Progress in Histo- and Cytochemistry, Vol. 26 W. Graumann / J. Drukker (Eds.), Histochemistry of Receptors © Fischer Verlag· Stuttgart· Jena· New York· 1992
2.6 Flow cytometric steroid receptor analysis B. SCHUlTE, H. M. E. SCHERES\ A. F. P. M. DE GOEIJ\ M.J. M. ROUSCH\ G. H. BLIJHAM2, F. T. BOSMAN3 , F. C. S. RAMAEKERS Departments of Molecular Cell Biology and Genetics, Ipathology, 2Internal Medicine, University of Limburg, Maastricht (The Netherlands), 3Department of Pathology, Erasmus University, Rotterdam (The Netherlands)
Introduction The estrogen and progesterone receptor content of breast cancer tissue is an important parameter in predicting short-term prognosis and the response to endocrine treatment (MCGUIRE 1986; LIPPMANN 1988; OSBORNE 1985). However, there is considerable variation in the course of the disease between patients with tumors which have been shown to express hormone receptors (LIPPMANN 1988). About one third of the cases of receptor positive breast cancers do not respond to endocrine therapy. Furthermore, tumors which are initially sensitive to hormonal treatment, frequently lose their hormone-sensitivity as the disease progresses (MCGUIRE 1986; LIPPMANN 1988). Hence new approaches in steroid receptor analysis are needed if the prediction of tumor behaviour and response to endocrine therapy of breast cancer patients are to be improved. The growth potential of a tumor is reflected in the percentage of cells present in the S phase of the cell cycle. Along with tumor cell ploidy this is a useful prognostic parameter (MCGUIRE 1986). DNA flow cytometry is a reliable tool for studying these parameters. Concomitant DNA flow cytometry and steroid receptor analysis of breast cancer cells could provide an even more reliable set of parameters for prediction of tumor cell behaviour. In several reports parallel analyses of steroid receptor expression and proliferation characteristics have been described. Some studies showed that slowly proliferating tumors contain higher amounts of ER than rapidly growing tumors (MCGUIRE 1986; KALLIONIEMI et al. 1987; PARADISO et al. 1988; MEYER et al. 1977; KUTE et al. 1981; SILVESTRINI et aI1979). In an other study, however, only a weak relationship was observed (HEDLEY et al. 1987). A bivariate analysis would allow the study of the direct relationship between hormone receptor expression and cell kinetics for individual cells. The technique would also allow modulation of tumour growth by hormonal factors to be tested, and it might be more helpful in establishing criteria for selecting patients likely to respond to endocrine treatment or chemotherapy. Although some reports have described the application of fluoresceinated estrone (17FE) and other fluorochrome labeled ligands such as estradiol-bovine serum albumin-FITC in flow cytometry (VAN et aI. 1984; BENZ et al. 1985), receptor detection by these probes lack sufficient specificity (BERNS et al. 1984; DE GOEIJ et al. 1986).
Flow cytometric steroid receptor analysis . 69
In this report we therefore describe the development of a technique for the flow cytometric detection of estrogen and progesterone receptors. The receptors are visualized in breast cancer cells and permeabilized with detergent, using monoclonal anti-receptor antibodies in an indirect immunofluorescence assay. Counterstaining of the cells with propidium iodide allows simultaneous DNA analysis.
Materials and methods Cell lines The human breas cancer cell lines T47-D, MCF-7, Esva-T were obtained from the American Type Culture Collection in Rockville, USA. The T47-D G3 clone was established in our institute by limiting dilution. The ZR-75 cell line and the clones derived from this cell line (ZR-75-Z11 and ZR-75-Z48) were kindly provided by Dr. J. Foekens, Department of Biochemistry, the Daniel den Hoed Cancer Centre, Rotterdam, The Netherlands. All cell lines were routinely cultured in Dulbecco's modified eagles medium (DMEM) (GIBCO, Zwanenburg, The Netherlands), supplemented with 10% heat-inactivated fetal calf serum (FCS) (GIBCO), except for the Zr-75 cells which were cultured in RPMI 1640 (GIPCO), supplemented with 10% FCS, penicillin (100 IU/ml) and streptomycin (100 I-tg/ml) (GIBCO). The cultures were maintained at 37°C in humidified atmosphere containing 5% CO2 and the medium was changed every 48 h. Prior to harvesting the cell lines were cultured for 24 h in medium with 5% FCS and stripped by dextran coated charcoal (DCC) to remove hormones. The cells were harvested by trypsinisation.
