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Fluoresceinated Estrone Binding by Human and Mouse Breast Cancer Cells¹

Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

A proportion of single, intact, viable cells derived from mouse and human breast cancers, when incubated with 17-fluoresceinated estrone, displayed fluorescence in all instances. The percentage of cells which were marker positive in mouse mammary tumors was remarkably uniform (35%), whereas in human breast cancers, it varied considerably (3 to 58%). While fluorescence of the entire cell most commonly occurred, staining of either the nucleus or the cytoplasm was observed frequently. Mouse uterus and liver were also marker positive. The proportion of such cells decreased in incidence following in vitro incubation with estradiol and tamoxifen or following administration of tamoxifen prior to tissue removal, findings indicative of 17-fluoresceinated estrone binding to estrogen receptor (ER). A variety of incubation times and temperatures and the use of ligand concentrations between 10^-10 and 10^-5 M failed to alter the proportion of marker-positive cells present in tumor. The latter indicates that nonspecific binding, should it occur as a consequence of high concentrations of ligand, is limited to cells containing ER. Consequently, the conclusion by others that nonspecific binding by cytochemical methods (should it occur) destroys the efficacy of the procedure as an indicator of patient prognosis or as a predictor of response to therapy may be unjustified, since nonspecific binding fails to alter results obtained by the cytochemical method.

When tumor ER values obtained biochemically were compared to the proportions of marker-positive cells in the same tumors, no direct quantitative relationship was evident. Consideration is given as to why such a lack of correlation does not of necessity indicate that one or the other method is inappropriate. Only if all marker cells possessed the same number of ER binding sites would a direct relationship between the two methods be apt to occur. Despite lack of a quantitative correlation, it was found that 81% of tumors with ER values of 10 to 99 fmol were associated with >10% marker-positive cells. The poorest association (39%) involved tumors with negative or borderline ER or those having inordinately high ER values, e.g., 412, >700 fmol (56%). Marker-positive cells ranged between 20 and 51% in the former and <10% in the latter. Experience with the cytochemical method as used in the present studies leads us to conclude that this is a technique which may be readily and rapidly carried out and is one which deserves more widespread evaluation. Since standard biochemical methods determine total ER in tumor cytosol and since cytochemical techniques measure the porportion of cells containing ER, the two are complimentary. When used simultaneously, they may provide more definitive information than is obtainable from either alone.

¹ Supported by USPHS Grants CA-26004 and CA-14972 from the National Cancer Institute.

² To whom requests for reprints should be addressed, at 914 Scaife Hall, University of Pittsburgh, 3550 Terrace Street, Pittsburgh, Pa. 15261.

³ Recipient of an award provided by the International Cancer Research Technology Transfer with the International Union Against Cancer.

⁴ Visiting scholar from Zhejiang Medical University, Hangzhou, China.

Received 7/27/81. Accepted 10/26/81.

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