JOURNAL OF PATHOLOGY, VOL.
162: 223-230 ( 1 990)
IN SITU DISTRIBUTION OF TRANSFORMING GROWTH FACTOR a IN NORMAL HUMAN TISSUES A N D IN MALIGNANT TUMOURS OF THE OVARY FRIEDRICH KOMMOSS, HANNS OLOF WINTZER*, SABINE VON KLEIST*, MANUELA KOHLER?, ROGER WALKER:, BEATRICE LANGTON:, KIM VAN TRANS, ALBRECHT PFLEIDERER AND THOMAS BAUKNECHT
Universirars-Frauenklinik,Albert-Ludwigs- Universitat Freiburg, Hugstetterstr. 55,D-7800 Freiburg. F. R.G.: */nstitut .fur Immunbiologie, Albert-Ludwigs- Universitat Freiburg, Stefan-Meier-Str. 8. D- 7800 Freiburg, F. R.G.; tlnstitut,fur Biologie II, Albert-Ludwigs-Universitat Freiburg, Schanzlestr. I , 0-7800 Freiburg, F.R.G.; $Triton Biosciences, Inc., Alumedu, California, U.S.A. Received 30 March 1990 Accepted4 June 1990
SUMMARY The distribution of transforming growth factor a (TGF-a) in human normal tissues from the uterus, Fallopian tube, ovary, small and large intestine, lung, spleen, kidney, and skin was studied by immunohistochemistry. TGF-a was found in epidermis, bronchial epithelium, intestinal mucosa, renal tubules, endo- as well as in exocervical and endometrial epithelium, and in the serous epithelium of the Fallopian tube. N o TGF-a was detected in the stromal components of any of the tissues nor in any of the pre- and post-menopausal ovaries studied. Twenty-nine ovarian tumours including 23 ovarian carcinomas, one malignant mixed Muellerian tumour, two ovarian metastases of gastrointestinal carcinomas, one dysgerminoma, one sarcoma, and one fibroma were studied for TGF-a by the same immunohistochemical method. In 25 cases, specific cytoplasmic staining for TGF-a of epithelial tumour cells could be demonstrated. The pattern and intensity of the TGF-a immunostain varied among the TGF-a-positive tumours. N o TGF-a was found by immunohistochemistry in the remaining four cases nor in the stromal tumour components of any of the lesions studied. Northern blot analysis for TGF-a mRNA was performed on 12 of the tumours. While the immunohistochemistry and blotting results correlated well in ten cases, discordant results were obtained in two lesions. KEY
WORDS-Growth factors, TGF-a. normal human tissues, ovarian carcinoma. immunohistochemistry, RNA analysis.
INTRODUCTION
GFs, transforming growth factor a (TGF-a) and vaccinia virus growth factor (VVGF), which possess Cell proliferation and differentiation are regulated similar biological properties, bind to EGF-R.9.’o by growth factors (GFs) and G F receptors (GF-Rs) The discovery of TGF-a, a 50 amino acid singlewhich are known to be proto-oncogene products.’ chain polypeptide synthesized as part of a larger Oncogene activation or an inappropriate expres- 160 amino acid precursor,” which was originally sion of GFs, which is often found in malignant detected in the culture media of several retrovirally tumours,24 may influence the biological and clinical transformed fibroblasts” as well as in human phenotype of a t ~ m o u r . ~ -The ’ epidermal growth tumours,”-” supported the concept that growth factor (EGF) is one of the best characterized growth control may be lost through autocrine stimulation.’‘ factors and interacts with specific cell membrane- The biological actions of TGF-a are mediated bound receptors (EGF-Rs).*At least two additional through reaction with the cell membrane-bound epidermal growth factor receptor (EGF-R)“ which Addressee for correspondence: F. Kommoss, Universitats- increases its tyrosine kinase activity and triggers the Frauenklinik, Albert-Ludwigs-Universitat Freiburg, Hugstetterstr. mitogenic signal transduction.’’ TGF-a was also found in embryonic tissues2G23 55. D-7800 Freiburg, F.R.G. and several normal 0022-341 7/90/11022348 $05.00 0 1990 by John Wiley & Sons, Ltd.
