Immunohistochemical Determination of Five Somatostatin Receptors in Meningioma Reveals Frequent Overexpression of Somatostatin Receptor Subtype Sst2a

Vol. 6, 1865–1874, May 2000 Clinical Cancer Research 1865

Immunohistochemical Determination of Five Somatostatin Receptors in Meningioma Reveals Frequent Overexpression of Somatostatin 1 Receptor Subtype sst2A

Stefan Schulz, Steffen Ulrich Pauli, expression was determined using Western blot analysis. Solveig Schulz, Manuela Ha¨ndel, Whereas sst2A was readily detectable as a broad band migrating at M 70,000 in 12 (75%) of these tumors, 8 tumors Knut Dietzmann, Raimond Firsching, and r (50%) showed particularly high levels of immunoreactive Volker Ho¨llt2 sst2A receptors. There was an excellent correlation (P < Departments of Pharmacology and Toxicology [St. S., M. H., V. H.], 0.001) between the level of sst protein expression detected Neurosurgery [S. U. P., R. F.], Obstetrics and Gynecology [So. S.], 2A and Neuropathology [K. D.], Otto-von-Guericke-University, 39120 in Western blots and the sst2A- immunoreactive staining Magdeburg, Germany seen in tissue sections. Thus, the frequent overexpression of

the sst2A receptor may explain the high tracer uptake often observed in meningioma patients during somatostatin recep- ABSTRACT tor scintigraphy. Moreover, this simple immunohistochem- Meningioma is one of a variety of human tumors that ical method could prove useful in identifying those cases of exhibit a very high density of somatostatin receptors and in recurrent disease that may possibly respond to therapy with many cases show a true positive somatostatin receptor scin- sst2-selective agonists. tigraphy. However, the level of expression of individual somatostatin receptor proteins in meningioma has not been investigated. We have recently developed a panel of soma- INTRODUCTION tostatin receptor subtype-specific antibodies that effectively It is well known that many human tumors can express stain formalin-fixed, paraffin-embedded tumor tissue (S. somatostatin receptors (1). This is the molecular basis for the Schulz et al., Clin. Cancer Res., 4: 2047–2052, 1998). In the application of long-acting somatostatin analogues, i.e., oct- present study, we have used these antibodies to determine reotide, for therapeutic and diagnostic purposes (2–4). Whereas the somatostatin receptor status of 40 randomly selected unlabeled octreotide has been used successfully in the treatment 111 1 meningiomas. Immunoreactive staining for all somatostatin of neuroendocrine malignancies, [ In-DTPA-D-Phe ]octreotide receptors was clearly located at the plasma membrane of the has proven useful for in vivo imaging of somatostatin receptor- tumor cells and completely blocked with antigenic peptide. positive tumors including intestinal and bronchial carcinoid tumors, The vast majority of tumors (29 cases; 70%) were positive malignant lymphoma, and meningioma (5–9). Among brain tu- for sst2A immunoreactivity; among these, 20 (69%) tumors mors, meningiomas show the highest incidence of somatostatin showed high levels of sst2A immunoreactivity. In contrast, all receptor expression, and somatostatin receptor scintigraphy is of other somatostatin receptors were only detected sporadi- value in the differentiation of meningiomas from other brain cally, and none of these cases revealed a particularly strong tumors (10–13). Treatment of meningioma with somatostatin an- staining. However, it is uncertain to what extent somatosta- alogues has also been attempted (14–16). tin receptor-immunoreactive staining intensity may trans- Recently, five subtypes of somatostatin receptors desig- late into somatostatin receptor protein expression on the nated sst1–5 have been identified (17). Two isoforms of sst2 have tumor cells. Therefore, in a prospective study, 16 surgically been isolated, sst2A and sst2B, which differ in size and the removed meningiomas were collected, and the level of sst2A sequence of their intracellular COOH-terminal domain (18, 19). All receptors bind natural somatostatin with high affinity but differ in their binding characteristics to various long-acting

somatostatin analogues (20). Whereas sst2, sst3, and sst5 exhibit high affinity for the synthetic somatostatin analogues seglitide Received 11/2/99; revised 1/24/00; accepted 1/31/00. The costs of publication of this article were defrayed in part by the (MK 678) and octreotide (SMS 201-995), sst1 and sst4 do not payment of page charges. This article must therefore be hereby marked bind these compounds. There is also evidence for different but advertisement in accordance with 18 U.S.C. Section 1734 solely to not mutually exclusive pathways of intracellular signaling of indicate this fact. somatostatin receptor subtypes. Whereas the antiproliferative 1 Supported by Grant SCHU 924/4-1 (to St. S.) from the Deutsche action of octreotide has been linked to stimulation of sst - Forschungsgemeinschaft, Grant QRTL-1999-00908 (to St. S.) from the 2 European Commission, Grant 1908A/0025 (to St. S.) from the Kul- associated tyrosine phosphatases, perturbation of the sst3 recep- tusministerium des Landes Sachsen/Anhalt, Grant I/75 172 from the tor is believed to induce apoptosis in human tumor cells (21– Volkswagen-Stiftung (to St. S.), a grant from Novartis, Germany (to 24). Furthermore, the antiproliferative effects of somatostatin So. S.), and a grant from the Fonds der Chemischen Industrie (to V. H.). analogues seem to require high numbers of somatostatin recep- 2 To whom requests for reprints should be addressed, at Department of Pharmacology and Toxicology, Otto-von-Guericke-University, Leip- tors, whereas the antihormonal effects occur in the presence of ziger Strasse 44, 39120 Magdeburg, Germany. Fax: 49-391-671-5869; a relatively low number of receptors. It is therefore crucial to E-mail: [email protected]. determine the pattern of somatostatin receptor subtype expres-

