Abstract PO-013: Irradiation induced expression of cytosolic DNA sensors and cytokines is associated with HPV status of pharyngeal squamous cell carcinoma cells
Authors: Levpuscek Kristina , Jesenko Tanja , Strojan Primoz , Sersa Gregor , Cemazar Maja 
Published: April 2021

Abstract
Human papillomavirus (HPV) positive pharyngeal squamous cell carcinoma (PSCC) appears to differ from HPV negative PSCC regarding several different characteristics due to the expression of HPV viral oncoproteins E6 and E7. Clinical studies suggest that HPV positive PSCC are more radiosensitive than HPV negative ones, however the exact mechanisms are still not known. It was previously shown that after X-ray radiation DNA accumulates in the cytosol of survived cells and can be sensed by specific pattern recognition receptors, i.e. cytosolic DNA sensors, which can modulate immune response and thus radiosensitivity of tumors. Therefore, the aim of our study was first to investigate the radiosensitivity and involvement of DNA sensing pathways in response to radiation in HPV positive and HPV negative PSCC in vitro. For determination of the radiosensitivity, HPV negative (FaDu and UM-SCC-6) and HPV positive (2A3 and UPCI:SCC090) PSCC cell lines were plated in 96 well plates and irradiated with different single doses (0, 2, 4, 6, 8 and 10 Gy). After 4 cell’s division PrestoBlue™ Cell Viability Reagent was used to determine the survival of cells taking into account different growth rate of the cells. For determination of DNA sensors and cytokines mRNA levels, PSCC cell lines were plated and irradiated with different single doses (0, 4 and 8 Gy). After 24 and 48 hours RNA was isolated using peqGOLD Total RNA Kit (VWR, West Chester, PA, USA) and reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Thermo Fisher Scientific). qRT-PCR was used to determine viral load (expression of HPV oncoproteins E6 and E7 mRNAs), expression of DNA sensors (cGAS, STING, RIG-I, DAI, IFI16, DDX60) and cytokines (IFNb, TNFa, IL1b). The expression of cytosolic DNA sensors and cytokines was dose- and time-dependent in both types of cells. The expression of DNA sensors DAI and RIG-I after irradiation was upregulated in HPV negative cell lines but not in HPV positive ones. The expression of cytokines IFN β and IL 1B was also upregulated only in HPV negative cell lines. Between the two HPV negative cell lines FaDu and SCC6, the expression profile did not differ, while the expression of DNA sensors and cytokines was higher in HPV positive cell line 2A3 compared to UM-SCC-6 cell lines, which has a higher level of viral proteins E6 and E7. In conclusion, our results indicate that on the in vitro level, DNA sensors are differentially expressed in response to irradiation in PSCC cell lines based on HPV infection but does not affect the radiosensitivity of the cells. Further in vivo studies are needed to elucidate the involvement of immune system in the response.
Citation Format: Kristina Levpuscek, Tanja Jesenko, Primoz Strojan, Gregor Sersa, Maja Cemazar. Irradiation induced expression of cytosolic DNA sensors and cytokines is associated with HPV status of pharyngeal squamous cell carcinoma cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-013.