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tissues.'"'60nly limited information has been available on the in situ distribution of TGF-a in normal and tumour tissues. We have described the occurrence of EGF-R and EGF-like factors (EGF-Fs). which are currently believed to consist of TGF-a and EGF. in gynaecological carcinoma^.',^','^ Both EGF-Rs and EGF-Fs seem to influence biological behaviour and prognosis in such tumours. In view of the suspected tumourigenic potential of TGF-u,'~ we studied a series of malignant ovarian tumours and a variety of normal human tissues using a monoclonal antibody against TGF-a in order to determine its cellular distribution, and compared the results with those of TGF-a mRNA analysis. MATERIALS AND METHODS Normal tissues The tissues studied were from surgical specimens obtained during surgery at the University of Freiburg. Material was taken from macroscopically normal areas, and sections stained with haematoxylin and eosin were cut in each case. Only normal tissues without evidence of inflammation or infiltration were included in this study. Tumour material The tumour tissues studied were obtained during operations on patients with ovarian tumours who were seen for surgical and further treatment at the Department of Obstetrics and Gynecology, University of Freiburg. Of the 29 tumours studied, there were 23 adenocarcinomas (15 serous papillary, 4 endometrioid, 1 clear cell, 1 mucinous tumour, 9 solid undifferentiated carcinomas), I malignant mixed mesodermal tumour, 1 dysgerminoma, I sarcoma, 2 metastatic carcinomas ( I with a gastric, 1 with a colonic primary), and 1 benign fibroma of he ovary. TAh 8 monoclonal antibod!, Preparation-Balblc mice were immunized P. with E. coli recombinant TGF-a conjugated to KLH (20 pg of peptide) emulsified 1: 1 in comp :te Freund's adjuvan; and boosted every 2 weeks with the conjugated peptide emulsified in incomplete adjuvant until a positive RIA titre to TGF-a was observed. A final i.v. boost was given and 4 days later spleen cells were fused with P3-X63Ag.653 myeloma cells at a ratio of 2 . 5 : l with PEG 4000 as
previously described.30 Hybridoma supernatants were tested for positive reactivity in an RIA assay. Stable subclones were obtained by two-fold limiting dilution. and secreting clones were injected into pristine primed Balblc mice for ascites production. Ascites were purified by HPLC on a Bakerbond ABx column (Baker, Philippsburg, NJ, U.S.A.) and then purified monoclonal antibody (designated TAb 8) was dialysed against PBS and stored at - 20°C. Reacririty-TAb 8 was determined to be of the IgG2b sub-isotype by radial immunodiffusion and greater than 90 per cent pure by SDS-PAGE followed by gel scan densitometry. TAb 8 binds to refolded E. coli recombinant TGF-a with an RIA titre of approximately 40 ng/ml (at 30 per cent of maximal binding). but fails to bind to the individual synthesized and refolded loops of TGF-a or to a carboxyamidomethylated linear TGF-a molecule. Using Geysen technology, no linear epitope in a series of 4-mers, 6-mers, or 8-mers encompassing the whole molecule, has been identified." No crossreactivity with EGF has been observed, and TAb 8 competes for the binding of TGF-a to the EGF-R of A431 cells in a radioreceptor assay with 5 per cent residual TGF-a activity at 10 ngjml peptide per 10 ,ug/ml antibody. Irnrnunohis tochernist rj.
Samples of the tumours studied were snap-frozen in liquid nitrogen and stored at -70°C. Cryostat sections were cut at 6 pm, air-dried at room temperature for 2 h, and fixed in acetone for 15 min at room temperature. Sections were stored at - 20°C until staining, which was performed within 14 days. Sections were then allowed to reach room temperature, thereby preventing condensation of cooled air on the slides. After another fixation in acetone for 10 rnin at room temperature, sections were rehydrated briefly in 0 . 0 0 4 ~PBS (pH 7.4), overlaid with the appropriately (1:40) diluted TAb 8, and incubated at 37°C for 30 rnin in a humidified chamber. Next, incubation with a secondary biotinylated horse anti-mouse IgG antibody (Vector LaboratoriesAtlanta, Heidelberg, F.R.G.) was performed for 30 rnin at room temperature. Finally, sections were incubated with ABC (Vector Laboratories) for 45 rnin at room temperature. ABC was obtained by mixing avidin and biotinylated peroxidase 30 min before use. Sections were washed three times in PBS following each incubation step. Peroxidase was
TGF-n IN NORMAL TISSUES AND IN OVARIAN TUMOURS
visualized by incubation with DAB chromogen (3,3-diaminobenzidine-tetrahydrochloride,0.05 per cent in PBS) in 0.03per cent hydrogen peroxide for 10 min. DAB staining was intensified by incubation for 5 min in 0.5 per cent CuSO, in PBS. After washing in water, sections were counterstained lightly with Mayer’s Haemalaun, dehydrated through alcohol, cleared with xylene, and mounted with Vitro-Clud (Langenbrinck, Emmendingen, F.R.G.). Negative controls were performed for each specimen using PBS instead of primary antibody. Sections of normal human skin known to contain TGF-a3’ served as a positive control. Semi-quantitative scores for the specific staining were created by multiplying the staining intensity (0-3) by the percentage of positive epithelial cells (0-33per cent: factor 1; 34-66 per cent: factor 2; and 67-100 per cent: factor 3), thus obtaining scores ranging from 0 to 9. In TGF-a-positive tumours, the type of staining distribution was assessed as described by Kommoss et type A tumours showing uniform intense staining of all epithelial tumour cells, while type B carcinomas had both clearly negative and distinctly positive cells. Finally, type C tumours showed a wide spectrum of staining intensity from negative to intensely positive. R N A analysis