Immunocytochemistry For purposes of flow cytometrical analysis approx. 2xl0 7 cells were fixed in paraformaldehyde (1 % w/v in saline, pH 7.6) for 2 min at room temperature. After centrifugation, the pellet was resuspended in 200 III phosphate buffered saline (PBS), and 2 ml of absolute methanol (-20°C) was added under constant agitation. The cells were kept at -20°C for 5 min, centrifuged and resuspended in citrate buffer (3 mM trisodiumcitrate, 0.5 mM Tris, 1.5 mM spermine, 0.4% v/v Triton X-I00 and 1 mg/ml bovine serum albumine). 25 III Anti-hormone receptor antibody (rat anti-estrogen or anti-progesterone receptor monoclonal antibody, Abbott Laboratories, Chicago, IL, USA) was added to approx. lxl0 6 cells in 100 III buffer. In the negative control the primary antibody was omitted. After overnight incubation at 4°C, the cells were washed twice in citrate buffer and incubated for 2 h at 4°C with appropriately diluted FITC-conjugated Fab 2 fragments of goat anti-rat Ig (Caltag, San Francisco, USA). The cells were washed twice and the pellet was resuspended in 0.5 ml citrate buffer supplemented with propidium iodide (PI, 10 Ilg/ml, Calbiochem, USA) and RNase (100 Ilg/ml, Serva, The Netherlands) 15 min prior to flow cytometric analysis. For purposes of determining ER immunoreactivity on cytocentrifuge preparations the Abbott ER-ICA protocol was used according to the manufacturer's instructions. For the determination of PR immunoreactivity the ABC method was used. In brief, cytocentrifuge preparations were incubated with anti-PR antibody overnight at 4°C, rinsed twice in PBS and then incubated with biotinylated rabbit anti-rat Ig (DAKOPATTS, Denmark) for 1 h at room temperature. After rinsing twice in PBS, the cytocentrifuge preparations were incubated with appropriately diluted
70 . B. Schutte et al.
peroxidase conjugated avidin biotin complex for 1 h at room temperature. Peroxidase activity was detected according to SCHERES et al. (1988). 0.4% Triton X-I00 was included in the incubation buffers for both ER and PR staining.
Radioligand binding assay Radioligand binding assay was performed on cytocentrifuge preparations using a modification of a technique for ER determination in cryostat sections as described previously (DE GOEl] et al. 1984; DE GOEl] et al. 1988; SCHERES et al. 1988). Briefly, 2xl05 cells were centrifuged onto gelatin chromalum coated coverslips, mounted on to microscopic slides and allowed to airdry. The cytocentrifuge preparations were overlayed with 100 III of buffer containing 0.015 M K2 HP0 4 / KH2 P0 4 pH 7.4, 0.0015 M EDTA, 0.003 M NaN}, 0.003 M monothioglycerol and 10% glycerol. For detection of total hormone binding eH] labeled estradiol (specific activity 140 Ci/mmol, Amersham, UK) or R5020 (specific activity 86 Ci/mmol, New England Nuclear, UK) were added at various concentrations ranging from 0.25 to 4.0 nM. Nonspecific binding was assessed in the presence of a hundredfold excess of cold diethylstilbestrol (DES) or promegestone (R5020). For PR determination the buffers for total and nonspecific binding also contained 80 nM nonradiolabeled dihydro-testosterone (DHT) to prevent binding of R5020 to androgen receptors. All incubations were performed in triplicate overnight in a humidified atmosphere at 4°C. For the detection of soluble receptor, 75 III of the overlay buffer was added to 75 III EORTC buffer containing 0.5% DCC. After mixing, the tubes were left for 10 min at 4°C and centrifuged at 12,000 g for 1 min. Aliquots of 75 III of the supernatant were mixed with 3 ml scintillation solution and counted. For the detection of cell bound receptor the covers lips were removed from the slides and washed three times with cold EORTC buffer at intervals of 15 min. Finally the coverslipbound radioactivity was determined by liquid scintillation counting. Aliquots of the cell suspensions were used for protein determination according to BRADFORD 1976. Affinity constants and the number of specific hormone binding sites/cell were obtained from a 5 point Scatchard analysis (SCATCHARD 1949). The sum of soluble and cell bound binding sites was used for the calculation of the total number of binding sites per cell.
