Sequential Colocalization of ERa, PR, and AR Hormone Receptors Using Confocal Microscopy Enables New Insights into Normal Breast and Prostate Tissue and Cancers

SIMPLE SUMMARY: At present, platforms for multiplex immunohistochemistry (e.g., Opal) identify markers in distinct cell populations within a tissue section using multispectral fluorescence and optic microscopy. However, the optic resolution is not enough to colocalize markers at the subcellular level in the main epithelial or cancer population. We use confocal microscopy in multiplex detection of nuclear hormone receptors since they are an important part of the diagnosis and treatment of breast and prostate cancer. Moreover, we increased the quantitative dynamic range and resolution through increasing the signal/noise ration through reducing autofluorescence and increased longer antibody incubations. ColNu mIHCF identified distinct patterns of nuclear receptor colocalization in breast cancers. Furthermore, in prostate cancer all cancer epithelium was positive for ERa at the plasma membrane; and in normal prostate a small ERa+/p63+/AR− basal population suggest stem cell commitment to differentiation. ColNu mIHCF could be used for improving diagnosis and treatment in cancer. ABSTRACT: Multiplex immunohistochemistry (mIHC) use markers staining different cell populations applying widefield optical microscopy. Resolution is low not resolving subcellular co-localization. We sought to colocalize markers at subcellular level with antibodies validated for clinical diagnosis, including the single secondary antibody (combination of anti-rabbit/mouse-antibodies) used for diagnostic IHC with any primary antibody, and confocal microscopy. We explore colocalization in the nucleus (ColNu) of nuclear hormone receptors (ERa, PR, and AR) along with the baseline marker p63 in paired samples of breast and prostate tissues. We established ColNu mIHCF as a reliable technique easily implemented in a hospital setting. In ERa+ breast cancer, we identified different colocalization patterns (nuclear or cytoplasmatic) with PR and AR on the luminal epithelium. A triple-negative breast-cancer case expressed membrane-only ERa. A PR-only case was double positive PR/p63. In normal prostate, we identified an ERa+/p63+/AR-negative distinct population. All prostate cancer cases characteristically expressed ERa on the apical membrane of the AR+ epithelium. We confirmed this using ERa IHC and needle-core biopsies. ColNu mIHCF is feasible and already revealed a new marker for prostate cancer and identified sub-patterns in breast cancer. It could be useful for pathology as well as for functional studies in normal prostate and breast tissues.