Total cellular RNA was isolated from frozen tissues by the guanidium-isothiocyanate-caesium chloride m e t h ~ d . ~ RNA ~ , ~ ’ (10 pg per lane) was electrophoresed in a 1 per cent agarose 2.2 M formaldehyde stained in ethidium bromide in order to control the amount of RNA loaded onto the gel, and transferred to nylon membranes (Zeta Probe, BIO RAD, Miinchen, F.R.G.) according to the manufacturer’s instructions. Membranes were then hybridized for 24 h at 42°C in the presence of 50 per cent formamide/lO per cent dextran sulphate/l M NaCI/l per cent NaDodSO,. An EcoRI (Bohringer Mannheim, F.R.G.) 1.4 kb fragment of human TGF-a cDNA, obtained from R. Walker (Triton Biosciences, Inc., Alameda CA, U.S.A.) was labelled by random primer extension3’ to a specific activity of 5 x 1O8dpm/ng. After hybridization, membranes were washed in 2 x SSCjO.5 per cent NaDodSO, for 30 min at room temperature and twice in 0.2 x SSCjO.1 per cent NaDodSO, for 30 min at 55°C. Membranes were then exposed to XAR-5 X-ray film (Kodak) using an intensifying screen for 10 days at -70°C. The degree of expression of the TGF-a in the tissues
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studied was then assessed semi-quantitatively using scores from 0 for TGF-a-negative tumours to 4 for strongly TGF-a-positive cases. RESULTS Immunocytochemical staining for TGF-a was performed using tissues from ten hysterectomy and salpingo-oophorectomy specimens, five intestinal biopsies, one lung biopsy, two spleen biopsies, five nephrectomy specimens, and several skin biopsies. TGF-a was found in the squamous epithelium of the uterine exocervix as well as in both endocervical epithelium and endocervical glands. TGF-a specific staining was also detected in both proliferative and secretory endometrial glands and in the serous epithelium of the Fallopian tube (Fig. 1). No such staining was found in cervical or endometrial stroma, nor in the myometrium of any of the uteri examined. Likewise, no TGF-a was present in the muscular layers of the Fallopian tube. No TGF-a could be detected in any of the five pre- or five postmenopausal ovaries studied. In all three colonic biopsies (Fig. 2) as well as in the specimen from the small bowel, the intestinal epithelium showed strong specific staining for TGF-a. Bronchial epithelium could be shown to be strongly TGF-a positive, whereas the biopsies from the spleen were TGF-a negative. In the five renal biopsies studied, TGF-a was consistently found in the epithelium of renal tubules, whereas glomeruli were TGF-a negative in all cases. Finally, specific staining was found in the parabasal layers of the keratinizing squamous epithelium of all of the skin biopsies studied. Immunocytochemical staining for TGF-a was performed using tissues from all 29 ovarian tumours (Table I). For technical reasons, TGF-a mRNA analysis could be performed only in 12 cases (Table 11). In all cases, specific staining for TGF-a was limited to the epithelial tumour components. Among the TGF-a-positive epithelial tumour cells, specific staining was exclusively found in the cytoplasm (Fig. 3). Within the TGF-a-positive cytoplasm, specific staining was diffusely distributed in a majority of cases (Figs 3 and 4) whereas in a few cases, reaction product deposits were concentrated along the cytoplasmic membrane (Fig. 6). Thirteen out of the 14serous papillary carcinomas were found to be TGF-a positive (Figs 3 and 4),as were three out of four endometrioid carcinomas
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F. KOMMOSS ETAL.
Fig. I-TGF-a specific staining in the serous epithelium of the Fallopian tube
Fig. 2 4 l a n d u l a r epithelium in colonic mucosa is TGF-a positive
Fig. 3-Moderately well differentiated serous papillary adenocarcinoma of the ovary (ID No. 513). TGF-a immunohistochemistry is strongly positive with a score of 9 and type-A staining pattern. TGF-a mRNA analysis is strongly positive (score of4, Fig. 7a)
Fig. &Poorly differentiated serous papillary adenocarcinoma ofthe ovary (ID No. 483). TGF-a immunohistochemistry is positive with a score of 6 and type-C staining pattern. TGF-a mRNA analysis is positive (score of 2, Fig. 7b)
Fig. >-Sarcoma of the ovary (ID No. 123). Both TGF-a immunohistochemistry and TGF-a mRNA analysis (Fig. 7c) are negative
Fig. &Positive TGF-a immunostain in a poorly differentiated endometrioid adenocarcinoma of the ovary (ID No. 301). Staining is concentrated along cytoplasmic borders of epithelial tumour cells
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TGF-a IN NORMAL TISSUES AND IN OVARIAN TUMOURS
Table I-Histological diagnosis and results of TGF-a immunohistochemistryin 29 tumours of the ovary
ID No. 583 277 505 513 564 566 I57 159 209 310 363 483 484 488 506 207 301 329 445 292 359 378 387 480 455 123 258 286 360
Immunohistochemistry Score Type*
Histological diagnosis Ca, papillary G 1 Ca, papillary G2 Ca, papillary G2 Ca, papillary G2 Ca, papillary G2 Ca, papillary G2 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, endometnoid G3 Ca, endometrioid G3 Ca, endometrioid G3 Ca, endometrioid G3 Ca, clear cell G2 Ca, mucinous G1 Ca, solid G3 Ca, solid G3 Mixed mesodermal tumour Dysgerminoma Sarcoma Metastatic gastric Ca Metastatic colonic Ca Fibroma
6 3 6 9 6 3 0 2 I 6 6 6 3 6 1
0 6 4 3 6 6 6 1
3 4 0 1 .-
6 0
A A A A A A -
C C
ct B
C C C A
-
Bt
ct C A A C A A C
-
C A -
'As described in Materials and Methods. tSpecific staining along cytoplasmic membranes.
