Abstract 1391: Establishment and validation of a high-content imaging assay with a 14-day incubation for the testing of epigenetic target-based therapeutics
Authors: Parry Jesse J. , Norman Vanessa L. , Wageman Charles R. , Cavedine Lee R. , Sindelar Timothy J. , Croff Alyssa M. , Garner Steven M. , Epkins Brogan A. , Robinson Natiya E. , Dempsey Kristin C. , Warrior Usha , King Alastair J. 
Published: August 2018

Abstract
Given the importance and significance of targeting mutations and epigenetic modification in cancer therapy, we sought to expand on our ability to test drugs with epigenetic modulatory abilities by increasing the exposure time of drugs against cell lines by the OncoPanel™ cell-based profiling service to 14 days. Using historic internal control data, generated in OncoPanel™ cell proliferation assays for 3-, 5-, and 10-day exposures, we were able to calculate the theoretical number of doublings in 14 days for each cell line. This information was used to calculate the seeding density at the time of cell plating that would provide the same endpoint per well density for a select number of cell lines from the panel. Due to the issue of increasing coefficient of variation (CV) with decreasing starting number of cells per well, we eliminated cell lines that had a calculated seeding density of &lt;15 cells per well. For cell line validation, cells were plated at the calculated theoretical density for each cell line in 384-well plates and incubated overnight. A time zero (T0) plate was also seeded for each cell line to allow calculation of the number of doublings in the assay. Test compounds (staurosporine, entinostat, and vorinostat) were added to the assay plates over a range of 10 concentrations, in triplicate, using acoustic liquid transfer. The assay plates were incubated continuously for 7 days, upon which the growth media were removed and replaced with fresh media, followed by re-dosing with compounds. Assay plates were then incubated continuously for an additional 7 days, after which the cells were fixed and stained to allow high-content, fluorescent imaging of nuclei. Data were analyzed as the relative cell count, where the measured fluorescence intensity was transformed to percent of control, as compared with a vehicle-treated control. Cellular response parameters were calculated, using nonlinear regression to a sigmoidal single-site dose response model. The criteria for each cell line to pass quality control included a vehicle CV of &lt;30%, endpoint well confluence, tightness of curve-fit, and an empirically-determined doubling number that was comparable with the calculated number of doublings. Comparison of the doubling numbers was striking and illustrated very high accuracy, adding to the consistency of the assay's performance. In summary, we have validated 110 human cancer cell lines, comprising 16 tissue types, in the 14-day OncoPanel™ proliferation assay, and show comparative analysis of each compound's activity profile from 3-, 5-, 10-, and 14-day incubations. The extended incubation time is highly relevant and useful for testing epigenetic target modulators, which may require a longer-term exposure to show full efficacy, with a greater potential therapeutic outcome.
Citation Format: Jesse J. Parry, Vanessa L. Norman, Charles R. Wageman, Lee R. Cavedine, Timothy J. Sindelar, Alyssa M. Croff, Steven M. Garner, Brogan A. Epkins, Natiya E. Robinson, Kristin C. Dempsey, Usha Warrior, Alastair J. King. Establishment and validation of a high-content imaging assay with a 14-day incubation for the testing of epigenetic target-based therapeutics [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 1391.