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

Johnston, Rebecca L., Wockner, Leesa, McCart Reed, Amy E., Wiegmans, Adrian, Chenevix-Trench, Georgia, Khanna, Kum Kum, Lakhani, Sunil R. and Smart, Chanel E. (2016) High content screening application for cell-type specific behaviour in heterogeneous primary breast epithelial subpopulations. Breast Cancer Research, 18 18: 1-18. doi:10.1186/s13058-016-0681-9


Author Johnston, Rebecca L.
Wockner, Leesa
McCart Reed, Amy E.
Wiegmans, Adrian
Chenevix-Trench, Georgia
Khanna, Kum Kum
Lakhani, Sunil R.
Smart, Chanel E.
Title High content screening application for cell-type specific behaviour in heterogeneous primary breast epithelial subpopulations
Journal name Breast Cancer Research   Check publisher's open access policy
ISSN 1465-542X
1465-5411
Publication date 2016-02-09
Year available 2016
Sub-type Article (original research)
DOI 10.1186/s13058-016-0681-9
Open Access Status DOI
Volume 18
Issue 18
Start page 1
End page 18
Total pages 18
Place of publication London, United Kingdom
Publisher BioMed Central
Collection year 2017
Language eng
Formatted abstract
Background
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.

Methods
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.

Results
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.

Conclusions
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.
Keyword High-content immunofluorescence
Breast
Mixed cell cultures
DNA damage
Ionising radiation
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: UQ Centre for Clinical Research Publications
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