A novel dual antibody staining assay to measure estrogen receptor transcriptional activity

Abstract

Activity of the canonical estrogen receptor (ER) pathway is equivalent to functional activity of the nuclear ER transcription factor. To assess transcriptional activity of ER, for biomedical research and diagnostic purposes ER monoclonal antibodies are routinely used to identify nuclear ER staining in cells and tissue samples, however it remained unclear whether this is sufficiently predictive for transcriptional activity of ER, and thus for ER pathway activity. Using ER positive breast cancer cell lines (MCF7 and T47D) in which the transcriptional activity status of ER was quantified using an mRNA based ER pathway activity assay, the relation between ER activity and nuclear ER staining with ER monoclonal antibodies (MoAb) was investigated. While the presence of ER in the cell nucleus is a prerequisite for ER activity, it was not predictive for ER transcriptional activity, confirming earlier findings. There were remarkable differences in behaviour of the used MoAbs: EP1 and 1D5 MoAbs showed reduced nuclear staining when ER was transcriptionally active, while staining with H4624 MoAb was independent of ER activity. To improve discrimination between active and inactive nuclear ER based on ER staining, a method was developed which consists of dual ER MoAb immunofluorescent staining, followed by generation of a digital image with a standard digital pathology scanner, and application of a cell nucleus detection algorithm and per cell calculation of the nuclear H4624/EP1 fluorescence intensity ratio, where a high H4624/EP1 ratio predicts an active ER. In this method the EP1 MoAb can in principle be replaced by the 1D5 MoAb. We hypothesize that the EP1 and 1D5 monoclonal antibodies (MoAb) recognize an ER epitope which becomes hidden upon transcriptional activation of ER, while the H4624 MoAb binds an ER epitope which remains accessible when ER is activated. The method is expected to be of value to better assess ER activity by means of staining.