Journal of Surgical Oncology 10:221-224 (1978)
lmmunocytological Localization of Estrogen in Human Mammary Carcinoma Cells by Horseradish-Anti-Horseradish Peroxidase Complex .......................................................................................... .......................................................................................... LUNA GHOSH, M.D., BIMAL C. GHOSH, M.D., and TAPAS K. DAS GUPTA, M.D., Ph.D. Tissues from 2 5 female and 2 male patients of mammary carcinoma were examined by the horseradish-anti-horseradish peroxidase complex to localize the presence of estrogen. Carcinoma cells of I 0 female and 2 male patients showed positive staining by this technique. The nucleus and the cytoplasmic membrane showed the reaction. This method is useful in identifying the exact location of the hormones or hormone-like substances in the malignant cells and, perhaps, will be of great help in selecting the patients for management.
.......................................................................................... .......................................................................................... Key words: immunocytological, horseradish, anti-horseradish peroxidase, estrogen, mammary carcinoma
INTRODUCTION Although a relationship between estrogen and human mammary carcinoma is known (Block et al., 1975; Dao, 1972; Jensen et al., 1971), the exact mechanism and the site of the influence in the malignant cells still remain matters of some dispute. The mammary carcinomas are responsive to different hormones of which estrogen exerts the maximum effects (Braunsberg et al., 1971; Hahnet et al., 1971; Jensen et al., 1971; Johansson et al., 1970;McGuire, 1975;Mobbs, 1971). In the past much had been investigated about the hormonal receptor sites by autoradiographic technique (Block et al., 1975; Folca et al., 1961 ;Hahnet e t al., 1971). To the best of our knowledge, no study has been made for cytological localization of hormones within the mammary carcinoma cells. Our objective was to localize estrogen within the human mammary carcinoma cells by using the unlabeled antibody enzyme technique with horseradish--anti-horseradish peroxidase complex (Mason et al., 1969; Sternberger et al., 1970).
From the Division of Surgical Oncology, Department of Surgery, and Department of Pathology, University of Illinois at the Abraham Lincoln School of Medicine and Cook County Hospital, Chicago, Illinois. Address reprint requests to Dr. Bimal C. Ghosh, 1825 West Harrison Street, Division of Surgical Oncology, Chicago, IL 60612.
0022-4790/78/1003-0221$01.10@ 1 9 7 8 Alan R. Liss, I n c
222
Ghosh, Ghosh, a n d Das G u p t a
MATERIALS AND METHODS Tissues obtained from 25 female, ages from 40 to 55 years, and 2 male patients, ages 65 and 68 years, of infiltrating duct carcinoma were fixed in 0.2% calcium acetate formalin, pH 7.4, for 4 hr, then dehydrated through graded alcohol, embedded in paraffin, section at 3 p thickness, and mounted on glass slides. Normal female breast tissue from 10 patients with benign breast disease was similarly treated, as a control. For estrogen localization, sections were immersed in phosphate-buffered saline (PBS), pH 7.2, for 30 min before treatment with antisera. Two drops of sheep antiestrddiol serum in different dilutions (1 :lo, 150, 1 :100) were added to each section and the sections were kept in a moist chamber at 37°C for 1 hr. The excess serum was then drained and the sections were washed for 30 min in three changes of PBS. Rabbit antisheep gamma globulin was then added to each section. The sections were kept at 37°C in a moist chamber for 1 hr, then washed in PBS for 30 min with three changes. Sheep peroxidase-anti-peroxidase (PAP) complex was added t o the sections for 1 hr at 37°C. The sections were washed again in PBS for 30 min, then incubated in the substrate solution for 5 min at room temperature. The substrate solution was composed of 30 mg% of 3’3’-diaminobenzidine tetrahydrochloride in Tris-buffered PBS. The buffer solution contained 600 mg% of tris(hydroxymethy1)aminoniethane in PBS adjusted to pH 7.6. The substrate solution was freshly prepared and a few drops of 3% H2 O2 were added just before use. Following enzymatic reaction the sections were washed for 15 min in 2% O s 0 4 . The sections were again rinsed in distilled water, then dehydrated and mounted in Permount.
Fig. 1. Male mammary carcinoma cells demonstrating positive reaction for estrogen by immunoperoxidase staining. x 300.
