Evaluating Chromosome Instability and Genotoxicity Through Single Cell Quantitative Imaging Microscopy.

Abstract

Across eukaryotes, genome stability is essential for normal cell function, physiology, and species survival. Aberrant expression of key genes or exposure to genotoxic agents can have detrimental effects on genome stability and contribute to the development of various diseases, including cancer. Chromosome instability (CIN), or ongoing changes in chromosome complements, is a frequent form of genome instability observed in cancer and is a driver of genetic and cell-to-cell heterogeneity that can be rapidly detected and quantitatively assessed using surrogate markers of CIN. For example, single cell quantitative imaging microscopy (QuantIM) can be used to simultaneously identify changes in nuclear areas and micronucleus formation. While changes in nuclear areas are often associated with large-scale changes in chromosome complements (i.e., ploidy), micronuclei are small extra-nuclear bodies found outside the primary nucleus that have previously been employed as a measure of genotoxicity of test compounds. Here, we present a facile QuantIM approach that allows for the rapid assessment and quantification of CIN associated phenotypes and genotoxicity. First, we provide protocols to optimize and execute CIN and genotoxicity assays. Secondly, we present the critical imaging settings, optimization steps, downstream statistical analyses, and data visualization strategies employed to obtain high quality and robust data. These approaches can be easily applied to assess the prevalence of CIN associated phenotypes and genotoxic stress for a myriad of experimental and clinical contexts ranging from direct tests to large-scale screens of various genetic contexts (i.e., aberrant gene expression) or chemical compounds. In summary, this QuantIM approach facilitates the identification of novel CIN genes and/or genotoxic agents that will provide greater insight into the aberrant genes and pathways underlying CIN and genotoxicity.