Table 1 Somatostatin receptor-like immunoreactivity in meningioma

a Patient Age (yrs) Gender Diagnosis Histological grade sst1 sst2A sst2B sst3 sst4 sst5 150m T G1 Ϫb ϩ Ϫ ϪϩϪ 249f T G1 ϪϩϩϪ ϩϪϪ 374f T G1 Ϫ Ϫ ϩ ϪϩϪ 45fF G1 Ϫ Ϫ Ϫ ϪϪϪ 572f T G1 Ϫ ϩ Ϫ ϪϪϪ 661m M G1 Ϫ Ϫ Ϫ ϪϪϪ 786f M G1 Ϫ ϩϩϩ Ϫ Ϫ Ϫ Ϫ 868f M G1 Ϫ ϩϩϩ Ϫ ϩ ϩ Ϫ 968f F G1 ϪϩϩϪ ϩϪϪ 10 67 m T G1 Ϫ ϩϩϩ Ϫ ϩ Ϫ Ϫ 11 47 f M G1 ϩ ϩϩϩ ϩ ϩ ϩ ϩ 12 65 f T G1 Ϫ Ϫ Ϫ ϪϪϪ 13 73 f T G1 Ϫ ϩ Ϫ ϪϪϪ 14 64 m T G1 Ϫ ϩϩϩ Ϫ Ϫ Ϫ ϩ 15 47 m M G1 Ϫ Ϫ Ϫ ϩϩϪ 16 42 f T G1 Ϫ ϩϩϩ Ϫ ϩ Ϫ Ϫ 17 62 f MC G1 Ϫϩϩϩ ϪϪϪ 18 80 f T G1 ϪϩϩϪ ϪϪϪ 19 61 m M G1 Ϫ ϩ Ϫ ϪϪϪ 20 74 f M G1 Ϫ Ϫ Ϫ ϪϪϪ 21 58 f T G1 Ϫ ϩ Ϫ ϪϪϪ 22 61 f F G1 ϪϩϩϪ ϪϪϪ 23 65 f F G1 Ϫ Ϫ Ϫ ϪϪϪ 24 60 f F G1 Ϫ ϩ Ϫ ϪϪϪ 25 82 f A G2 ϩ ϩϩϩ Ϫ Ϫ ϩ Ϫ 26 44 m T G1 Ϫ ϩϩϩ Ϫ Ϫ Ϫ Ϫ 27 46 f T G1 ϪϩϩϪ ϪϪϩ 28 56 f T G1 ϪϩϩϪ ϪϪϪ 29 51 f F G1 Ϫ ϩ ϩ ϪϪϪ 30 51 f F G1 Ϫ Ϫ Ϫ ϪϪϪ 31 64 f T G1 Ϫ ϩϩϩϩϩϪ 32 64 f F G1 ϪϩϩϪ ϪϪϪ 33 61 f M G1 Ϫ ϩ Ϫ ϪϪϩ 34 75 f PS G1 ϪϩϩϪ ϪϪϩ 35 62 f M G1 Ϫ ϩϩϩ ϩ ϩ ϩ Ϫ 36 64 m M G1 Ϫ Ϫ Ϫ ϪϩϪ 37 30 f M G1 Ϫ Ϫ Ϫ ϪϪϪ 38 46 f T G1 Ϫ ϩϩϩ Ϫ Ϫ ϩ Ϫ 39 64 f A G1 Ϫ Ϫ Ϫ ϪϪϪ 40 64 f A G1 ϪϩϩϪ ϩϩϪ a T, transitional (fibroblastic and meningothelial) meningioma; F, fibroblastic meningioma; M, meningothelial meningioma; MC, microcystic meningioma; A, atypical meningioma; PS, psammon body rich meningioma. b ϩϩϩ, strong; ϩϩ, moderate; ϩ, weak; Ϫ, negative immunostaining.