Estrogen i n Human Mammary Carcinoma
223
Fig. 2. Female mammary carcinoma cells for estrogen. T h e staining is also prominent in t h e nucleus and cytoplasmic membrane. Immunoperoxidase stain. x 300.
RESULTS The cytoplasm of the adenocarcinoma cells was stained when diluted sheep antiestradiol serum was used. Positive staining of tumor cells occurred with all the dilutions of antiserum. The reactive cells had a finely granular dark brown appearance. The nuclear membrane and the nucleus showed positive staining. The specificity of the bridge technique was verified by using other organs. Sections from muscle and thyroid failed to show any staining. In a second set, the sections were stained either by normal rabbit serum or by normal sheep serum instead of rabbit antigrowth hormone or sheep anti-estradiol sera. Both failed to show any demonstrable staining. An additional control experiment was done by substituting normal sheep serum for sheep anti-rabbit serum, and some sections were incubated in diaminobenzidine peroxidase only; neither had a reaction. Both male patients and 10 female patients (40%) showed positive reactions and proved, to have estrogen in the carcinoma cells by cytochemical localization.
COMMENT The influence of hormones on the growth and spread of mammary carcinoma is known; however, the exact mechanism and the site of influence in the malignant cells are controversial. With the use of tissue sections (Block et al., 1975; Jensen et al., 1971) and tissue homogenate autoradiography (Folca et al., 1961;Hahnet et al., 1971), the presence
224
Ghosh, Ghosh, and Das Gupta
of receptor sites of hormones has been determined. The problem in autoradiographic determination is that with this method it is not possible to ascertain which cellular component of a given tumor is either influenced by or is the site of storage of the hormone. Therefore, it is logical to attempt to use a technique that might selectively localize the site of activity of estrogen within the malignant cells. Although localization of estrogen in the tumor cells is a step toward understanding its role in mammary carcinoma, it raises the inevitable question of whether these are true receptor sites or whether some of these malignant cells independently secrete estrogenic hormones or hormone-like substances. This technique is useful in determining the exact location of the hormones and perhaps will be a great help in clinical practice for selecting patients for different modalities of treatment.
REFERENCES Block GE, Jensen LV, Polley TZ Jr: T h e prediction of hornional dependency of mammary cancer. Ann Surg 182:342-352, 1975. Braunsberg H, Carter AE, lrvine WT, James I:, James VHT: Estrogens in breast tumors. Nat Cancer Inst Monogr 34:71-75,1971. Dao T L : Ablation therapy for hormone dependent tumors. Annu Rev Med 23:l-18, 1972. Folca PJ, Glascock R F , Irvine WT: Studies with tritium-labeled hexaestrol in advanced breast cancer. Lancet 2:796-798, 1961. Hahnet R, Twaddle I:: Estrogen receptors in human breast cancer. 1. Methodology and characterization of receptors. Steroids 18:653-680, 1971. Hahnet R , Twaddle E, Vivian AB: Estrogen receptors in human breast cancer. 2. In vitro binding of estradiol by benign and malignant tumors. Steroids 18:681-708, 1971. Jensen EV, Block GI:, Smith S, Kyser K, De'Sombre E: Estrogen receptors and breast cancer response to adrenaleclomy. Nat Cancer Inst Monogr 34:55-70, 1971. Johansson H, Terenius L, Thoren 1.: T h e binding of estradiol 17 beta to human breast cancers and other tissues in vitro. Cancer Res 30:692-698, 1970. Mason EE, Phifer R F , Spicer SS, Swallow R A , Dreskin RS: An immunoglobulin-enzyme bridge method for localizing tissue antigens. J Histochem Cytochem 1 7 5 6 3 - - 5 6 9 , 1969. McGuire WL: Current status of estrogen receptors in human breast cancer. Cancer 36:638-644, 1975. Mobbs BG: Lstradiol uptake by induced rat mammary tumors and its implications for the treatment of breast cancer. Nat Cancer lnst Monogr 34:33-37, 1971. Scheike 0, Svenstrup B, k'randsen VA: Male breast carcinoma. 11. Metabolism of estradiol-17p in men with breast cancer. J Steroid Biochem 4:489-501, 1973. Sternberger LA, Hardy PH, Cuculis J J , Meyer HG: The unlabeled antibody enzyme method of immunohistochemistry . Preparation and properties of soluble antigen-antibody complex (horscradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315-333, 1970.