Flow cytometry Cells were analysed using a FACS IV Cell Sorter (Becton & Dickinson, Sunnyvale, CA) equipped with a 4 Watt Argon Ion Laser (Spectra Physics, model 164-05) tuned to 488 nm at 400 mW output. A 520 lp filter was used to block scattered light. Green fluorescence was measured through a 540 sp filter and recorded as a measure of the amount of bound anti-steroid receptor antibody. Red DNA fluorescence was measured through a 620 lp filter and was recorded as a measure of the amount of bound propidium iodide. The number of hormone receptor positive cells was determined by an arbitrary threshold setting allowing 5% of positive counts in the negative control. The relative fluorescence intensity (RFI) was calculated as the ratio of the average fluorescence intensity of sample and negative controls.
Flow cytometric steroid receptor analysis . 71
Results The conditions of DNA and receptor staining procedures were partly based on the procedures used for staining of cell suspensions for the nuclear protein PCNA and subsequent flow cytometric analysis as described by KURKI et al. (1988). Receptor staining strongly increased after overnight incubation with the anti-receptor antibody as compared to a short incubation step (1-2 h). After 20 h of incubation no further increase of specific receptor immunofluorescence was observed. Using this protocol, ER and PR of T47-D cells were found to be localized exclusively in the nucleus, as shown in the micrographs of Fig. 1. In metaphase cells a weak but specific staining of the cytoplasm was observed. Fig. 2 shows the flow cytometrically obtained immunofluorescence histograms and the Scatchard analyses for ER and PRo T47-D cells showed intense PR fluorescence in the majority of the nuclei, clearly separated from the negative control values. Similar observations were made in the immunoperoxidase stained cytocentrifuge preparations of the same cells. In contrast, the ER immunofluorescence signal partially overlapped with that of the negative control due to a low intensity of ER staining in this cell line. The parallel im-
Fig. 1. Immunocytochemistry of estrogen and progesterone receptors in T47-D cells using fluorescence and peroxidase immunostaining procedures. Immunofluorescence pattern of ER (a) and PR (b) after staining of cells in suspension. Immunoperoxidase staining pattern of ER (c), PR (d) and negative control (e) of cytocentrifuge preparations.
72 . B. Schutte et al.