(Fig. 6 ) , both undifferentiated carcinomas, the malignant mixed mesodermal tumour, and both metastatic carcinomas. In the dysgerminoma, the malignant germ cells were positive, but no staining was found in the tumour stroma which was densely infiltrated by lymphocytes. Among the TGF-a-positive tumours, the intensity of the TGF-a immunostain varied from weak to very strong, and all three staining patterns described (A, B, and C) were present (see Materials and Methods, and Table I) Both the ovarian sarcoma (Fig. 5) and the fibroma were TGF-a negative. Among the 12 tumours on which TGF-a mRNA analysis was performed, eight were found to contain
varying amounts of TGF-a mRNA (Table 11, and Fig. 7). Seven of the latter tumours were confirmed to be TGF-a positive by immunohistochemistry, and the respective semi-quantitative scores correlated well (Table 11). One poorly differentiated endometrioid carcinoma which had been shown to contain moderate amounts of TGF-a mRNA was TGF-a negative by immunohistochemistry. In the remaining four cases, no specific RNA transcripts for TGF-a could be detected. The negative results of TGF-a Northern blot analysis were confirmed by immunohistochemistry in three of these cases (papillary carcinoma, sarcoma, and fibroma); however, in the remaining papillary
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F. KOMMOSS ETAL.
Table 11-Comparison of TGF-a immunostaining scores and banding intensities of TGF-a Northern blots in 12 ovarian tumours
ID No. 513 483 292 286 378 217 207 359 I23 157 360 30 1
TGF-a immunostaining scores (0-9) 9 6 6 6 6 3 0 6 0 0 0 6
TGF-a mRNA Northern blot banding intensities (0-4)
4 2 2 2 2 2 2 1
0 0
0 0
28s I) TGF a I )
c Fig. 7-TGF-a
mRNA analysis of the cases shown in Figs 3-5
carcinoma, which was TGF-a negative by mRNA analysis, immunohistochemistry revealed focal strong TGF-a specific staining. DISCUSSION In this study we used monoclonal antibody and an indirect immunoperoxidase technique to localize TGF-a in different normal human adult tissues.
TGF-a immunoreactivity was present exclusively in epithelial cells of the tissues studied. Both stromal cells and inflammatory cells were completely TGF-a negative. Coffey et al. described TGF-a expression in the epidermis using in situ hybridization and immunohistochemistry.25 Our results indicate a similar distribution of TGF-a in the normal skin. EGF-R production in normal squamous epithelium of the exocervix has been demonstrated by us3*to be located in the basal cells. EGF and TGF-a have been found to induce proliferation of epidermal k e r a t i n o c y t e ~contributing , ~ ~ ~ ~ ~ to the normal proliferation of the skin. Parabasal TGF-a-producing cells may thus regulate proliferation of the EGF-Rexpressing basal cells. EGF-Rs are also synthesized by epithelial cells in the bowel,41 kidney, lung, endometrium, and Fallopian tube.42Since we found TGF-a in all of the latter tissues, it may be concluded that TGF-a regulates the proliferation of many different tissues in an auto- or paracrine way. In contrast to the results of Kudlow et who reported immunoreactive TGF-a to be present in the theca cells of rat ovaries, we were unable to detect TGF-a specific staining in pre- o r postmenopausal human ovaries. This discrepancy remains unclear, but may be solved by further experiments in which we will study TGF-a mRNA expression in human ovaries. We also investigated TGF-a expression in 29 ovarian tumours by immunohistochemistry and Northern blot mRNA analysis. Using immunocytochemical methods, we found TGF-a in the epithelial components of 87 per cent of the ovarian carcinomas, one malignant mixed mesodermal tumour, one dysgerminoma, and of two ovarian metastases of gastrointestinal carcinomas. TGF-a-specific immunostain was diffusely distributed in the cytoplasm of tumour cells in most cases but was concentrated along the cytoplasmic membrane in three cases. No TGF-a was present in two ovarian carcinomas, nor in two mesenchymal tumours of the ovary. This supports the results of Coffey et and indicates, together with our own findings in normal human tissues, that TGF-a is expressed only in epithelial tissues. Generally semi-quantitative analysis by immunohistochemistry of the TGF-a tissue content and TGF-a mRNA analysis showed comparable results. However, in a G3 endometrioid carcinoma which was TGF-a positive by immunohistochemistry, no TGF-a mRNA was found by Northern blot analysis. Degradation of the RNA preparation of this tumour was highly unlikely, since RNA specific for c-myc and EGF-R was found