sion for a specific tumor to select one or more somatostatin for the development of specific anti-somatostatin receptor anti- analogues for optimal therapeutic effect. bodies is exemplified by the fact that at the same time, several The expression of somatostatin receptors in human tumors other laboratories have reported very similar protocols for

has previously been detected using binding autoradiography, in the detection of sst2A in human endocrine tumors (26–28). In situ hybridization, or reverse transcription-PCR. However, the the present study, we have generated antibodies directed against

diagnostic value of these methods is limited because the subtype the COOH-terminal sequences of sst4 and sst5 and determined selectivity of ligands available for binding autoradiography is the complete somatostatin receptor status of 40 meningiomas, not high enough to discriminate between individual somatosta- one of a variety of human tumors known to exhibit particularly tin receptors. Moreover, it is often uncertain whether transcripts high levels of somatostatin binding sites. detected in reverse transcription-PCR originate from tumor cells or from adjacent normal tissue. Progress on this front has been hampered by the lack of specific antibodies for immunohisto- MATERIALS AND METHODS chemical detection of somatostatin receptor proteins. We have Patients, Tumors, and Tissue Preparation. Meningio- recently generated antibodies that exert selective specificity for mas from 40 patients were studied. All patients were initially

the somatostatin receptor subtypes sst1, sst2A, sst2B, and sst3 treated by surgical tumor resection between 1996 and 1998 at (25). We have also developed an immunohistochemical protocol the Department of Neurosurgery, Otto-von-Guericke University that allows efficient staining of formalin-fixed, paraffin-embed- (Magdeburg, Germany). Pertinent data from patient histories ded human tumor tissue using these antibodies (25). The need (age, gender, diagnosis, and histological grade) are given in

Table 1. Tumor specimens were fixed in phosphate-buffered 4% selection of individual clones. Cells were then grown on cov- formalin for a minimum of 24 h. After dehydration through erslips overnight and fixed with 4% paraformaldehyde and 0.2% graded percentages of ethanol and xylene, the tissue was em- picric acid in 0.1 M phosphate buffer (pH 6.9) for1hatroom bedded in paraffin wax. In addition, 16 specimens were frozen temperature. Cells were washed several times in TPBS3 and Ϫ immediately in liquid N2 and stored at 70°C until analysis. preincubated with TPBS containing 0.3% Triton X-100 and 3% Generation of Anti-peptide Antisera. Production and NGSfor1hatroom temperature. Cells were then incubated

characterization of anti-sst1 (4819), anti-sst2A (6291), anti-sst2B with anti-sst1 (4819), anti-sst2A (6291), anti-sst3 (4823), anti- (4820), and anti-sst3 (4823) antisera has been described previ- sst4 (6002), or anti-sst5 (6006) antibodies at a 1:5000 dilution in ously. In this study, polyclonal antisera were generated against TPBS containing 0.3% Triton X-100 and 1% NGS at 4°C

the COOH-terminal tails of sst4 and sst5. The identity of the overnight. For homologous and heterologous adsorption con- peptides was CQQEPVQAEPGCKQVPFTKTTTF, which cor- trols, antisera were preincubated with 10 ␮g/ml peptides. Bound

responds to residues 362–384 of the mouse sst4 receptor, and primary antibody was detected with biotinylated secondary an- QEATRPRTAAANGLMQTSK, which corresponds to residues tibodies (1:1000 dilution; Vector Laboratories, Burlingame,

345–364 of the human sst5 receptor. Peptides were custom- CA) followed by cyanin 3.18-conjugated streptavidin (1:400 synthesized by Gramsch Laboratories (Schwabhausen, Germa- dilution; Amersham). Cells were then dehydrated, cleared in ny), purified by high-performance liquid chromatography, and xylol, and permanently mounted in DPX (Fluka, Neu-Ulm,

coupled via an NH2-terminally added cysteine and a succinimi- Germany). Specimens were examined using a Leica TCS-NT dyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate linker to laser scanning confocal microscope equipped with a krypton/ keyhole limpet hemocyanin. The conjugates were mixed 1:1 argon laser. Cyanin 3.18 was imaged with 568 nm excitation with Freund’s adjuvant and injected into groups of two rabbits and 570–630 nm bandpass emission filters.

(6001–6002 for anti-sst4 and 6005–6006 for anti-sst5 antisera Western Blot Analysis. Membranes were prepared from production). Animals were injected at 4-week intervals, and stably transfected HEK-293 cells as well as 16 meningiomas, serum was obtained 2 weeks after immunizations beginning and glycoproteins were partially purified using wheat germ with the second injection. lectin-agarose (Vector Laboratories) essentially as described Immunodot-Blot Analysis. The specificity of the anti- previously (30). Briefly, tissue was lysed in homogenization sera as well as possible cross-reactivity with other somatostatin buffer [5 mM EDTA, 3 mM EGTA, 250 mM sucrose, and 10 mM receptor subtypes was initially tested in dot-blot assays. Serial Tris-HCl (pH 7.6) containing 1 mM phenylmethylsulfonyl fluo- dilutions of the unconjugated peptides corresponding to the ride, 1 ␮M pepstatin, 10 ␮g/ml leupeptin, and 2 ␮g/ml aproti- ϫ COOH-terminal sequences of sst1, sst2A, sst2B, sst3, sst4, and nin]. The homogenate was spun at 500 g for 5 min at 4°C to sst5 were blotted onto nitrocellulose membranes. The identity remove unbroken cells and nuclei. Membranes were then pel- of the peptides was: (a) CRNGTCTSRITTL, which corre- leted at 20,000 ϫ g for 30 min at 4°C. Membranes were then

