Abstract 854: Validation of a viability assay for assessing radiation response and investigating drug/radiation combinations

Abstract

Abstract Purpose: Radiotherapy (RT) is frequently used either alone or in combination with chemotherapy for curative treatment of squamous cell carcinomas (SCC) originating from the head and neck, lung, esophagus, and cervix. Treatment outcomes for SCCs are heterogeneous, in part due to variable degrees of resistance to ionizing radiation and modest benefit of existing radiosensitizing drugs. Preclinical research into intrinsic cancer cell radiosensitivity biomarkers and novel radiosensitizing drugs has been hampered by a lack of robust high throughput assays of radiation response. We sought to validate a recently published high throughput viability assay as a surrogate of clonogenic survival and extend its use to drug-radiation combinations. Experimental Methods: Clonogenic assays—the gold standard for assessing radiation survival in vitro—and viability assays were performed on 19 SCC cell lines (16 head and neck, 3 esophageal). The clonogenic assay was performed according to published procedures (Franken et al. Nat. Protoc, 2006) with colonies counted using ImageJ. The viability assay was adapted from Abazeed et al. (Cancer Res., 2013). Cells were seeded with a range of densities in 96 well plates then treated with a range of radiation doses (0, 2, 4, and 8 Gy); viability was measured using CellTitre Glo reagent after 9 days. Agreement between clonogenic and viability assays was measured using modified concordance index (C-index) and Pearson correlation. For the addition of drug, cells were allowed to adhere to plates for 2 hours before drug administration. Cells were irradiated 16 hours later again at a range of doses (0, 2, 4, and 6 Gy for drug analysis), and then quantified as above. All assays were performed with technical and biological triplicates. Results: There was a high degree of agreement between the area under the curve with the clonogenic assay and the viability assay (C-index = 0.80, p = 3.03x10-7; Pearson r = 0.70, p = 1.10x10–3). Significant concordance was also seen at 2 Gy and 4 Gy dose points (Pearson r = 0.62 and 0.73 respectively), however not at 8 Gy (Pearson r<0.5). The viability assay decreases the overall time for the experiment, as it has a 9-day endpoint rather than the 14 days typical for the clonogenic assay. The viability assay also eliminates cell counting time, as results can be analyzed in minutes. Survival curves with and without radiation with clinically utilized chemotherapeutic drugs cisplatin and paclitaxel were developed which showed an additive effect for drug combinations, demonstrating the ability of the assay to be combined with chemotherapeutic agents and allows for the investigation into dose-response effects. Conclusion: The proliferative assay recapitulates the clonogenic assay within our cohort of SCC cell lines. Novel higher throughput methods to analyze radiation response will allow for a more efficient measure of drug and radiation combinations. Citation Format: Meghan Lambie, Venkata S. Manem, Benjamin Haibe-Kains, Scott V. Bratman. Validation of a viability assay for assessing radiation response and investigating drug/radiation combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 854.