How Intelligent Is xCELLigence? Classification of Doxorubicin Cytotoxicity Based on Kinetic Profiling

This mini-review provides a comprehensive analysis of kinetic data from cytotoxicity testing of the conventional anti-proliferative compound doxorubicin (DOX). The value of information provided by xCELLigence Real Time Cell Analysis (RTCA) for performing simple, robust in vitro quantitative kinetic profiling has been estimated, in contrast to the currently most widely used static endpoint assay, such as the MTT assay. It has exquisite sensitivity with easy study of a wide repertoire of label-free molecules and cells, and it is well suited for combination therapy research. Despite numerous publications on xCELLigence technology, focusing on compoundmediated cytotoxicity, combination therapy, cell adhesion, receptor activation, and more, there is a lack of uniformity in descriptions, and the data analysis is insufficient. To identify those gaps and attempt to unify data analysis, DOX was chosen as the reference standard for classification in this mini-review. All results from its cytotoxicity tests across various cell types were analyzed. Three typical scenarios of cell responses (cell index, CI) following APC addition were proposed. Type 1, "normal response", shows CI declining. Type 2, "hump", shows gradual CI increase above control, then declining. Type 3, "plateau" - delayed declining response. Variations of kinetic profiles of CI were analyzed for: DOX itself; DOX in combination with other drugs; physically and chemically modified DOX. The next direction for xCELLigence RTCA method development is a kinetic analysis of changes in cell physiology in drug-treated cells, focusing on early and delayed responses. This focus displacement could reveal novel drug mechanisms of action and kinetics.