sponds to residues 382–391 of the human sst1 receptor; (b) dissolved in lysis buffer [150 mM NaCl, 5 mM EDTA, 3 mM ETQRTLLNGDLQTSI, which corresponds to residues 355–369 EGTA, and 20 mM HEPES (pH 7.4) containing 4 mg/ml dode- ␤ of the human sst2A receptor; (c) FRNNKNRKK, which corre- cyl- -maltoside and proteinase inhibitors as described above] ␮ sponds to residues 348–356 of the human sst2B receptor; (d) and incubated with 150 l of wheat germ lectin-agarose beads CQERPPSRVA, which corresponds to residue 384–393 of the for 90 min at 4°C. Beads were washed five times in lysis buffer,

human sst3 receptor; (e) CQQEALQPEPGRKRIPLTRTTTF, and adsorbed glycoproteins were eluted with SDS-sample buffer which corresponds to residues 366–388 of the human sst4 for 60 min at 37°C. The protein content was determined using receptor; (f) CQQEPVQAEPGCKQVPFTKTTTF, which corre- the BCA method according to the instructions of the manufac-

sponds to residues 362–384 of the mouse sst4 receptor; and (g) turer (Pierce), and aliquots of each sample containing equal QEATRPRTAAANGLMQTSKL, which corresponds to resi- amounts of protein were subjected to 8% SDS-PAGE and im-

dues 345–364 of the human sst5 receptor. Membranes were then munoblotted onto nitrocellulose. Another aliquot of each sample incubated with the antisera at dilutions ranging from 1:1,000 to was run on a duplicate gel that was then stained with Coomassie 1:20,000 for 60 min at room temperature. Blots were developed Blue, and equal loading was verified by densitometric analysis

using the enhanced chemiluminescence method (Amersham, as described below. Blots were incubated with anti-sst1 (4819), Braunschweig, Germany. For subsequent analysis, either these anti-sst2A (6291), anti-sst2B (4820), anti-sst3 (4823), anti-sst4 crude antisera or affinity-purified antibodies were used. Anti- (6002), or anti-sst5 (6006) antibodies either crude at a dilution of bodies were affinity-purified against their immunizing peptides 1:20,000 or after affinity purification at a concentration of 1 using the Sulfo-Link coupling gel (Pierce, Rockford, IL) accord- ␮g/ml overnight at 4°C. Blots were developed using peroxidase- ing to the instructions of the manufacturer. conjugated secondary antibodies purchased either from Sigma Immunocytochemistry. Human embryonic kidney (A-9169;1:5,000 dilution) or from Amersham (NA 934; 1:5,000

HEK-293 cells were stably transfected with either sst1, sst2A, dilution) and enhanced chemiluminescence. Densitometric anal- sst3, sst4,orsst5 (all human) using the calcium phosphate ysis of Western blots exposed in the linear range of the X-ray precipitation method as described previously (29). Plasmids were kindly provided by Dr. F. Raulfs (Novartis, Basel, Swit- zerland). Approximately 1.5 ϫ 106 cells were transfected with

20 ␮g of plasmid DNA. Cells were selected in the presence of 3 The abbreviations used are: TPBS, 10 mM Tris, 10 mM phosphate ␮ 500 g/ml G418 (Life Technologies, Inc., Eggenstein, Germa- buffer, 137 mM NaCl, and 0.05% thimerosal (pH 7.4); NGS, normal ny), and the whole pool of resistant cells was used without goat serum.

Menzel, Braunschweig, Germany) for immunohistochemical staining. Sections were dewaxed three times in xylene and rehydrated in a graded series of ethanol. After rinsing in TPBS,

sections were incubated in methanol containing 0.3% H2O2 for 30 min at room temperature. Sections were transferred into TPBS and subsequently microwaved in 10 mM citric acid (pH 6.0) for 20 min at 600 W. Specimens were then allowed to cool to room temperature, washed in TPBS, and preincubated in TPBS containing 3% NGS for1hatroom temperature. Sections