TGF-a IN NORMAL TISSUES A N D I N OVARIAN TUMOURS
229
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IN SITU DISTRIBUTION OF TRANSFORMING GROWTH FACTOR a IN NORMAL HUMAN TISSUES A N D IN MALIGNANT TUMOURS OF THE OVARY FRIEDRICH KOMMOSS, HANNS OLOF WINTZER*, SABINE VON KLEIST*, MANUELA KOHLER?, ROGER WALKER:, BEATRICE LANGTON:, KIM VAN TRANS, ALBRECHT PFLEIDERER AND THOMAS BAUKNECHT
Universirars-Frauenklinik,Albert-Ludwigs- Universitat Freiburg, Hugstetterstr. 55,D-7800 Freiburg. F. R.G.: */nstitut .fur Immunbiologie, Albert-Ludwigs- Universitat Freiburg, Stefan-Meier-Str. 8. D- 7800 Freiburg, F. R.G.; tlnstitut,fur Biologie II, Albert-Ludwigs-Universitat Freiburg, Schanzlestr. I , 0-7800 Freiburg, F.R.G.; $Triton Biosciences, Inc., Alumedu, California, U.S.A. Received 30 March 1990 Accepted4 June 1990
SUMMARY The distribution of transforming growth factor a (TGF-a) in human normal tissues from the uterus, Fallopian tube, ovary, small and large intestine, lung, spleen, kidney, and skin was studied by immunohistochemistry. TGF-a was found in epidermis, bronchial epithelium, intestinal mucosa, renal tubules, endo- as well as in exocervical and endometrial epithelium, and in the serous epithelium of the Fallopian tube. N o TGF-a was detected in the stromal components of any of the tissues nor in any of the pre- and post-menopausal ovaries studied. Twenty-nine ovarian tumours including 23 ovarian carcinomas, one malignant mixed Muellerian tumour, two ovarian metastases of gastrointestinal carcinomas, one dysgerminoma, one sarcoma, and one fibroma were studied for TGF-a by the same immunohistochemical method. In 25 cases, specific cytoplasmic staining for TGF-a of epithelial tumour cells could be demonstrated. The pattern and intensity of the TGF-a immunostain varied among the TGF-a-positive tumours. N o TGF-a was found by immunohistochemistry in the remaining four cases nor in the stromal tumour components of any of the lesions studied. Northern blot analysis for TGF-a mRNA was performed on 12 of the tumours. While the immunohistochemistry and blotting results correlated well in ten cases, discordant results were obtained in two lesions. KEY
WORDS-Growth factors, TGF-a. normal human tissues, ovarian carcinoma. immunohistochemistry, RNA analysis.
INTRODUCTION
GFs, transforming growth factor a (TGF-a) and vaccinia virus growth factor (VVGF), which possess Cell proliferation and differentiation are regulated similar biological properties, bind to EGF-R.9.’o by growth factors (GFs) and G F receptors (GF-Rs) The discovery of TGF-a, a 50 amino acid singlewhich are known to be proto-oncogene products.’ chain polypeptide synthesized as part of a larger Oncogene activation or an inappropriate expres- 160 amino acid precursor,” which was originally sion of GFs, which is often found in malignant detected in the culture media of several retrovirally tumours,24 may influence the biological and clinical transformed fibroblasts” as well as in human phenotype of a t ~ m o u r . ~ -The ’ epidermal growth tumours,”-” supported the concept that growth factor (EGF) is one of the best characterized growth control may be lost through autocrine stimulation.’‘ factors and interacts with specific cell membrane- The biological actions of TGF-a are mediated bound receptors (EGF-Rs).*At least two additional through reaction with the cell membrane-bound epidermal growth factor receptor (EGF-R)“ which Addressee for correspondence: F. Kommoss, Universitats- increases its tyrosine kinase activity and triggers the Frauenklinik, Albert-Ludwigs-Universitat Freiburg, Hugstetterstr. mitogenic signal transduction.’’ TGF-a was also found in embryonic tissues2G23 55. D-7800 Freiburg, F.R.G. and several normal 0022-341 7/90/11022348 $05.00 0 1990 by John Wiley & Sons, Ltd.
