High content screening application for cell-type specific behaviour in heterogeneous primary breast epithelial subpopulations.

ABSTRACT: The complex interaction between multiple cell types and the microenvironment underlies the diverse pathways to carcinogenesis and necessitates sophisticated approaches to in vitro hypotheses testing. The combination of mixed culture format with high content immunofluorescence screening technology provides a powerful platform for observation of cell type specific behavior.We have developed a versatile, high-throughput method for assessing cell-type specific responses. In addition to the specificity and sensitivity offered traditionally by immunofluorescent detection in flow cytometry, the 'in-cell' analysis method we describe provides the added benefits of higher throughput and the ability to analyse protein subcellular localisation in situ. Furthermore, elimination of the cell dissociation step allows for more immediate analysis of responses to specific extrinsic stimuli. We applied this method to investigate ionising radiation treatment response in normal breast epithelial cells, measuring growth rate, cell cycle response and double-strand DNA breaks.The 'in-cell' analysis approach elucidated several interesting donor and cell-type specific differences. Notably, in response to ionizing radiation we observed differential expression in luminal and basal-like cells of a member of the APOBEC enzyme family, recently identified as a critical driver of an oncogenic signature. Our findings suggest that this enzyme is active in the normal breast epithelium during DNA damage response.We demonstrate the practical application of a new method for assessing cell-type specific change in mixed cultures, especially the analysis of normal primary cultures, overcoming a major technical issue of dissecting the response of multiple cell types in a heterogeneous population.

SUBMITTER: Johnston RL 

PROVIDER: S-EPMC4748588 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature