Flow cytometric analysis of estrogen receptor expression in isolated nuclei and cells from mammary cancer tissues

Background: Cellular expression of receptors for the hormones estrogen and progesterone in human mammary tumors is of diagnostic and prognostic value. Ligand binding assays have been replaced by immunohistochemical analysis o f receptor expression. However, both of these techniques are slow, and in t he ligand-binding assay it is difficult to measure heterogeneity of recepto r expression in individual cells. Flow cytometry has been used extensively for monitoring the expression of cellular receptors in hematopoietic tumors but has been of limited value in the analysis of mammary tumors, which are difficult to disaggregate into single cells for flow analysis. Hormone rec eptors have a predominant nuclear localization, and it is relatively easy t o isolate nuclei from paraffin-embedded archival tissues for flow cytometri c analysis of receptor expression. Methods: Thick sections from formalin-fixed paraffin-embedded archival mamm ary tumors were digested by different enzyme solutions for the isolation of single nuclei. Different fixatives were used to compare the results on sub sequent staining of the nuclei for estrogen receptor (CER) expression. Doub le staining with propidium iodide and fluorescein isothiocyanate labeled se condary antibodies for ER expression was used for multiparametric analysis of ER and DNA content. Results: Digestion of paraffin sections with low concentration of pepsin an d detergents was ideal for isolation of single nuclei. Fixation with parafo rmaldehyde in the presence of Triton X-100 improved staining of the cells. Isolated nuclei had enhanced immunoreactivity compared with the whole cells , and subpopulations differing in reactivity could be identified in the nuc lear fractions. Double staining of nuclei for ER expression and DNA content could allow for multiparametric analysis of these two important parameters . Conclusions: The procedures described can be used for processing of archiva l paraffin-embedded mammary tumors for monitoring of ER expression and aneu ploidy. These two parameters have important diagnostic and prognostic signi ficance in mammary tumors. Laser flow cytometry by providing multiparametri c analysis can allow for correlation of these cellular markers with other i mportant cellular and clinical parameters. Cytometry 36:131-139, 1999 (C) 1 999 Wiley-Liss,Inc.