Comparison of Growth Inhibition and Clonogenic Assays for Assessing Radiotherapy Responses in Breast Cancer Cell Lines

Background: In vitro assays are widely used to optimize emerging radiotherapy strategies before undertaking more resource-intensive in vivo or clinical studies. However, multiple assay types are currently employed, and it remains unclear whether they yield comparable or complementary measures of radiosensitivity, complicating cross-study comparisons. Purpose: This study provides the first direct comparison of two of the most widely used radiosensitivity assays, clonogenic survival and growth inhibition, using commonly reported but non-identical protocols. The goals are to determine whether the assay measurements are correlated and assess their repeatability in breast cancer cell lines. Methods: Five cancer cell lines (4T1, SKBR3, BT474, MCF7, and MDA-MB-231) underwent clonogenic and growth inhibition assays following X-ray irradiation at 2, 4, 6, and 8 Gy. Correlations between assay measurements were assessed using Spearman correlations. The entire experiment was repeated twice for each assay to assess repeatability. Results: At a given radiation dose, no significant correlations were observed between the clonogenic assay measures and the growth inhibition measures. Across cell lines, the radiation dose resulting in 50% growth inhibition for the growth inhibition assay (mean = 3.2 Gy) was significantly higher than the dose resulting in 50% survival for the clonogenic assay (mean 1.3 Gy; p = 0.02). The coefficient of variation between repeat percent of control measurements for growth inhibition assay was trending lower than for clonogenic assay in every experimental condition and was statistically significant at 8 Gy (p = 0.02). However, the coefficient of variation values for D50 measurements were not significantly different between the assays. Conclusions: This procedural comparison demonstrates that clonogenic survival and growth inhibition assays do not yield correlated measurements under the specific conditions tested and instead provide complementary information about cellular radiation response. These findings highlight important practical considerations for assay selection and suggest that the combined use of both assays may provide a more complete characterization of radiosensitivity.