Meningioma is one of a variety of human tumors that exhibit a very high density of somatostatin receptors and in many cases show a true positive somatostatin receptor scintigraphy. However, the level of expression of individual somatostatin receptor proteins in meningioma has not been investigated. We have recently developed a panel of somatostatin receptor subtype-specific antibodies that effectively stain formalin-fixed, paraffin-embedded tumor tissue (S. Schulz et al., Clin. Cancer Res., 4: 2047–2052, 1998). In the present study, we have used these antibodies to determine the somatostatin receptor status of 40 randomly selected meningiomas. Immunoreactive staining for all somatostatin receptors was clearly located at the plasma membrane of the tumor cells and completely blocked with antigenic peptide. The vast majority of tumors (29 cases;70%) were positive for sst2A immunoreactivity; among these, 20 (69%) tumors showed high levels of sst2Aimmunoreactivity. In contrast, all other somatostatin receptors were only detected sporadically, and none of these cases revealed a particularly strong staining. However, it is uncertain to what extent somatostatin receptor-immunoreactive staining intensity may translate into somatostatin receptor protein expression on the tumor cells. Therefore, in a prospective study, 16 surgically removed meningiomas were collected, and the level of sst2A expression was determined using Western blot analysis. Whereas sst2A was readily detectable as a broad band migrating at Mr 70,000 in 12 (75%) of these tumors, 8 tumors (50%) showed particularly high levels of immunoreactive sst2A receptors. There was an excellent correlation (P < 0.001) between the level of sst2A protein expression detected in Western blots and the sst2A- immunoreactive staining seen in tissue sections. Thus, the frequent overexpression of the sst2Areceptor may explain the high tracer uptake often observed in meningioma patients during somatostatin receptor scintigraphy. Moreover, this simple immunohistochemical method could prove useful in identifying those cases of recurrent disease that may possibly respond to therapy with sst2-selective agonists.

It is well known that many human tumors can express somatostatin receptors (1). This is the molecular basis for the application of long-acting somatostatin analogues, i.e.,octreotide, for therapeutic and diagnostic purposes (2, 3, 4). Whereas unlabeled octreotide has been used successfully in the treatment of neuroendocrine malignancies,[111In-DTPA-d-Phe1]octreotide has proven useful for in vivo imaging of somatostatin receptor-positive tumors including intestinal and bronchial carcinoid tumors, malignant lymphoma, and meningioma (5, 6, 7, 8, 9). Among brain tumors, meningiomas show the highest incidence of somatostatin receptor expression, and somatostatin receptor scintigraphy is of value in the differentiation of meningiomas from other brain tumors (10, 11, 12, 13). Treatment of meningioma with somatostatin analogues has also been attempted (14, 15, 16).

Recently, five subtypes of somatostatin receptors designated sst1–5 have been identified (17). Two isoforms of sst2 have been isolated,sst2A and sst2B, which differ in size and the 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 (MK 678) and octreotide (SMS 201-995),sst1 and sst4 do not bind these compounds. There is also evidence for different but not mutually exclusive pathways of intracellular signaling of somatostatin receptor subtypes. Whereas the antiproliferative action of octreotide has been linked to stimulation of sst2-associated tyrosine phosphatases, perturbation of the sst3 receptor is believed to induce apoptosis in human tumor cells (21, 22, 23, 24). Furthermore, the antiproliferative effects of somatostatin analogues seem to require high numbers of somatostatin receptors, whereas the antihormonal effects occur in the presence of a relatively low number of receptors. It is therefore crucial to determine the pattern of somatostatin receptor subtype expression for a specific tumor to select one or more somatostatin analogues for optimal therapeutic effect.

The expression of somatostatin receptors in human tumors has previously been detected using binding autoradiography, in situhybridization, or reverse transcription-PCR. However, the diagnostic value of these methods is limited because the subtype selectivity of ligands available for binding autoradiography is not high enough to discriminate between individual somatostatin receptors. Moreover, it is often uncertain whether transcripts 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 immunohistochemical detection of somatostatin receptor proteins. We have recently generated antibodies that exert selective specificity for the somatostatin receptor subtypes sst1, sst2A,sst2B, and sst3(25). We have also developed an immunohistochemical protocol that allows efficient staining of formalin-fixed,paraffin-embedded human tumor tissue using these antibodies (25). The need for the development of specific anti-somatostatin receptor antibodies is exemplified by the fact that at the same time, several other laboratories have reported very similar protocols for the detection of sst2A in human endocrine tumors (26, 27, 28). In the present study, we have generated antibodies directed against the COOH-terminal sequences of sst4 and sst5and determined the complete somatostatin receptor status of 40 meningiomas, one of a variety of human tumors known to exhibit particularly high levels of somatostatin binding sites.

Patients, Tumors, and Tissue Preparation.

