Cytofluorometric analysis for estrogen receptors using fluorescent estrogen probes

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Abstract
Estrogen receptor (ER) analysis of breast cancer tissue has been shown to be very useful in predicting which patients will respond to hormone therapy and have a better prognosis. The ER assay is, however, tedious and time consuming. Measurement of ER by flow cytometry would be rapid and based on either an average fluorescence‐E2 probe intensity per cell or the percentage of the ER+ cells per cell suspension. Analysis of E2 modified structures for relative binding affinity to the ER determined by competition studies and for fluorescence uptake into cell suspensions determined by flow cytometry was performed. Lack of high affinity to the ER and purity of the compound were major problems for the fluorescein‐labeled estrogen in fluorescein‐E2 compounds demonstrated by HPLC very little estradiol derivative in the parent compounds compared to total components present. A second type of fluoresceinated estrogen which has a peptide bond between the steroid and the chromophore was also tested. It was less contaminated but was unable to get into the cell and showed no binding activity to the ER. A pure plant fluorescent estrogen, coumestrol, has Ka of 6 × 108 M−1 for the ER and is a single component as determined by HPLC. Specific fluroescent uptake of coumestrol was performed on ER+ and ER− viable cell suspensions. When these coumestrol‐cell suspensions were excited at 350–360 nm and the blue emission was measured using flow cytometry, the result was a fluroescence uptake that was not highly displaceable by exess nonfluorescence E2 probes. Further studies on the modification of several parameters of the incubation step to enhance specific fluorescent uptake were also unsuccessful. Coumestrol was, however, a good competitor for both cellular and nuclear uptake of 3H‐E2.