lmmunocytological Localization of Estrogen in Human Mammary Carcinoma Cells by Horseradish-Anti-Horseradish Peroxidase Complex .......................................................................................... .......................................................................................... LUNA GHOSH, M.D., BIMAL C. GHOSH, M.D., and TAPAS K. DAS GUPTA, M.D., Ph.D. Tissues from 2 5 female and 2 male patients of mammary carcinoma were examined by the horseradish-anti-horseradish peroxidase complex to localize the presence of estrogen. Carcinoma cells of I 0 female and 2 male patients showed positive staining by this technique. The nucleus and the cytoplasmic membrane showed the reaction. This method is useful in identifying the exact location of the hormones or hormone-like substances in the malignant cells and, perhaps, will be of great help in selecting the patients for management.
.......................................................................................... .......................................................................................... Key words: immunocytological, horseradish, anti-horseradish peroxidase, estrogen, mammary carcinoma
INTRODUCTION Although a relationship between estrogen and human mammary carcinoma is known (Block et al., 1975; Dao, 1972; Jensen et al., 1971), the exact mechanism and the site of the influence in the malignant cells still remain matters of some dispute. The mammary carcinomas are responsive to different hormones of which estrogen exerts the maximum effects (Braunsberg et al., 1971; Hahnet et al., 1971; Jensen et al., 1971; Johansson et al., 1970;McGuire, 1975;Mobbs, 1971). In the past much had been investigated about the hormonal receptor sites by autoradiographic technique (Block et al., 1975; Folca et al., 1961 ;Hahnet e t al., 1971). To the best of our knowledge, no study has been made for cytological localization of hormones within the mammary carcinoma cells. Our objective was to localize estrogen within the human mammary carcinoma cells by using the unlabeled antibody enzyme technique with horseradish--anti-horseradish peroxidase complex (Mason et al., 1969; Sternberger et al., 1970).
From the Division of Surgical Oncology, Department of Surgery, and Department of Pathology, University of Illinois at the Abraham Lincoln School of Medicine and Cook County Hospital, Chicago, Illinois. Address reprint requests to Dr. Bimal C. Ghosh, 1825 West Harrison Street, Division of Surgical Oncology, Chicago, IL 60612.
0022-4790/78/1003-0221$01.10@ 1 9 7 8 Alan R. Liss, I n c
222
Ghosh, Ghosh, a n d Das G u p t a
MATERIALS AND METHODS Tissues obtained from 25 female, ages from 40 to 55 years, and 2 male patients, ages 65 and 68 years, of infiltrating duct carcinoma were fixed in 0.2% calcium acetate formalin, pH 7.4, for 4 hr, then dehydrated through graded alcohol, embedded in paraffin, section at 3 p thickness, and mounted on glass slides. Normal female breast tissue from 10 patients with benign breast disease was similarly treated, as a control. For estrogen localization, sections were immersed in phosphate-buffered saline (PBS), pH 7.2, for 30 min before treatment with antisera. Two drops of sheep antiestrddiol serum in different dilutions (1 :lo, 150, 1 :100) were added to each section and the sections were kept in a moist chamber at 37°C for 1 hr. The excess serum was then drained and the sections were washed for 30 min in three changes of PBS. Rabbit antisheep gamma globulin was then added to each section. The sections were kept at 37°C in a moist chamber for 1 hr, then washed in PBS for 30 min with three changes. Sheep peroxidase-anti-peroxidase (PAP) complex was added t o the sections for 1 hr at 37°C. The sections were washed again in PBS for 30 min, then incubated in the substrate solution for 5 min at room temperature. The substrate solution was composed of 30 mg% of 3’3’-diaminobenzidine tetrahydrochloride in Tris-buffered PBS. The buffer solution contained 600 mg% of tris(hydroxymethy1)aminoniethane in PBS adjusted to pH 7.6. The substrate solution was freshly prepared and a few drops of 3% H2 O2 were added just before use. Following enzymatic reaction the sections were washed for 15 min in 2% O s 0 4 . The sections were again rinsed in distilled water, then dehydrated and mounted in Permount.
Fig. 1. Male mammary carcinoma cells demonstrating positive reaction for estrogen by immunoperoxidase staining. x 300.