Meningiomas from 40 patients were studied. All patients were initially treated by surgical tumor resection between 1996 and 1998 at the Department of Neurosurgery, Otto-von-Guericke University (Magdeburg,Germany). Pertinent data from patient histories (age, gender,diagnosis, and histological grade) are given in Table 1. Tumor specimens were fixed in phosphate-buffered 4% formalin for a minimum of 24 h. After dehydration through graded percentages of ethanol and xylene, the tissue was embedded in paraffin wax. In addition, 16 specimens were frozen immediately in liquid N2 and stored at−70°C until analysis.

Generation of Anti-peptide Antisera.

Production and characterization of anti-sst1(4819), anti-sst2A (6291),anti-sst2B (4820), and anti-sst3 (4823) antisera has been described previously. In this study, polyclonal antisera were generated against the COOH-terminal tails of sst4 and sst5. The identity of the peptides was CQQEPVQAEPGCKQVPFTKTTTF, which corresponds to residues 362–384 of the mouse sst4 receptor, and QEATRPRTAAANGLMQTSK,which corresponds to residues 345–364 of the human sst5 receptor. Peptides were custom-synthesized by Gramsch Laboratories (Schwabhausen, Germany), purified by high-performance liquid chromatography, and coupled via an NH2-terminally added cysteine and a succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate linker to keyhole limpet hemocyanin. The conjugates were mixed 1:1 with Freund’s adjuvant and injected into groups of two rabbits (6001–6002 for anti-sst4 and 6005–6006 for anti-sst5 antisera production). Animals were injected at 4-week intervals, and serum was obtained 2 weeks after immunizations beginning with the second injection.

Immunodot-Blot Analysis.

The specificity of the antisera as well as possible cross-reactivity with other somatostatin receptor subtypes was initially tested in dot-blot assays. Serial dilutions of the unconjugated peptides corresponding to the COOH-terminal sequences of sst1, sst2A,sst2B, sst3,sst4, and sst5 were blotted onto nitrocellulose membranes. The identity of the peptides was: (a) CRNGTCTSRITTL, which corresponds to residues 382–391 of the human sst1 receptor;(b) ETQRTLLNGDLQTSI, which corresponds to residues 355–369 of the human sst2A receptor;(c) FRNNKNRKK, which corresponds to residues 348–356 of the human sst2B receptor; (d) CQERPPSRVA,which corresponds to residue 384–393 of the human sst3 receptor; (e)CQQEALQPEPGRKRIPLTRTTTF, which corresponds to residues 366–388 of the human sst4 receptor; (f)CQQEPVQAEPGCKQVPFTKTTTF, which corresponds to residues 362–384 of the mouse sst4 receptor; and (g)QEATRPRTAAANGLMQTSKL, which corresponds to residues 345–364 of the human sst5 receptor. Membranes were then incubated with the antisera at dilutions ranging from 1:1,000 to 1:20,000 for 60 min at room temperature. Blots were developed using the enhanced chemiluminescence method (Amersham, Braunschweig, Germany. For subsequent analysis, either these crude antisera or affinity-purified antibodies were used. Antibodies were affinity-purified against their immunizing peptides using the Sulfo-Link coupling gel (Pierce,Rockford, IL) according to the instructions of the manufacturer.

Immunocytochemistry.

Human embryonic kidney HEK-293 cells were stably transfected with either sst1, sst2A,sst3, sst4, or sst5 (all human) using the calcium phosphate precipitation method as described previously (29). Plasmids were kindly provided by Dr. F. Raulfs (Novartis, Basel,Switzerland). Approximately 1.5 × 106 cells were transfected with 20 μg of plasmid DNA. Cells were selected in the presence of 500 μg/ml G418 (Life Technologies, Inc., Eggenstein,Germany), and the whole pool of resistant cells was used without selection of individual clones. Cells were then grown on coverslips overnight and fixed with 4% paraformaldehyde and 0.2% picric acid in 0.1 m phosphate buffer (pH 6.9) for 1 h at room temperature. Cells were washed several times in TPBS3 and preincubated with TPBS containing 0.3% Triton X-100 and 3% NGS for 1 h at room temperature. Cells were then incubated with anti-sst1 (4819),anti-sst2A (6291),anti-sst3 (4823), anti-sst4(6002), or anti-sst5 (6006) antibodies at a 1:5000 dilution in TPBS containing 0.3% Triton X-100 and 1% NGS at 4°C overnight. For homologous and heterologous adsorption controls,antisera were preincubated with 10 μg/ml peptides. Bound primary antibody was detected with biotinylated secondary antibodies (1:1000 dilution; Vector Laboratories, Burlingame, CA) followed by cyanin 3.18-conjugated streptavidin (1:400 dilution; Amersham). Cells were then dehydrated, cleared in xylol, and permanently mounted in DPX (Fluka, Neu-Ulm, Germany). Specimens were examined using a Leica TCS-NT laser scanning confocal microscope equipped with a krypton/argon laser. Cyanin 3.18 was imaged with 568 nm excitation and 570–630 nm bandpass emission filters.

