Abstract 6055: Cell confluence growth tracking as a supplement assay for clonogenic assay in prostate cancer radiotherapy combination evaluation
Authors: Jiang Wen , Brennen W. Nathaniel , Denmeade Samuel R. , Thorek Daniel L. 
Published: June 2022

Abstract
Background: Radiation therapy is a pillar of cancer care, and over 50% of all cancer patients will receive a form of this treatment. There is great interest in the capacity to improve radiation response and to de-escalate dose through combination pharmacological strategies. Evaluating these methods is a persistent challenge to the field. The clonogenic assay has been the gold standard assay in radiobiology research to evaluate the ability of a single cell to grow into a colony after different radiation treatments. However, the assay is laborious, time consuming and sensitive to environmental conditions. Also, the low cell seeding density may pose challenges for cells to proliferate, and extra conditioned culture medium is required for certain cell lines. Here, we have used the Celigo imaging cytometer to track cell confluence growth day by day, to demonstrate its comparable ability with the clonogenic assay to reveal differences in cell proliferation ability after different radiation treatments.
Methods: Firstly, confluence measurement consistency was validated by repeatedly measuring the same wells for 6 times (Celigo 200 BFFL). Next, cell confluence growth tracking was compared with the clonogenic assay using 4 prostate cancer cell lines (LNCaP, PC3, 22Rv1, and LN95), after X-ray treatment and X-ray in combination with androgen receptor (AR)-inhibiting enzalutamide treatment. Treated cells were collected and seeded to 12-well plates followed by daily checks with Celigo. The Celigo imaging cytometer can image each well and conduct automatic image segmentation to calculate cell confluence reported as a percentage of well area. Daily cell confluence data was used to draw cell growth curves.
Results: The consistency of cell confluence measurements was validated with a coefficient of variation less than 5%. In comparing cell confluence growth with clonogenic assay, with the increase of X-ray dose (0Gy, 2Gy, and 4Gy), the degenerating cell proliferation ability was consistently observed in all 4 cell lines with both assays. Additionally, X-ray in combination with enzalutamide treated LNCaP cells demonstrated decreased cell proliferation ability than X-ray alone treated cells, as demonstrated by both cell confluence tracking and clonogenic assay. However, enzalutamide did not affect the proliferation ability of X-ray treated AR-negative PC3 cells, and was confirmed in both assays. The radiopotentiation effect of enzalutamide was also observed in AR-expressing 22Rv1 and LN95 cells with clonogenic assay, but not in 22Rv1 cells with cell confluence tracking.
Conclusion: Cell confluence tracking with Celigo allows daily in situ monitoring of cells and a higher cell seeding density than the clonogenic assay. It is able to provide additional evidence of cell proliferation to supplement the traditional clonogenic assay in radiobiology research.
Citation Format: Wen Jiang, W. Nathaniel Brennen, Samuel R. Denmeade, Daniel L. Thorek. Cell confluence growth tracking as a supplement assay for clonogenic assay in prostate cancer radiotherapy combination evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6055.