Abstract
BackgroundThe 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.MethodsWe 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.ResultsThe ‘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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0681-9) contains supplementary material, which is available to authorized users.
Highlights
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
Johnston et al Breast Cancer Research (2016) 18:18 studies of subpopulation-specific mammary epithelial biology indicate that the luminal progenitor compartment exhibits telomere dysfunction which might be a link to an unstable genome and tumourigenesis occurring in this cell type [8], which represents a great step forward in our understanding of the early steps of carcinogenesis in the breast
Using the GE IN Cell Analyser 2000 (INCA) we have optimised a method of distinguishing the different major subtypes of primary human mammary epithelial cells in short-term primary culture
Summary
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. Comparative studies of normal tissue adjacent to tumours show that neighbouring normal luminal and basal cells share the same initial genetic changes as the tumour [9]. This could imply that basal cells somehow ‘resist’ transformation, whilst luminal cells can accumulate further change and form a tumour [10]. This is interesting given that the different cell types are genetically identical and reside together in the same tissue, yet their inherently different biology and microenvironment might contribute to differences in transformation susceptibility. We can hypothesise that the interplay between microenvironment and cell type drives oncogenic change at different rates within a heterogeneous population
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