were then incubated either with anti-sst1 (4819), anti-sst2A (6291), anti-sst2B (4820), anti-sst3 (4823), anti-sst4 (6002), or anti-sst5 (6006) antibodies at a dilution of 1:500 (crude) or at a concentration of 1 ␮g/ml (affinity pure) in TPBS containing 1% NGS overnight. Primary antibody staining was detected using the biotin amplification procedure as described previously (25, 30, 32, 33). Briefly, tissue sections were transferred to biotinylated goat antirabbit IgG or biotinylated goat anti-guinea pig IgG (1:200; Vector Laboratories) for 1 h, incubated in AB solution (reagents from Vector Laboratories ABC Elite kit; 25 ␮l of A and 25 ␮l of B) for 60 min, and incubated in biotinylated tyramine (1:250 dilution; prepared as described in Ref. 32) for 30 min, followed by a final incubation in AB solution (12.5 ␮l of A and 12.5 of ␮l B). Tissue was rinsed and stained with 3,3Ј-diaminobenzidine-glucose oxidase for 30 min. All incubation steps were carried out at room temperature. The cell nuclei were lightly counterstained with hematoxylin. Sections were then dehydrated through several concentrations of alcohol, cleared in xylol, and coverslipped with DPX. For immunohistochemical controls, the primary antibody was either omitted, replaced by preimmune sera, or adsorbed with several concentrations (range, 1–10 ␮g/ml) of homologous or heterologous peptides for2hatroom temperature. A tumor known to stain Fig. 1 Immunodot-blot analysis of the specificity of anti-sst4 and anti- positively was included in each batch of staining as a positive sst antisera. Serial dilutions (0–2000 ng) of the peptides corresponding 5 control. to the COOH-terminal regions of hsst , hsst , hsst , hsst , msst , hsst , 1 2A 2B 3 4 4 Assessment of Staining Patterns. Immunohistochemi- or hsst5 were blotted onto nitrocellulose membranes and incubated either with anti-sst4 (6002; top panel) or anti-sst5 (6006; bottom panel) cal staining patterns were assessed as described previously (25), antisera at a dilution of 1:2000. Membranes were developed using the and all slides were evaluated by the same investigator. Briefly, enhanced chemiluminescence method. Note that both the anti-sst4 and the presence or absence of staining and the depth of color were the anti-sst5 antisera selectively detected the peptide corresponding to their cognate receptor but did not detect the peptides corresponding to noted, as well as the number of cells showing a positive reaction and whether or not the staining was localized to the plasma other somatostatin receptors. In addition, the anti-sst4 antibodies detected the sequence corresponding to the mouse as well as the sequence membrane. The depth of color was recorded as pale, medium, or corresponding to the human sst4 receptor. dark according to how easily it was seen. The tumors were then categorized as weak, moderate, or strong stainers according to the following criteria: (a) strong (ϩϩϩ), dark staining at the film was performed as described by Roth et al. (31). The amount plasma membrane that is easily visible with a low-power ob- of immunoreactive material in each lane was quantified by jective; (b) moderate (ϩϩ), medium staining that is visible with ϩ densitometric analysis of the sst2A-specific bands using NIH a low-power objective; (c) weak ( ), pale staining that is not Image 1.57 software (developed at the NIH and available on the easily seen under a low-power objective; and (d) negative (Ϫ), 4 internet). Extracts from sst2A-transfected HEK-293 cells were tumors that show none of the above. used as an internal control. For adsorption controls, antisera Statistical Evaluation. Data were analyzed by using the were preincubated with 10 ␮g/ml of their cognate peptide for SAS statistical program package (SAS Institute, Cary, NC). 2 h at room temperature. Data grouped into categories were analyzed for correlations Immunohistochemistry. Seven-␮m sections were cut with the ␹2 test, Fisher’s exact test, and Spearman test. and floated onto positively charged slides (SuperFrost*/Plus; RESULTS Characterization of Antibodies. Specificity of the anti- 4 NIH Image 1.57 is available on the internet at http://rsb.info.nih.gov/ sera was monitored using immunodot-blot analysis. After four nih-image. booster injections, one anti-sst4 antiserum and one rabbit anti-

Fig. 2 Characterization of anti-sst antisera using stably transfected HEK-293 cells. HEK-293 cells stably transfected to express either sst1, sst2A, sst3, sst4, or sst5 (vertical columns) were immunofluorescence stained with either anti-sst1 (4819), anti-sst2A (6291), anti-sst3 (4823), anti-sst4 (6002), or anti-sst5 (6006) antisera (horizontal columns). Note that prominent immunofluorescence localized at the level of the plasma membrane was seen only in HEK-293 cells bearing their cognate somatostatin receptor and not in HEK-293 cells transfected with other somatostatin receptors. Scale bar,10␮m.

sst5 antiserum developed a titer against their immunizing pep- ceptor-like bands only in cells transfected with their cognate tides. As shown in Fig. 1, the antisera 6002 (anti-sst4) and 6006 somatostatin receptor subtype and not in wild-type cells or in (anti-sst5) specifically detected quantities as low as 25 ng of HEK-293 cells transfected with other somatostatin receptors their cognate peptide but did not detect the peptides correspond- (Fig. 3). The molecular weight of the somatostatin receptors ing to other somatostatin receptor subtypes. Moreover, anti- expressed heterologously in HEK-293 cells was Mr 52,000– serum 6002, which was raised against the COOH terminus of 63,000 for sst1, Mr 62,000–72,000 for sst2A, Mr 60,000–75,000 the mouse sst4 receptor, detected not only the sequence of the for sst3, Mr 40,000–50,000 for sst4, and Mr 54,000–63,000 for mouse but also the corresponding sequence of the human sst4. sst5. These bands were no longer detected when the antisera Somatostatin receptor antisera were further characterized were preincubated with their cognate peptides (data not shown). using immunofluorescent staining of stably transfected HEK- Antibodies were then subjected to immunohistochemical

