamar Blue assay, were compared between different concentrations of irradiated LY2 protein as well as the different exposure types. Cell supernatants were also harvested for analysis of the T cell specific cytokines, IL-4, IL-2, and IFNg.
Results: LY2—HNSCC showed reduced viability in response ionizing radiation in a dose-dependent manner. The ideal, immunogenic range for harvested protein between 1.6µg and 8µg elicited an optimal T cell immune response. Additionally, Alamar Blue assay analysis demonstrated that LY2 proteins harvested from cells exposed to ionizing radiation or freeze/thaw cycling exhibited a proliferative response that was both dose-dependent and antigen specific, while cells exposed to UV light displayed no significant T cell proliferation.
Conclusion: These results confirm a dose-dependent, cytotoxic effect of ionizing radiation on HNSCC cells. Proteins from cells exposed to high doses of ionizing radiation and freeze/thaw cycles can be successfully extracted with preserved antigenic profiles. Future work will allow characterization of the cellular response and enhance the study of the anti-tumor immune mechanisms in new and exciting ways.
Citation Format: Travis Pero, Nathan Ryan, Kelvin Anderson, Steve Oghumu. Effect of radiation on oral cancer cell viability and anti-tumor T-cell responses [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO066.