Western Blot Analysis.

Membranes were prepared from stably transfected HEK-293 cells as well as 16 meningiomas, and glycoproteins were partially purified using wheat germ lectin-agarose (Vector Laboratories) essentially as described previously (30). Briefly, tissue was lysed in homogenization buffer [5 mm EDTA, 3 mm EGTA,250 mm sucrose, and 10 mm Tris-HCl (pH 7.6)containing 1 mm phenylmethylsulfonyl fluo-ride, 1μ m pepstatin, 10 μg/ml leupeptin, and 2 μg/ml aprotinin]. The homogenate was spun at 500 × g for 5 min at 4°C to remove unbroken cells and nuclei. Membranes were then pelleted at 20,000 × g for 30 min at 4°C. Membranes were then dissolved in lysis buffer [150 mmNaCl, 5 mm EDTA, 3 mm EGTA,and 20 mm HEPES (pH 7.4) containing 4 mg/ml dodecyl-β-maltoside and proteinase inhibitors as described above]and incubated with 150 μl of wheat germ lectin-agarose beads for 90 min at 4°C. Beads were washed five times in lysis buffer, and adsorbed glycoproteins were eluted with SDS-sample buffer for 60 min at 37°C. The protein content was determined using the BCA method according to the instructions of the manufacturer (Pierce), and aliquots of each sample containing equal amounts of protein were subjected to 8% SDS-PAGE and immunoblotted onto nitrocellulose. Another aliquot of each sample was run on a duplicate gel that was then stained with Coomassie Blue, and equal loading was verified by densitometric analysis as described below. Blots were incubated with anti-sst1 (4819),anti-sst2A (6291),anti-sst2B (4820),anti-sst3 (4823), anti-sst4(6002), or anti-sst5 (6006) antibodies either crude at a dilution of 1:20,000 or after affinity purification at a concentration of 1 μg/ml overnight at 4°C. Blots were developed using peroxidase-conjugated secondary antibodies purchased either from Sigma (A-9169;1:5,000 dilution) or from Amersham (NA 934; 1:5,000 dilution) and enhanced chemiluminescence. Densitometric analysis of Western blots exposed in the linear range of the X-ray film was performed as described by Roth et al.(31). The amount of immunoreactive material in each lane was quantified by densitometric analysis of the sst2A-specific bands using NIH Image 1.57 software (developed at the NIH and available on the internet).4Extracts from sst2A-transfected HEK-293 cells were used as an internal control. For adsorption controls, antisera were preincubated with 10 μg/ml of their cognate peptide for 2 h at room temperature.

Immunohistochemistry.

Seven-μm sections were cut and floated onto positively charged slides(SuperFrost*/Plus; 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 for 1 h at room 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 μg/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 μl of A and 25 μl 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 μl of A and 12.5 of μl B). Tissue was rinsed and stained with 3,3′-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 μg/ml) of homologous or heterologous peptides for 2 h at room temperature. A tumor known to stain positively was included in each batch of staining as a positive control.

Assessment of Staining Patterns.

Immunohistochemical staining patterns were assessed as described previously (25), and all slides were evaluated by the same investigator. Briefly, the presence or absence of staining and the depth of color were noted, as well as the number of cells showing a positive reaction and whether or not the staining was localized to the plasma membrane. The depth of color was recorded as pale, medium, or 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 (+++), dark staining at the plasma membrane that is easily visible with a low-power objective;(b) moderate (++), medium staining that is visible with a low-power objective; (c) weak (+), pale staining that is not easily seen under a low-power objective; and (d) negative(−), tumors that show none of the above.

Statistical Evaluation.

Data were analyzed by using the SAS statistical program package (SAS Institute, Cary, NC). Data grouped into categories were analyzed for correlations with the χ2 test, Fisher’s exact test, and Spearman test.

Characterization of Antibodies.

Specificity of the antisera was monitored using immunodot-blot analysis. After four booster injections, one anti-sst4 antiserum and one rabbit anti-sst5 antiserum developed a titer against their immunizing peptides. As shown in Fig. 1, the antisera 6002(anti-sst4) and 6006(anti-sst5) specifically detected quantities as low as 25 ng of their cognate peptide but did not detect the peptides corresponding to other somatostatin receptor subtypes. Moreover,antiserum 6002, which was raised against the COOH terminus of the mouse sst4 receptor, detected not only the sequence of the mouse but also the corresponding sequence of the human sst4.