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tissues.'"'60nly limited information has been available on the in situ distribution of TGF-a in normal and tumour tissues. We have described the occurrence of EGF-R and EGF-like factors (EGF-Fs). which are currently believed to consist of TGF-a and EGF. in gynaecological carcinoma^.',^','^ Both EGF-Rs and EGF-Fs seem to influence biological behaviour and prognosis in such tumours. In view of the suspected tumourigenic potential of TGF-u,'~ we studied a series of malignant ovarian tumours and a variety of normal human tissues using a monoclonal antibody against TGF-a in order to determine its cellular distribution, and compared the results with those of TGF-a mRNA analysis. MATERIALS AND METHODS Normal tissues The tissues studied were from surgical specimens obtained during surgery at the University of Freiburg. Material was taken from macroscopically normal areas, and sections stained with haematoxylin and eosin were cut in each case. Only normal tissues without evidence of inflammation or infiltration were included in this study. Tumour material The tumour tissues studied were obtained during operations on patients with ovarian tumours who were seen for surgical and further treatment at the Department of Obstetrics and Gynecology, University of Freiburg. Of the 29 tumours studied, there were 23 adenocarcinomas (15 serous papillary, 4 endometrioid, 1 clear cell, 1 mucinous tumour, 9 solid undifferentiated carcinomas), I malignant mixed mesodermal tumour, 1 dysgerminoma, I sarcoma, 2 metastatic carcinomas ( I with a gastric, 1 with a colonic primary), and 1 benign fibroma of he ovary. TAh 8 monoclonal antibod!, Preparation-Balblc mice were immunized P. with E. coli recombinant TGF-a conjugated to KLH (20 pg of peptide) emulsified 1: 1 in comp :te Freund's adjuvan; and boosted every 2 weeks with the conjugated peptide emulsified in incomplete adjuvant until a positive RIA titre to TGF-a was observed. A final i.v. boost was given and 4 days later spleen cells were fused with P3-X63Ag.653 myeloma cells at a ratio of 2 . 5 : l with PEG 4000 as
previously described.30 Hybridoma supernatants were tested for positive reactivity in an RIA assay. Stable subclones were obtained by two-fold limiting dilution. and secreting clones were injected into pristine primed Balblc mice for ascites production. Ascites were purified by HPLC on a Bakerbond ABx column (Baker, Philippsburg, NJ, U.S.A.) and then purified monoclonal antibody (designated TAb 8) was dialysed against PBS and stored at - 20°C. Reacririty-TAb 8 was determined to be of the IgG2b sub-isotype by radial immunodiffusion and greater than 90 per cent pure by SDS-PAGE followed by gel scan densitometry. TAb 8 binds to refolded E. coli recombinant TGF-a with an RIA titre of approximately 40 ng/ml (at 30 per cent of maximal binding). but fails to bind to the individual synthesized and refolded loops of TGF-a or to a carboxyamidomethylated linear TGF-a molecule. Using Geysen technology, no linear epitope in a series of 4-mers, 6-mers, or 8-mers encompassing the whole molecule, has been identified." No crossreactivity with EGF has been observed, and TAb 8 competes for the binding of TGF-a to the EGF-R of A431 cells in a radioreceptor assay with 5 per cent residual TGF-a activity at 10 ngjml peptide per 10 ,ug/ml antibody. Irnrnunohis tochernist rj.
Samples of the tumours studied were snap-frozen in liquid nitrogen and stored at -70°C. Cryostat sections were cut at 6 pm, air-dried at room temperature for 2 h, and fixed in acetone for 15 min at room temperature. Sections were stored at - 20°C until staining, which was performed within 14 days. Sections were then allowed to reach room temperature, thereby preventing condensation of cooled air on the slides. After another fixation in acetone for 10 rnin at room temperature, sections were rehydrated briefly in 0 . 0 0 4 ~PBS (pH 7.4), overlaid with the appropriately (1:40) diluted TAb 8, and incubated at 37°C for 30 rnin in a humidified chamber. Next, incubation with a secondary biotinylated horse anti-mouse IgG antibody (Vector LaboratoriesAtlanta, Heidelberg, F.R.G.) was performed for 30 rnin at room temperature. Finally, sections were incubated with ABC (Vector Laboratories) for 45 rnin at room temperature. ABC was obtained by mixing avidin and biotinylated peroxidase 30 min before use. Sections were washed three times in PBS following each incubation step. Peroxidase was
TGF-n IN NORMAL TISSUES AND IN OVARIAN TUMOURS
visualized by incubation with DAB chromogen (3,3-diaminobenzidine-tetrahydrochloride,0.05 per cent in PBS) in 0.03per cent hydrogen peroxide for 10 min. DAB staining was intensified by incubation for 5 min in 0.5 per cent CuSO, in PBS. After washing in water, sections were counterstained lightly with Mayer’s Haemalaun, dehydrated through alcohol, cleared with xylene, and mounted with Vitro-Clud (Langenbrinck, Emmendingen, F.R.G.). Negative controls were performed for each specimen using PBS instead of primary antibody. Sections of normal human skin known to contain TGF-a3’ served as a positive control. Semi-quantitative scores for the specific staining were created by multiplying the staining intensity (0-3) by the percentage of positive epithelial cells (0-33per cent: factor 1; 34-66 per cent: factor 2; and 67-100 per cent: factor 3), thus obtaining scores ranging from 0 to 9. In TGF-a-positive tumours, the type of staining distribution was assessed as described by Kommoss et type A tumours showing uniform intense staining of all epithelial tumour cells, while type B carcinomas had both clearly negative and distinctly positive cells. Finally, type C tumours showed a wide spectrum of staining intensity from negative to intensely positive. R N A analysis
