[CANCER RESEARCH 43, 4956-4965, October 1983] New Approach for Visualizing Estrogen Receptors in Target Cells Using Inherently Fluorescent Ligands and Image Intensification1'2 Pierre M. Martin,2 Henri P. Magdelenat,3 BahíaBenyahia, Odile Rigaud, and John A. Katzenellenbogen4 Laboratoire des RécepteursHormonaux, Facultéde Medicine Nord, 13326 Marseille Cedex 3, France [P. M. M.]; Laboratoire de Ftadiopathologie, Institut Curie, 26 Rue d'Ulm, 75231 Paris Cedex 5, France [H. P. M., B. B., O. R.]; and School of Chemical Sciences, University of Illinois, Urbana, Illinois 61801 [J. A. K.J ABSTRACT managing the disease (7,8,24,34). Biochemical assays, as they are used currently, give only an average value for receptor Four fluorescent estrogen ligands were investigated as agents concentration and do not address an essential question concern for visualization of estrogen receptors in cells: 2-{2,4-dihydroxy- ing the proportion of cells among the total tumor cell population phenyl)-6-hydroxy-3-benzofurancarboxylic acid 6-lactone (cou- that contain receptor and thus may retain steroid sensitivity. mestrol) and 9(11)-dehydro-12-oxoestradiol [12-oxo-1,3,5- Methods for the assessment of cell subpopulations having dif (KtyQCIIJ-estratetraene-S.IT^-diol] (12-oxoestradiol), which ferent binding capacities for sex steroid hormones would provide are inherently fluorescent compounds; and tamoxifen j(Z)-1-[4- valuable information about the cellular heterogeneity of normal (2-dimethylaminoethoxy)phenyl]-1,2-diphenyl-1 -butène) and 4- or pathological hormone-dependent tissues. hydroxytamoxifen {(Z)-1-[4-(2-dimethylaminoethoxy)phenyl]-1 - Cellular heterogeneity is a particularly important issue in the (4-hydroxyphenyl)-2-phenyl-1-butènej, which become maximally case of human breast tumors, where it has been used to explain fluorescent only after ultraviolet irradiation. By conventional flu the varied clinical response to hormone therapy in receptor- orescence techniques, these agents can be detected down to 10"8 M in water, but only to 10~6 to 10"7 M in protein solutions; positive cases (37). Furthermore, it is thought that a proper knowledge of the cellular heterogeneity of a tumor would be however, by photon-counting spectrofluorimetry, coumestrol and useful in predicting the nature of the response. Until recently, 12-oxoestradiol can be detected in protein solutions down to 5 x 10~10M. Three of these compounds have good affinity for the only autoradiography using tritiated ligands with high specific activity has been used to visualize steroid-binding cell subpopu estrogen receptor: coumestrol (20%); 12-oxoestradiol (12%); lations at hormone concentrations of physiological significance and 4-hydroxytamoxifen (37%), relative to estradiol (100%). Un (10"9 to 10~10M). However, autoradiography requires the prep der conditions where autoradiographic controls indicate that aration of 4-u.m frozen sections and long exposure times at low most of the estrogen receptor of MCF-7 human breast cancer temperatures. These procedural drawbacks make this technique cells is in the nucleus, we could demonstrate nuclear fluores cence using 10~9 M concentrations of coumestrol, 12-oxoestra inappropriate for widespread clinical use. Recently, promising methodologies based on monoclonal estrogen receptor antibod diol, and 4-hydroxytamoxifen. This nuclear fluorescence was ies have been emerging (9,11). abolished by a 200-fold excess of diethylstilbestrol and could The use of steroid hormones conjugated with fluorescent only be observed through a fluorescence microscope equipped molecules is under investigation as an approach to measuring with a microchannel image intensifier and a video camera detec tor that together provide a sensitivity enhancement of ~104. the cellular heterogeneity of receptors (15,26,29,30,32). Often, however, the derivatization of the steroid ligand with the bulky These studies indicate that the estrogen receptor in breast fluorophore results in a large structural perturbation that reduces cancer cells can be visualized by fluorescence techniques, pro affinity for the receptor drastically (6, 16, 23, 31). Attempts to vided that the visualizing ligands have adequate affinity and use these agents at higher concentrations raise the possibility specificity for the receptor and appropriate fluorescence char that sites other than the receptor are being detected (6, 23, 31). acteristics, and provided that the fluorescence instrument has In this paper, we describe the use of ligands for the estrogen adequate sensitivity to observe fluorescence emission from cells receptor that are inherently or latently fluorescent and that have treated with nw concentrations of the fluorescent agents. high affinity for the estrogen