293 cells. When HEK-293 cells stably expressing human sst1, staining of a panel of human tumor tissues including primary sst2A, sst3, sst4,orsst5 were stained with either anti-sst1 (4819), breast cancer, carcinoid tumor, pituitary adenoma, and menin- anti-sst2A (6291), anti-sst3 (4823), anti-sst4 (6002), or anti-sst5 gioma. The antisera 4819 (anti-sst1), 6291 (anti-sst2A), 4820 (6006) antisera, prominent immunofluorescence localized at the (anti- sst2B), 4823 (anti-sst3), 6002 (anti-sst4), and 6006 (anti- level of the plasma membrane was seen only in HEK-293 cells sst5) yielded prominent staining that was predominantly local- bearing their cognate somatostatin receptor (Fig. 2) and not in ized to the plasma membrane of the tumor cells and were used HEK-293 cells transfected with other somatostatin receptors. throughout this study. The staining intensity for each antibody This staining was completely blocked by preincubation of the varied greatly between individual tumors, giving consistently antisera with homologous but not heterologous peptides (data different sample-specific patterns of somatostatin receptor sub- not shown). Next, the antisera were tested for possible cross- type expression under otherwise identical conditions. Immuno- reactivity with other proteins present in HEK-293 cells. When staining for each antiserum was completely abolished by pre- membrane preparations from stably transfected HEK-293 cells absorption with 10 ␮g/ml immunizing peptides. were separated electrophoretically and blotted onto nitrocellu- Somatostatin Receptor Immunohistochemical Staining lose, the antisera 4819 (anti-sst1), 6291 (anti-sst2A), 4823 (anti- in Meningioma. A series of 40 meningiomas was stained im- sst3), 6002 (anti-sst4), and 6006 (anti-sst5) revealed broad re- munohistochemically with polyclonal anti-sst1 (4819), anti-sst2A

was present in 10 tumors (25%), unequivocal staining for sst4 was

present in 11 tumors (28%), and unequivocal staining for sst5 was

present in 4 tumors (11%). Interestingly, the majority of sst2A- positive tumors showed moderate to strong immunostaining. In contrast, none of the other somatostatin receptor subtypes revealed

a particularly strong staining, indicating that sst2A is the predominant somatostatin receptor subtype expressed in meningioma. In the vast majority of positively stained tumors, somatostatin receptor immunoreactivity was uniformly present on nearly all tumor cells. Both the level and the pattern of expression of somatostatin receptor subtypes varied greatly between individual tumors. No staining

for sst2A or other somatostatin receptor subtypes was observed in normal meninges. Thus, sst2A overexpression in meningiomas ap- pears to reflect a tumor-specific phenotype. Somatostatin receptor staining patterns were analyzed for correlation of each subtype with patient age, gender, diagnosis, and histological grade. No correlations among these data groups were found. The lack of correlation of somatostatin receptor subtype expression and patient age, gender, diagnosis, and histopathological grade indicates that somatostatin receptor subtype expression was regulated independently of these variables.

Correlation between sst2A-immunoreactive Staining and sst2A Protein Expression. The clinical utility of octreotide depends on the number of sst2A receptors on the tumor cells. However, to what extent sst2A-immunoreactive staining intensity translates into sst2A protein expression is uncertain. Thus, we have collected 16 surgically removed meningiomas

and analyzed immunoreactive sst2A receptors in paraffin sections as well as in immunoblots. sst2A immunohistochemical staining was evaluated according to the criteria described in “Materials and Methods.” Typical staining patterns are shown in

Fig. 4. On Western blots, the sst2A receptor was readily detectable as a broad band migrating at Mr 70,000 (Fig. 5). In some tumor lysates, an additional band was detected at Mr 110,000. However, this band appeared to originate from nonspecific binding of the secondary antibody (Sigma) because it was neither detected with a secondary antibody from a different manufacturer (Amersham) nor completely blocked by preincubation with antigenic peptide. As shown in Fig. 6, top panel, the

level of sst2A protein expression varied greatly between individual tumors. In fact, eight tumors (50%) revealed particularly

high levels of immunoreactive sst2A receptors. In the remaining Fig. 3 Western blot analysis of the specificity of anti-sst antisera. tumors, the sst2A-specific band was either weak or not detecta- Membrane preparations from HEK-293 cells stably transfected to ex- ble. Similar immunoreactive staining for sst2A was scored mod- press either sst , sst , sst , sst ,orsst were separated on an 8% 1 2A 3 4 5 erate to strong in 7 of the 16 tumors (44%). Nine tumors were SDS-polyacrylamide gel and blotted onto nitrocellulose membranes. Membranes were then incubated with either anti-sst (4819), anti-sst scored negative or weak. Interestingly, there was an excellent 1 2A Ͻ ϭ (6291), anti-sst3 (4823), anti-sst4 (6002), or anti-sst5 (6006) antisera at a correlation (P 0.001; r 0.8622) between the densitometric dilution of 1:20,000. Blots were developed using enhanced chemilumi- analysis of sst2A band intensity and the sst2A-immunoreactive nescence. Ordinate, migration of protein molecular weight markers staining score (Fig. 6, bottom panel). No other somatostatin (M ϫ 10Ϫ3). r receptors were unequivocally detected by Western blot analysis in these 16 tumors.