Total cellular RNA was isolated from frozen tissues by the guanidium-isothiocyanate-caesium chloride m e t h ~ d . ~ RNA ~ , ~ ’ (10 pg per lane) was electrophoresed in a 1 per cent agarose 2.2 M formaldehyde stained in ethidium bromide in order to control the amount of RNA loaded onto the gel, and transferred to nylon membranes (Zeta Probe, BIO RAD, Miinchen, F.R.G.) according to the manufacturer’s instructions. Membranes were then hybridized for 24 h at 42°C in the presence of 50 per cent formamide/lO per cent dextran sulphate/l M NaCI/l per cent NaDodSO,. An EcoRI (Bohringer Mannheim, F.R.G.) 1.4 kb fragment of human TGF-a cDNA, obtained from R. Walker (Triton Biosciences, Inc., Alameda CA, U.S.A.) was labelled by random primer extension3’ to a specific activity of 5 x 1O8dpm/ng. After hybridization, membranes were washed in 2 x SSCjO.5 per cent NaDodSO, for 30 min at room temperature and twice in 0.2 x SSCjO.1 per cent NaDodSO, for 30 min at 55°C. Membranes were then exposed to XAR-5 X-ray film (Kodak) using an intensifying screen for 10 days at -70°C. The degree of expression of the TGF-a in the tissues
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studied was then assessed semi-quantitatively using scores from 0 for TGF-a-negative tumours to 4 for strongly TGF-a-positive cases. RESULTS Immunocytochemical staining for TGF-a was performed using tissues from ten hysterectomy and salpingo-oophorectomy specimens, five intestinal biopsies, one lung biopsy, two spleen biopsies, five nephrectomy specimens, and several skin biopsies. TGF-a was found in the squamous epithelium of the uterine exocervix as well as in both endocervical epithelium and endocervical glands. TGF-a specific staining was also detected in both proliferative and secretory endometrial glands and in the serous epithelium of the Fallopian tube (Fig. 1). No such staining was found in cervical or endometrial stroma, nor in the myometrium of any of the uteri examined. Likewise, no TGF-a was present in the muscular layers of the Fallopian tube. No TGF-a could be detected in any of the five pre- or five postmenopausal ovaries studied. In all three colonic biopsies (Fig. 2) as well as in the specimen from the small bowel, the intestinal epithelium showed strong specific staining for TGF-a. Bronchial epithelium could be shown to be strongly TGF-a positive, whereas the biopsies from the spleen were TGF-a negative. In the five renal biopsies studied, TGF-a was consistently found in the epithelium of renal tubules, whereas glomeruli were TGF-a negative in all cases. Finally, specific staining was found in the parabasal layers of the keratinizing squamous epithelium of all of the skin biopsies studied. Immunocytochemical staining for TGF-a was performed using tissues from all 29 ovarian tumours (Table I). For technical reasons, TGF-a mRNA analysis could be performed only in 12 cases (Table 11). In all cases, specific staining for TGF-a was limited to the epithelial tumour components. Among the TGF-a-positive epithelial tumour cells, specific staining was exclusively found in the cytoplasm (Fig. 3). Within the TGF-a-positive cytoplasm, specific staining was diffusely distributed in a majority of cases (Figs 3 and 4) whereas in a few cases, reaction product deposits were concentrated along the cytoplasmic membrane (Fig. 6). Thirteen out of the 14serous papillary carcinomas were found to be TGF-a positive (Figs 3 and 4),as were three out of four endometrioid carcinomas
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Fig. I-TGF-a specific staining in the serous epithelium of the Fallopian tube
Fig. 2 4 l a n d u l a r epithelium in colonic mucosa is TGF-a positive
Fig. 3-Moderately well differentiated serous papillary adenocarcinoma of the ovary (ID No. 513). TGF-a immunohistochemistry is strongly positive with a score of 9 and type-A staining pattern. TGF-a mRNA analysis is strongly positive (score of4, Fig. 7a)
Fig. &Poorly differentiated serous papillary adenocarcinoma ofthe ovary (ID No. 483). TGF-a immunohistochemistry is positive with a score of 6 and type-C staining pattern. TGF-a mRNA analysis is positive (score of 2, Fig. 7b)
Fig. >-Sarcoma of the ovary (ID No. 123). Both TGF-a immunohistochemistry and TGF-a mRNA analysis (Fig. 7c) are negative
Fig. &Positive TGF-a immunostain in a poorly differentiated endometrioid adenocarcinoma of the ovary (ID No. 301). Staining is concentrated along cytoplasmic borders of epithelial tumour cells
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TGF-a IN NORMAL TISSUES AND IN OVARIAN TUMOURS
Table I-Histological diagnosis and results of TGF-a immunohistochemistryin 29 tumours of the ovary
ID No. 583 277 505 513 564 566 I57 159 209 310 363 483 484 488 506 207 301 329 445 292 359 378 387 480 455 123 258 286 360
Immunohistochemistry Score Type*
Histological diagnosis Ca, papillary G 1 Ca, papillary G2 Ca, papillary G2 Ca, papillary G2 Ca, papillary G2 Ca, papillary G2 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, papillary G3 Ca, endometnoid G3 Ca, endometrioid G3 Ca, endometrioid G3 Ca, endometrioid G3 Ca, clear cell G2 Ca, mucinous G1 Ca, solid G3 Ca, solid G3 Mixed mesodermal tumour Dysgerminoma Sarcoma Metastatic gastric Ca Metastatic colonic Ca Fibroma
6 3 6 9 6 3 0 2 I 6 6 6 3 6 1
0 6 4 3 6 6 6 1
3 4 0 1 .-
6 0
A A A A A A -
C C
ct B
C C C A
-
Bt
ct C A A C A A C
-
C A -
'As described in Materials and Methods. tSpecific staining along cytoplasmic membranes.