Estrogen i n Human Mammary Carcinoma
223
Fig. 2. Female mammary carcinoma cells for estrogen. T h e staining is also prominent in t h e nucleus and cytoplasmic membrane. Immunoperoxidase stain. x 300.
RESULTS The cytoplasm of the adenocarcinoma cells was stained when diluted sheep antiestradiol serum was used. Positive staining of tumor cells occurred with all the dilutions of antiserum. The reactive cells had a finely granular dark brown appearance. The nuclear membrane and the nucleus showed positive staining. The specificity of the bridge technique was verified by using other organs. Sections from muscle and thyroid failed to show any staining. In a second set, the sections were stained either by normal rabbit serum or by normal sheep serum instead of rabbit antigrowth hormone or sheep anti-estradiol sera. Both failed to show any demonstrable staining. An additional control experiment was done by substituting normal sheep serum for sheep anti-rabbit serum, and some sections were incubated in diaminobenzidine peroxidase only; neither had a reaction. Both male patients and 10 female patients (40%) showed positive reactions and proved, to have estrogen in the carcinoma cells by cytochemical localization.
COMMENT The influence of hormones on the growth and spread of mammary carcinoma is known; however, the exact mechanism and the site of influence in the malignant cells are controversial. With the use of tissue sections (Block et al., 1975; Jensen et al., 1971) and tissue homogenate autoradiography (Folca et al., 1961;Hahnet et al., 1971), the presence
224
Ghosh, Ghosh, and Das Gupta
of receptor sites of hormones has been determined. The problem in autoradiographic determination is that with this method it is not possible to ascertain which cellular component of a given tumor is either influenced by or is the site of storage of the hormone. Therefore, it is logical to attempt to use a technique that might selectively localize the site of activity of estrogen within the malignant cells. Although localization of estrogen in the tumor cells is a step toward understanding its role in mammary carcinoma, it raises the inevitable question of whether these are true receptor sites or whether some of these malignant cells independently secrete estrogenic hormones or hormone-like substances. This technique is useful in determining the exact location of the hormones and perhaps will be a great help in clinical practice for selecting patients for different modalities of treatment.
REFERENCES Block GE, Jensen LV, Polley TZ Jr: T h e prediction of hornional dependency of mammary cancer. Ann Surg 182:342-352, 1975. Braunsberg H, Carter AE, lrvine WT, James I:, James VHT: Estrogens in breast tumors. Nat Cancer Inst Monogr 34:71-75,1971. Dao T L : Ablation therapy for hormone dependent tumors. Annu Rev Med 23:l-18, 1972. Folca PJ, Glascock R F , Irvine WT: Studies with tritium-labeled hexaestrol in advanced breast cancer. Lancet 2:796-798, 1961. Hahnet R, Twaddle I:: Estrogen receptors in human breast cancer. 1. Methodology and characterization of receptors. Steroids 18:653-680, 1971. Hahnet R , Twaddle E, Vivian AB: Estrogen receptors in human breast cancer. 2. In vitro binding of estradiol by benign and malignant tumors. Steroids 18:681-708, 1971. Jensen EV, Block GI:, Smith S, Kyser K, De'Sombre E: Estrogen receptors and breast cancer response to adrenaleclomy. Nat Cancer Inst Monogr 34:55-70, 1971. Johansson H, Terenius L, Thoren 1.: T h e binding of estradiol 17 beta to human breast cancers and other tissues in vitro. Cancer Res 30:692-698, 1970. Mason EE, Phifer R F , Spicer SS, Swallow R A , Dreskin RS: An immunoglobulin-enzyme bridge method for localizing tissue antigens. J Histochem Cytochem 1 7 5 6 3 - - 5 6 9 , 1969. McGuire WL: Current status of estrogen receptors in human breast cancer. Cancer 36:638-644, 1975. Mobbs BG: Lstradiol uptake by induced rat mammary tumors and its implications for the treatment of breast cancer. Nat Cancer lnst Monogr 34:33-37, 1971. Scheike 0, Svenstrup B, k'randsen VA: Male breast carcinoma. 11. Metabolism of estradiol-17p in men with breast cancer. J Steroid Biochem 4:489-501, 1973. Sternberger LA, Hardy PH, Cuculis J J , Meyer HG: The unlabeled antibody enzyme method of immunohistochemistry . Preparation and properties of soluble antigen-antibody complex (horscradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315-333, 1970.