receptor: the natural phytoestrogen coumestrol5; the estradiol derivative 12-oxoestradiol (13); and INTRODUCTION the antiestrogens TAM and 4-OH-TAM. Because the low con centrations of these ligands required to label the high-affinity The determination of steroid receptor concentrations in human binding sites specifically (10~9 to 10~10 M) make conventional breast tumor provides important prognostic information that is fluorescence microscopy unsuitable, we have used light amplifi useful in selecting the most appropriate therapeutic strategy for cation provided by a microchannel image intensifier and a video camera detector, and we report results obtained using this new 1Preliminary presentations of this work have been made at the Sixth Interna approach for steroid receptor visualization. tiona! Congress on Hormonal Steroids, Jerusalem, Israel, September 5 to 10,1982 (18), and at the Eighth Annual Meeting of the European Society for Medical Oncology, Nice, France, 1982 (19). 6 The abbreviations and trivial names used are: coumestrol, 2-(2,4-dihydroxy- 2 Recipient of Grant 118997 from the Institut National de la Sante et la Reserche phenyl)-6-hydroxy-3-benzofurancarboxylic acid Wactone; 12-oxoestradiol, 9(11)- Medicale and support from the Association de la Reserche Contre de le Cancer dehydro-12-oxoestradiol [12-oxo-1,3,5(10),9(11)-estratetraene-3,17/3-diol]; TAM, (Villejuif) and the Facultéde Medicine, Universite Aix Marseille. To whom requests tamoxifen |(Z>1 -[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenyl-1 -butène); 4-OH- for reprints should be addressed. TAM, 4-hydroxytamoxifen |(Z>1 -[4-(2-dimethylaminoethoxy)phenyl)-1 -(4-hydroxy- 3 Recipient of support from the Comitéde Coordination Institut Curie, Paris, and phenyl)-2-phenyl-1-butène); R2858, moxestrol [17a-ethynyl-110-methoxy-1,3,5- the Comitéde Paris de la Ligne Nationale FrançaiseContre le Cancer. (10)-estratetraene-3,17/3-diol]; PBS, phosphate-buffered saline (0.15 M NaCI:0.01 ' Recipient of USPHS Grant 5R01 AM15556 from the NIH. M sodium phosphate); MEM. minimal essential medium; OES, diethylstilbestrol |(E> Received November 29,1982; accepted July 11,1983. 3,4-bis(4-hydroxyphenyl)-3-hexene]. 4956 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1983 American Association for Cancer Research. Fluorescent Visualization of Estrogen Receptors MATERIALS AND METHODS (1:500 dilution of MEM), and spun down in 0.3 M sucrose in MEM. Washing with antisera to estrogens greatly reduced autoradiographic BiochemicalStudies background. The cell pellets were mounted on a brass stud with Tissue- tek II O.C.T. compound 4583 (Labteck, Miles) and frozen in liquified Chemicals. Coumestrol was purchased from Eastman Organic Chem propane. Four-^m sections were cut in a cryostat and mounted for icals (Rochester, N. Y.). Alcoholic solutions gave unimodal fluorescence autoradiography on emulsion-coated slides by the thaw-mount proce spectra. 12-Oxoestradiol (13, 26) was prepared by oxidation of estradiol dure (22). The slides were exposed at -15° for up to 6 weeks, photo with dichlorodicyanoquinone, according to the method of Bodenberger graphically processed, and stained with methyl green-pyronine. and Dannenberg (2). TAM and 4-OH-TAM were kindly provided by ICI Pharmaceuticals, Ltd. (Alderley Park, England), as well as [3H]-4-OH- Fluorescent Labeling TAM (45 Ci/mmol; mixed isomers, cis:trans, ~50:50). R2858 and [3H]R2858 (51 Ci/mmol) were from New England Nuclear (Boston, Incubation Conditions. Incubations were carried out on adherent cells Mass.). directly in the Labteck slides. The culture medium was discarded, and Unlabeled hormones and biochemicals were from Sigma (St. Louis, the cells were incubated 20 min at 20° with 10"8 M progesterone in Mo.), and gentamycin was from Unilabo (Paris, France). Other chemicals, Medium B (250 ^I/chamber) prior to fluorescent labeling. This preincu- including fluorescence-grade ethanol, were from Merck (Darmstadt, Ger bation with progesterone was shown to diminish nonspecific steroid many). Triple-distilled water was used for fluorescence photon-counting labeling, especially on the cell membrane, without interfering with the experiments. Culture medium and fetal calf serum were purchased from emission spectrum of the fluorescent labels.6 Cells were washed with Grand Island Biological Co. (Grand Island, N. Y.). PBS, and 250 ¡Aof either 10~8, 10~9, or 10~1°Mfluorescent ligands in Medium B were added to the Labteck chambers. Incubation was carried Fluorescence Spectra out for 50 min at 37°in 5% CO2:air. Cells were washed 3 times with PBS and air dried. The walls of the 4-chambered Labteck slides were Conventional Spectrofluorimetry. Fluorescence spectra down to 10~8 M ethanolic or aqueous solutions of ligands