(6291), anti-sst2B (4820), anti-sst3 (4823), anti-sst4 (6002), or anti- DISCUSSION sst5 (6006) antisera. The staining pattern of somatostatin receptor- In the present study, we have determined the pattern of like immunoreactivity is shown in Table 1. Unequivocal staining somatostatin receptor protein expression in human meningioma

for sst1 was present in 2 tumors (5%), unequivocal staining for using a panel of somatostatin receptor subtype-selective anti- sst2A was present in 29 tumors (70%), unequivocal staining for bodies that have been characterized extensively. Several lines of sst2B was present in 6 tumors (15%), unequivocal staining for sst3 evidence indicate that these antisera specifically detect their

Fig. 4 sst2A immunohistochemical staining in meningioma. Left panels, staining patterns for sst2A in typical meningiomas displaying either strong (ϩϩϩ), moderate (ϩϩ), weak (ϩ), or negative (Ϫ) staining. Right panels, corresponding peptide adsorption controls. Sections were dewaxed, treated with methanol-H2O2, microwaved in citric acid, and incubated with anti-sst2A (6291) antibodies at a dilution of 1:500. Sections were then sequentially treated with biotinylated antirabbit IgG, AB solution, biotinylated tyramine, and AB solution. Sections were then developed in 3,3Ј-diaminobenzidine- glucose oxidase and lightly counterstained with hematoxylin. The immunoreactive score was determined according to the criteria described in “Materials and Methods.” For adsorption controls, primary antibodies were preincubated with the immunizing peptide (10 ␮g/ml). Scale bar,10␮m.

Fig. 5 Western blot analysis of sst2A immunoreactivity in meningioma. Membrane preparations from four surgically removed meningiomas (patients A–D) were separated on an 8% SDS-polyacrylamide gel and blotted onto nitrocellulose membranes. Membranes were then incubated

with anti-sst2A antiserum (6291) at a dilution of 1:20,000 in the absence (Ϫ) or presence (ϩ) of peptide antigen (10 ␮g/ml). Blots were devel- Fig. 6 Correlation between sst2A-immunoreactive staining and sst2A oped using enhanced chemiluminescence. Note that in some tumor protein expression in meningioma. Top panel, membrane preparations

lysates, an additional band was detected at Mr 110,000. However, this from 16 surgically removed meningiomas (patients 1–16) were sepa- band appeared to originate from nonspecific binding of the secondary rated on an 8% SDS-polyacrylamide gel and blotted onto nitrocellulose antibody because it was not completely blocked by preincubation with membranes. Membranes were then incubated with anti-sst2A antiserum antigenic peptide. Ordinate, migration of protein molecular weight (6291) at a dilution of 1:20,000. Blots were developed using enhanced ϫ Ϫ3 markers (Mr 10 ). chemiluminescence. Ordinate, migration of protein molecular weight ϫ Ϫ3 markers (Mr 10 ). Bottom panel, the amount of immunoreactive sst2A receptors for each tumor was determined by densitometric analysis as described in “Materials and Methods.” The corresponding paraffin sections from patients 1–16 were stained immunohistochemically, and cognate receptor and do not cross-react. First, in immunodot- the immunoreactive score was determined according to the criteria blot assays, the anti-somatostatin receptor antisera specifically described in “Materials and Methods.” Note that there was an excellent Ͻ detected their cognate peptides but not the peptides correspond- correlation (P 0.001) between sst2A-immunoreactive staining and ing to the COOH-terminal region of other somatostatin receptor sst2A protein expression. The correlation coefficient and statistical sig- nificance were determined using the Spearman test. subtypes. Second, immunocytochemical staining of stably transfected HEK-293 cells revealed that the anti-somatostatin receptor antisera selectively stained cells expressing their targeted

receptor but did not stain wild-type cells or cells transfected presence and the level of sst2A expression on the tumor cell. with other somatostatin receptors. Third, in Western blots, the However, it is uncertain to what extent sst2A-immunoreactive antisera detected a band of the appropriate molecular weight staining intensity may translate into sst2A protein expression on only in those HEK-293 cells that expressed their cognate recep- the tumor cells. Therefore, we conducted a prospective study tor and not in cells that expressed other somatostatin receptor using 16 surgically removed meningiomas. Interestingly, there subtypes. Fourth, the anti-somatostatin receptor antibodies ef- was an excellent correlation (P Ͻ 0.001) between the level of

