The clonogenic assay: robustness of plating efficiency-based analysis is strongly compromised by cellular cooperation

Abstract

Background The clonogenic assay is a versatile and frequently used tool to quantify reproductive cell survival in vitro. Current state-of-the-art analysis relies on plating efficiency-based calculations which assume a linear correlation between the number of cells seeded and the number of colonies counted. The present study was designed to test the validity of this assumption and to evaluate the robustness of clonogenic survival results obtained. Methods A panel of 50 established cancer cell lines was used for comprehensive evaluation of the clonogenic assay procedure and data analysis. We assessed the performance of plating efficiency-based calculations and examined the influence of critical experimental parameters, such as cell density seeded, assay volume, incubation time, as well as the cell line-intrinsic factor of cellular cooperation by auto-/paracrine stimulation. Our findings were integrated into a novel mathematical approach for the analysis of clonogenic survival data. Results For various cell lines, clonogenic growth behavior failed to be adequately described by a constant plating efficiency, since the density of cells seeded severely influenced the extent and the dynamics of clonogenic growth. This strongly impaired the robustness of survival calculations obtained by the current state-of-the-art method using plating efficiency-based normalization. A novel mathematical approach utilizing power regression and interpolation of matched colony numbers at different irradiation doses applied to the same dataset substantially reduced the impact of cell density on survival results. Cellular cooperation was observed to be responsible for the non-linear clonogenic growth behavior of a relevant number of cell lines and the impairment of survival calculations. With 28/50 cell lines of different tumor entities showing moderate to high degrees of cellular cooperation, this phenomenon was found to be unexpectedly common. Conclusions Our study reveals that plating efficiency-based analysis of clonogenic survival data is profoundly compromised by cellular cooperation resulting in strongly underestimated assay-intrinsic errors in a relevant proportion of established cancer cell lines. This severely questions the use of plating efficiency-based calculations in studies aiming to achieve more than semiquantitative results. The novel approach presented here accounts for the phenomenon of cellular cooperation and allows the extraction of clonogenic survival results with clearly improved robustness.

Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon

“…Although DNA damage markers such as γ-H2AX are detectable within less than an hour after irradiation, the irradiated cells initiate DNA damage repair programs [20][21][22]; hence, the effect of ionizing radiation on the level of cellular viability is measurable following a significantly longer timescale. The 'classical' method for analysis of effect of ionizing radiation is the clonogenic assay which can be carried out even several weeks after the irradiation [23]. Here, we set out to explore whether the resazurin assay can be used for monitoring quicker responses on the level of cellular viability following irradiation, as shortening of assay times usually results in improved throughput.…”

Small-Molecule Gankyrin Inhibition as a Therapeutic Strategy for Breast and Lung Cancer

“…The clonogenic assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. It is often considered an essential 2D technique to confirm anti-proliferative activity previously observed in an MTT assay. − Figure A,B illustrates the ability of compounds 51b and 51d to inhibit the formation of MDA-MB-231 cell colonies after adding treatment for 48 h, followed by a 7 day incubation with fresh media. Treatment with 1.15 μM compound 51b resulted in 75.3% growth, and a 2.30 μM treatment resulted in 60.3% growth.…”

“…13 (d, J = 8.1 Hz, 2H), 3.90 (s, 3H), 3.17 (q, J = 6.4 Hz, 2H), 2.76 (t, J = 7.5 Hz, 2H), 1.93−1.77 (m, 2H). 13 Methyl (31). General procedure 2.…”

Biocompatibility and Cytotoxicity Study of Polydimethylsiloxane (PDMS) and Palm Oil Fuel Ash (POFA) Sustainable Super-Hydrophobic Coating for Biomedical Applications

“…Then, the well plate was incubated at room temperature for 15 min and the cells were washed with deionized water and air-dried in order to count the colonies. Colonies consisting of more than 50 cells were counted for the clonogenic assay [ 24 ].…”

“…Super-hydrophobic biomaterial coating for applications such as controlled drug release and reduction of bacterial interaction on implants requires the critical evaluation of cell behavior mediated by surface wettability and topography. However, the effects of surface energy and surface roughness on cell viability are often contradictory as cell behavior can be highly dependent on cell type [24]. To comprehensively evaluate the interaction of mammalian cells on the fabricated superhydrophobic coating, MTT assay was conducted to study the viability of the L929 cell line in differing concentrations of PDMS: SS coating with the utilization of zinc diethyldithiocarbamate (ZDEC) as a positive control.…”