munoperoxidase stained cytocentrifuge preparation exhibited a low number of weakly immunoreactive cells. The number of hormone binding sites per cell was 6750 and 184000 for ER and PR respectively. The dissociation constant was 0.09 nM for ER and 0.11 nM for PR respectively. In order to investigate in more detail the correlation between immunofluorescence intensity and hormone receptor content as determined by the radioligand binding assay, the relative fluorescence intensity was plotted against the total number of specific steroid hormone binding sites per cell. The results obtained for a series of cell lines and a number of derived clones are presented in Fig. 3. For PR a strong linear correlation between the two parameters was obtained (r=0.95, N = 10, p. < 0.0002). For ER a low relative fluorescence intensity was measured by flow cytometry in the same series of cell lines - which, as determined by the radioligand binding assay, had very low receptor·
700
700
a
eoo 500
500
ol::
b
eoo
~
=
300
4>
:I
300
co
~
200
100 0 0
\
10
200
100
20
30
40
eo eo
70
80
0
90
anti ER immunofluorescence intensity
0.40 0.8 0.30
.. .:
0.30
=
0.25
~
::a
=
0.6 0.5
:I 0.4
2.6 Flow cytometric steroid receptor analysis B. SCHUlTE, H. M. E. SCHERES\ A. F. P. M. DE GOEIJ\ M.J. M. ROUSCH\ G. H. BLIJHAM2, F. T. BOSMAN3 , F. C. S. RAMAEKERS Departments of Molecular Cell Biology and Genetics, Ipathology, 2Internal Medicine, University of Limburg, Maastricht (The Netherlands), 3Department of Pathology, Erasmus University, Rotterdam (The Netherlands)
Introduction The estrogen and progesterone receptor content of breast cancer tissue is an important parameter in predicting short-term prognosis and the response to endocrine treatment (MCGUIRE 1986; LIPPMANN 1988; OSBORNE 1985). However, there is considerable variation in the course of the disease between patients with tumors which have been shown to express hormone receptors (LIPPMANN 1988). About one third of the cases of receptor positive breast cancers do not respond to endocrine therapy. Furthermore, tumors which are initially sensitive to hormonal treatment, frequently lose their hormone-sensitivity as the disease progresses (MCGUIRE 1986; LIPPMANN 1988). Hence new approaches in steroid receptor analysis are needed if the prediction of tumor behaviour and response to endocrine therapy of breast cancer patients are to be improved. The growth potential of a tumor is reflected in the percentage of cells present in the S phase of the cell cycle. Along with tumor cell ploidy this is a useful prognostic parameter (MCGUIRE 1986). DNA flow cytometry is a reliable tool for studying these parameters. Concomitant DNA flow cytometry and steroid receptor analysis of breast cancer cells could provide an even more reliable set of parameters for prediction of tumor cell behaviour. In several reports parallel analyses of steroid receptor expression and proliferation characteristics have been described. Some studies showed that slowly proliferating tumors contain higher amounts of ER than rapidly growing tumors (MCGUIRE 1986; KALLIONIEMI et al. 1987; PARADISO et al. 1988; MEYER et al. 1977; KUTE et al. 1981; SILVESTRINI et aI1979). In an other study, however, only a weak relationship was observed (HEDLEY et al. 1987). A bivariate analysis would allow the study of the direct relationship between hormone receptor expression and cell kinetics for individual cells. The technique would also allow modulation of tumour growth by hormonal factors to be tested, and it might be more helpful in establishing criteria for selecting patients likely to respond to endocrine treatment or chemotherapy. Although some reports have described the application of fluoresceinated estrone (17FE) and other fluorochrome labeled ligands such as estradiol-bovine serum albumin-FITC in flow cytometry (VAN et aI. 1984; BENZ et al. 1985), receptor detection by these probes lack sufficient specificity (BERNS et al. 1984; DE GOEIJ et al. 1986).
Flow cytometric steroid receptor analysis . 69
In this report we therefore describe the development of a technique for the flow cytometric detection of estrogen and progesterone receptors. The receptors are visualized in breast cancer cells and permeabilized with detergent, using monoclonal anti-receptor antibodies in an indirect immunofluorescence assay. Counterstaining of the cells with propidium iodide allows simultaneous DNA analysis.
Materials and methods Cell lines The human breas cancer cell lines T47-D, MCF-7, Esva-T were obtained from the American Type Culture Collection in Rockville, USA. The T47-D G3 clone was established in our institute by limiting dilution. The ZR-75 cell line and the clones derived from this cell line (ZR-75-Z11 and ZR-75-Z48) were kindly provided by Dr. J. Foekens, Department of Biochemistry, the Daniel den Hoed Cancer Centre, Rotterdam, The Netherlands. All cell lines were routinely cultured in Dulbecco's modified eagles medium (DMEM) (GIBCO, Zwanenburg, The Netherlands), supplemented with 10% heat-inactivated fetal calf serum (FCS) (GIBCO), except for the Zr-75 cells which were cultured in RPMI 1640 (GIPCO), supplemented with 10% FCS, penicillin (100 IU/ml) and streptomycin (100 I-tg/ml) (GIBCO). The cultures were maintained at 37°C in humidified atmosphere containing 5% CO2 and the medium was changed every 48 h. Prior to harvesting the cell lines were cultured for 24 h in medium with 5% FCS and stripped by dextran coated charcoal (DCC) to remove hormones. The cells were harvested by trypsinisation.
