Abstract 3055: Clonogenic high-throughput assay for screening radiation modulators

Abstract

Abstract We present here the development of a high-throughput clonogenic methodology for the identification of novel radiation modulators, with critical and laborious steps automated for speed and ease of use. Clonogenic assays have long been the benchmark methodology for assessing reproductive cell death following treatment with ionizing radiation, alone or in combination with a variety of chemotherapeutic agents (cytotoxic, genotoxic, targeted therapies). However, owning to the large growth formats and intrinsic laborious nature of clonogenic assays heretofore large-scale screening for radiation modulators with this assay has been impractical. Further, presently available large-scale cellular growth screens (e.g. MTT type assays) have proven unreliable at reproducibly identifying clinically relevant radiation modulators. As a result, numerous attempts have been made at developing a streamlined clonogenic process, with modest degrees of success. Here we present the development of an automated miniaturized clonogenic methodology that finally provides for the high throughput screening of novel radiation modulators in cell lines representing seven cancer indications where radiation therapy is the standard of care (Esophageal cancer, Glioblastoma, Head and Neck cancer, metastatic Melanoma, Non-small-cell lung carcinoma, Rectal cancer, and Soft tissue sarcoma). First, we have identified multiple cell lines for each cancer indication that form consistently small and discrete colonies, making them amenable to clonogenic growth in 24- and/or 96-well formats. Second, we have identified conditions in which chemoradiation response is identical between traditional and miniaturized clonogenic formats. Third, we have developed fluorescent cell labeling protocols that allow for live cell colony counting, streamlining the assay and allowing for real-time monitoring over the course of each experiment. Fourth, we have developed software for the automated calling/quantification of clonogenic images, calculation of α/β ratio, dose modifying factor, and calling of hits in large scale screening. Fifth, we have developed an instrument that in large scale is capable of robotic liquid handling, irradiation, drug treatment, and incubation of clonogenic plates in an integrated manner. Preliminary screens with this methodology have demonstrated its ability to accurately identify known radiation modulators. Taken together these new technologies provide a platform to allow for the high-throughput clonogenic screening of drug libraries to identify novel radiation modulators. Further, this methodology is adaptable for identification of novel drug-drug combinations using a clonogenic endpoint. This work is funded in part by NCI SBIR Phase II contract No. 75N9109C0038. Citation Format: Barbara Frederick, Nathan Gomes, Tin Tin Su. Clonogenic high-throughput assay for screening radiation modulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3055.