(Fig. 6 ) , both undifferentiated carcinomas, the malignant mixed mesodermal tumour, and both metastatic carcinomas. In the dysgerminoma, the malignant germ cells were positive, but no staining was found in the tumour stroma which was densely infiltrated by lymphocytes. Among the TGF-a-positive tumours, the intensity of the TGF-a immunostain varied from weak to very strong, and all three staining patterns described (A, B, and C) were present (see Materials and Methods, and Table I) Both the ovarian sarcoma (Fig. 5) and the fibroma were TGF-a negative. Among the 12 tumours on which TGF-a mRNA analysis was performed, eight were found to contain
varying amounts of TGF-a mRNA (Table 11, and Fig. 7). Seven of the latter tumours were confirmed to be TGF-a positive by immunohistochemistry, and the respective semi-quantitative scores correlated well (Table 11). One poorly differentiated endometrioid carcinoma which had been shown to contain moderate amounts of TGF-a mRNA was TGF-a negative by immunohistochemistry. In the remaining four cases, no specific RNA transcripts for TGF-a could be detected. The negative results of TGF-a Northern blot analysis were confirmed by immunohistochemistry in three of these cases (papillary carcinoma, sarcoma, and fibroma); however, in the remaining papillary
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F. KOMMOSS ETAL.
Table 11-Comparison of TGF-a immunostaining scores and banding intensities of TGF-a Northern blots in 12 ovarian tumours
ID No. 513 483 292 286 378 217 207 359 I23 157 360 30 1
TGF-a immunostaining scores (0-9) 9 6 6 6 6 3 0 6 0 0 0 6
TGF-a mRNA Northern blot banding intensities (0-4)
4 2 2 2 2 2 2 1
0 0
0 0
28s I) TGF a I )
c Fig. 7-TGF-a
mRNA analysis of the cases shown in Figs 3-5
carcinoma, which was TGF-a negative by mRNA analysis, immunohistochemistry revealed focal strong TGF-a specific staining. DISCUSSION In this study we used monoclonal antibody and an indirect immunoperoxidase technique to localize TGF-a in different normal human adult tissues.
TGF-a immunoreactivity was present exclusively in epithelial cells of the tissues studied. Both stromal cells and inflammatory cells were completely TGF-a negative. Coffey et al. described TGF-a expression in the epidermis using in situ hybridization and immunohistochemistry.25 Our results indicate a similar distribution of TGF-a in the normal skin. EGF-R production in normal squamous epithelium of the exocervix has been demonstrated by us3*to be located in the basal cells. EGF and TGF-a have been found to induce proliferation of epidermal k e r a t i n o c y t e ~contributing , ~ ~ ~ ~ ~ to the normal proliferation of the skin. Parabasal TGF-a-producing cells may thus regulate proliferation of the EGF-Rexpressing basal cells. EGF-Rs are also synthesized by epithelial cells in the bowel,41 kidney, lung, endometrium, and Fallopian tube.42Since we found TGF-a in all of the latter tissues, it may be concluded that TGF-a regulates the proliferation of many different tissues in an auto- or paracrine way. In contrast to the results of Kudlow et who reported immunoreactive TGF-a to be present in the theca cells of rat ovaries, we were unable to detect TGF-a specific staining in pre- o r postmenopausal human ovaries. This discrepancy remains unclear, but may be solved by further experiments in which we will study TGF-a mRNA expression in human ovaries. We also investigated TGF-a expression in 29 ovarian tumours by immunohistochemistry and Northern blot mRNA analysis. Using immunocytochemical methods, we found TGF-a in the epithelial components of 87 per cent of the ovarian carcinomas, one malignant mixed mesodermal tumour, one dysgerminoma, and of two ovarian metastases of gastrointestinal carcinomas. TGF-a-specific immunostain was diffusely distributed in the cytoplasm of tumour cells in most cases but was concentrated along the cytoplasmic membrane in three cases. No TGF-a was present in two ovarian carcinomas, nor in two mesenchymal tumours of the ovary. This supports the results of Coffey et and indicates, together with our own findings in normal human tissues, that TGF-a is expressed only in epithelial tissues. Generally semi-quantitative analysis by immunohistochemistry of the TGF-a tissue content and TGF-a mRNA analysis showed comparable results. However, in a G3 endometrioid carcinoma which was TGF-a positive by immunohistochemistry, no TGF-a mRNA was found by Northern blot analysis. Degradation of the RNA preparation of this tumour was highly unlikely, since RNA specific for c-myc and EGF-R was found
TGF-a IN NORMAL TISSUES A N D I N OVARIAN TUMOURS
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