Immunocytochemistry For purposes of flow cytometrical analysis approx. 2xl0 7 cells were fixed in paraformaldehyde (1 % w/v in saline, pH 7.6) for 2 min at room temperature. After centrifugation, the pellet was resuspended in 200 III phosphate buffered saline (PBS), and 2 ml of absolute methanol (-20°C) was added under constant agitation. The cells were kept at -20°C for 5 min, centrifuged and resuspended in citrate buffer (3 mM trisodiumcitrate, 0.5 mM Tris, 1.5 mM spermine, 0.4% v/v Triton X-I00 and 1 mg/ml bovine serum albumine). 25 III Anti-hormone receptor antibody (rat anti-estrogen or anti-progesterone receptor monoclonal antibody, Abbott Laboratories, Chicago, IL, USA) was added to approx. lxl0 6 cells in 100 III buffer. In the negative control the primary antibody was omitted. After overnight incubation at 4°C, the cells were washed twice in citrate buffer and incubated for 2 h at 4°C with appropriately diluted FITC-conjugated Fab 2 fragments of goat anti-rat Ig (Caltag, San Francisco, USA). The cells were washed twice and the pellet was resuspended in 0.5 ml citrate buffer supplemented with propidium iodide (PI, 10 Ilg/ml, Calbiochem, USA) and RNase (100 Ilg/ml, Serva, The Netherlands) 15 min prior to flow cytometric analysis. For purposes of determining ER immunoreactivity on cytocentrifuge preparations the Abbott ER-ICA protocol was used according to the manufacturer's instructions. For the determination of PR immunoreactivity the ABC method was used. In brief, cytocentrifuge preparations were incubated with anti-PR antibody overnight at 4°C, rinsed twice in PBS and then incubated with biotinylated rabbit anti-rat Ig (DAKOPATTS, Denmark) for 1 h at room temperature. After rinsing twice in PBS, the cytocentrifuge preparations were incubated with appropriately diluted
70 . B. Schutte et al.
peroxidase conjugated avidin biotin complex for 1 h at room temperature. Peroxidase activity was detected according to SCHERES et al. (1988). 0.4% Triton X-I00 was included in the incubation buffers for both ER and PR staining.
Radioligand binding assay Radioligand binding assay was performed on cytocentrifuge preparations using a modification of a technique for ER determination in cryostat sections as described previously (DE GOEl] et al. 1984; DE GOEl] et al. 1988; SCHERES et al. 1988). Briefly, 2xl05 cells were centrifuged onto gelatin chromalum coated coverslips, mounted on to microscopic slides and allowed to airdry. The cytocentrifuge preparations were overlayed with 100 III of buffer containing 0.015 M K2 HP0 4 / KH2 P0 4 pH 7.4, 0.0015 M EDTA, 0.003 M NaN}, 0.003 M monothioglycerol and 10% glycerol. For detection of total hormone binding eH] labeled estradiol (specific activity 140 Ci/mmol, Amersham, UK) or R5020 (specific activity 86 Ci/mmol, New England Nuclear, UK) were added at various concentrations ranging from 0.25 to 4.0 nM. Nonspecific binding was assessed in the presence of a hundredfold excess of cold diethylstilbestrol (DES) or promegestone (R5020). For PR determination the buffers for total and nonspecific binding also contained 80 nM nonradiolabeled dihydro-testosterone (DHT) to prevent binding of R5020 to androgen receptors. All incubations were performed in triplicate overnight in a humidified atmosphere at 4°C. For the detection of soluble receptor, 75 III of the overlay buffer was added to 75 III EORTC buffer containing 0.5% DCC. After mixing, the tubes were left for 10 min at 4°C and centrifuged at 12,000 g for 1 min. Aliquots of 75 III of the supernatant were mixed with 3 ml scintillation solution and counted. For the detection of cell bound receptor the covers lips were removed from the slides and washed three times with cold EORTC buffer at intervals of 15 min. Finally the coverslipbound radioactivity was determined by liquid scintillation counting. Aliquots of the cell suspensions were used for protein determination according to BRADFORD 1976. Affinity constants and the number of specific hormone binding sites/cell were obtained from a 5 point Scatchard analysis (SCATCHARD 1949). The sum of soluble and cell bound binding sites was used for the calculation of the total number of binding sites per cell.