fectively stained formalin-fixed, paraffin-embedded tissue from sst2A protein expression detected in Western blots and the a variety of human tumors including meningioma, primary sst2A-immunoreactive staining seen in tissue sections. This breast cancer, and carcinoid tumor. Staining of all antisera was finding highlights yet another advantage of this simple immu- completely neutralized by preincubation with homologous pep- nohistochemical procedure. Compared with currently available tides but not with heterologous peptides. Finally, the COOH- somatostatin receptor detection methods, e.g., binding autora- terminal peptides are likely to have served as somatostatin diography, in situ hybridization, or reverse transcription-PCR, it receptor-specific immunogens because these peptides were is less time consuming, suitable for paraffin-embedded tissues, found to have minimal homologies to other peptide sequences completely subtype selective, and provides information about when aligned to current entries in the European Molecular the level of somatostatin receptor expression on the tumor cell Biology Laboratory databases using BLASTp or FASTa. (25–28).

In our series of 40 randomly selected meningiomas, sst2A It has been well documented that many cases of meningi- was clearly the predominant somatostatin receptor subtype. The oma show a particularly high tracer uptake during somatostatin 111 1 sst2A receptor was not only the most frequently detected recep- receptor imaging using [ In-DTPA-D-Phe ]octreotide (10–13). tor but also yielded the most prominent staining of all soma- In fact, somatostatin receptor scintigraphy is often of value in tostatin receptor subtypes. It is believed that the antiproliferative the differentiation of meningiomas from other brain tumors

effects of somatostatin analogues require high numbers of so- (10–13). Octreotide, which binds preferentially to sst2 and sst5, matostatin receptors, whereas the antihormonal effects occur in has also been implicated in the treatment of meningioma (14– the presence of a relatively low number of receptors. Thus, in a 16). Moreover, at the mRNA level, several studies have detected

clinical setting, it may prove useful to determine both the a particularly high expression of sst2A in the majority of men-

ingiomas (7, 9). Thus, our observation that sst2A protein is 7. Reubi, J-C., Schaer, J. C., Waser, B., and Mengod, G. Expression and frequently overexpressed in human meningioma corresponds localization of somatostatin receptor SSTR1, SSTR2, and SSTR3 mes- well to these findings and would thus explain the high rate of senger RNA in primary human tumors using in situ hybridization. Cancer Res., 54: 3455–3459, 1994. true positive somatostatin receptor scintigraphy of this tumor. 8. Reubi, J. C., Maurer, R., Klijn, J. G., Stefanko, S. Z., Foekens, J. A., What are the implications of immunohistochemical soma- Blaauw, G., Blankenstein, M. A., and Lamberts, S. W. High incidence tostatin receptor determination for the treatment of meningi- of somatostatin receptors in human meningiomas: biochemical charac- oma? Surgical removal of the tumor is clearly the first option; terization. J. Clin. Endocrinol. Metab., 63: 433–438, 1986. however, some cases of unresectable tumor or recurrent disease 9. Dutour, A., Kumar, U., Panetta, R., Ouafik, L., Fina, F., Sasi, R., and exist that demand further attention. Knowledge of the soma- Patel, Y. C. Expression of somatostatin receptor subtypes in human tostatin receptor status of these tumors may help in identifying brain tumors. Int. J. Cancer, 76: 620–627, 1998. those cases that may possibly respond to therapy with octreotide 10. Hildebrandt, G., Scheidhauer, K., Luyken, C., Schicha, H., Klug, N., Dahms, P., and Krisch, B. High sensitivity of the in vivo detection or other sst2A-selective ligands. It should be noted, however, of somatostatin receptors by 111indium (DTPA-octreotide)-scintigraphy that the effectiveness of octreotide in the treatment of meningi- in meningioma patients. Acta Neurochir., 126: 63–71, 1994. oma is expected to be limited. Although some cases of success- 11. Barth, A., Haldemann, A. R., Reubi, J. C., Godoy, N., Rosler, H., ful treatment of meningioma with octreotide have been reported Kinser, J. A., and Seiler, R. W. Noninvasive differentiation of menin- 111 (14–16), somatostatin analogues have also been shown to stim- giomas from other brain tumours using combined indium-octreotide/ 99mtechnetium-DTPA brain scintigraphy. Acta Neurochir., 138: 1179– ulate the growth of cultured human meningioma cells in vitro 1185, 1996. (34, 35). Nevertheless, novel nonpeptide agonists for all soma- 12. Bohuslavizki, K. H., Brenner, W., Braunsdorf, W. E., Behnke, A., tostatin receptors as well as cytotoxic and radiolabeled soma- Tinnemeyer, S., Hugo, H. H., Jahn, N., Wolf, H., Sippel, C., Clausen, tostatin analogues are currently being developed and may pro- M., Mehdorn, H. M., and Henze, E. Somatostatin receptor scintigraphy vide further options for treatment (36–41). in the differential diagnosis of meningioma. Nucl. Med. 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Stefan Schulz, Steffen Ulrich Pauli, Solveig Schulz, et al.

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