Flow cytometry Cells were analysed using a FACS IV Cell Sorter (Becton & Dickinson, Sunnyvale, CA) equipped with a 4 Watt Argon Ion Laser (Spectra Physics, model 164-05) tuned to 488 nm at 400 mW output. A 520 lp filter was used to block scattered light. Green fluorescence was measured through a 540 sp filter and recorded as a measure of the amount of bound anti-steroid receptor antibody. Red DNA fluorescence was measured through a 620 lp filter and was recorded as a measure of the amount of bound propidium iodide. The number of hormone receptor positive cells was determined by an arbitrary threshold setting allowing 5% of positive counts in the negative control. The relative fluorescence intensity (RFI) was calculated as the ratio of the average fluorescence intensity of sample and negative controls.
Flow cytometric steroid receptor analysis . 71
Results The conditions of DNA and receptor staining procedures were partly based on the procedures used for staining of cell suspensions for the nuclear protein PCNA and subsequent flow cytometric analysis as described by KURKI et al. (1988). Receptor staining strongly increased after overnight incubation with the anti-receptor antibody as compared to a short incubation step (1-2 h). After 20 h of incubation no further increase of specific receptor immunofluorescence was observed. Using this protocol, ER and PR of T47-D cells were found to be localized exclusively in the nucleus, as shown in the micrographs of Fig. 1. In metaphase cells a weak but specific staining of the cytoplasm was observed. Fig. 2 shows the flow cytometrically obtained immunofluorescence histograms and the Scatchard analyses for ER and PRo T47-D cells showed intense PR fluorescence in the majority of the nuclei, clearly separated from the negative control values. Similar observations were made in the immunoperoxidase stained cytocentrifuge preparations of the same cells. In contrast, the ER immunofluorescence signal partially overlapped with that of the negative control due to a low intensity of ER staining in this cell line. The parallel im-
Fig. 1. Immunocytochemistry of estrogen and progesterone receptors in T47-D cells using fluorescence and peroxidase immunostaining procedures. Immunofluorescence pattern of ER (a) and PR (b) after staining of cells in suspension. Immunoperoxidase staining pattern of ER (c), PR (d) and negative control (e) of cytocentrifuge preparations.
72 . B. Schutte et al.
munoperoxidase stained cytocentrifuge preparation exhibited a low number of weakly immunoreactive cells. The number of hormone binding sites per cell was 6750 and 184000 for ER and PR respectively. The dissociation constant was 0.09 nM for ER and 0.11 nM for PR respectively. In order to investigate in more detail the correlation between immunofluorescence intensity and hormone receptor content as determined by the radioligand binding assay, the relative fluorescence intensity was plotted against the total number of specific steroid hormone binding sites per cell. The results obtained for a series of cell lines and a number of derived clones are presented in Fig. 3. For PR a strong linear correlation between the two parameters was obtained (r=0.95, N = 10, p. < 0.0002). For ER a low relative fluorescence intensity was measured by flow cytometry in the same series of cell lines - which, as determined by the radioligand binding assay, had very low receptor·
700
700
a
eoo 500
500
ol::
b
eoo
~
=
300
4>
:I
300
co
~
200
100 0 0
\
10
200
100
20
30
40
eo eo
70
80
0
90
anti ER immunofluorescence intensity
0.40 0.8 0.30
.. .:
0.30
=
0.25
~
::a
=
0.6 0.5
:I 0.4