Somatostatin receptor antisera were further characterized using immunofluorescent staining of stably transfected HEK-293 cells. When HEK-293 cells stably expressing human sst1,sst2A, sst3,sst4, or sst5 were stained with either anti-sst1 (4819),anti-sst2A (6291),anti-sst3 (4823), anti-sst4(6002), or anti-sst5 (6006) antisera, prominent immunofluorescence localized at the level of the plasma membrane was seen only in HEK-293 cells bearing their cognate somatostatin receptor(Fig. 2) and not in HEK-293 cells transfected with other somatostatin receptors. This staining was completely blocked by preincubation of the antisera with homologous but not heterologous peptides (data not shown). Next, the antisera were tested for possible cross-reactivity with other proteins present in HEK-293 cells. When membrane preparations from stably transfected HEK-293 cells were separated electrophoretically and blotted onto nitrocellulose, the antisera 4819 (anti-sst1),6291 (anti-sst2A), 4823(anti-sst3), 6002(anti-sst4), and 6006(anti-sst5) revealed broad receptor-like bands only in cells transfected with their cognate somatostatin receptor subtype and not in wild-type cells or in HEK-293 cells transfected with other somatostatin receptors (Fig. 3). The molecular weight of the somatostatin receptors expressed heterologously in HEK-293 cells was Mr52,000–63,000 for sst1, Mr 62,000–72,000 for sst2A, Mr60,000–75,000 for sst3, Mr 40,000–50,000 for sst4, and Mr54,000–63,000 for sst5. These bands were no longer detected when the antisera were preincubated with their cognate peptides (data not shown).

Antibodies were then subjected to immunohistochemical staining of a panel of human tumor tissues including primary breast cancer, carcinoid tumor, pituitary adenoma, and meningioma. The antisera 4819(anti-sst1), 6291(anti-sst2A), 4820 (anti-sst2B), 4823 (anti-sst3),6002 (anti-sst4), and 6006(anti-sst5) yielded prominent staining that was predominantly localized to the plasma membrane of the tumor cells and were used throughout this study. The staining intensity for each antibody varied greatly between individual tumors, giving consistently different sample-specific patterns of somatostatin receptor subtype expression under otherwise identical conditions. Immunostaining for each antiserum was completely abolished by preabsorption with 10μg/ml immunizing peptides.

Somatostatin Receptor Immunohistochemical Staining in Meningioma.

A series of 40 meningiomas was stained immunohistochemically with polyclonal anti-sst1 (4819),anti-sst2A (6291),anti-sst2B (4820),anti-sst3 (4823), anti-sst4(6002), or anti-sst5 (6006) antisera. The staining pattern of somatostatin receptor-like immunoreactivity is shown in Table 1. Unequivocal staining for sst1was present in 2 tumors (5%), unequivocal staining for sst2A was present in 29 tumors (70%),unequivocal staining for sst2B was present in 6 tumors (15%), unequivocal staining for sst3 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 appears 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 sst2Areceptors 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 sst2Areceptors. In the remaining tumors, the sst2A-specific band was either weak or not detectable. Similar immunoreactive staining for sst2A was scored moderate to strong in 7 of the 16 tumors (44%). Nine tumors were scored negative or weak. Interestingly, there was an excellent correlation (P <0.001; r = 0.8622) between the densitometric analysis of sst2A band intensity and the sst2A-immunoreactive staining score (Fig. 6, bottom panel). No other somatostatin receptors were unequivocally detected by Western blot analysis in these 16 tumors.

In the present study, we have determined the pattern of somatostatin receptor protein expression in human meningioma using a panel of somatostatin receptor subtype-selective antibodies that have been characterized extensively. Several lines of evidence indicate that these antisera specifically detect their cognate receptor and do not cross-react. First, in immunodot-blot assays, the anti-somatostatin receptor antisera specifically detected their cognate peptides but not the peptides corresponding to the COOH-terminal region of other somatostatin receptor 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 with other somatostatin receptors. Third, in Western blots, the antisera detected a band of the appropriate molecular weight only in those HEK-293 cells that expressed their cognate receptor and not in cells that expressed other somatostatin receptor subtypes. Fourth, the anti-somatostatin receptor antibodies effectively stained formalin-fixed, paraffin-embedded tissue from a variety of human tumors including meningioma, primary breast cancer, and carcinoid tumor. Staining of all antisera was completely neutralized by preincubation with homologous peptides but not with heterologous peptides. Finally,the COOH-terminal peptides are likely to have served as somatostatin receptor-specific immunogens because these peptides were found to have minimal homologies to other peptide sequences when aligned to current entries in the European Molecular Biology Laboratory databases using BLASTp or FASTa.

In our series of 40 randomly selected meningiomas,sst2A was clearly the predominant somatostatin receptor subtype. The sst2A receptor was not only the most frequently detected receptor but also yielded the most prominent staining of all somatostatin receptor subtypes. It is believed that the antiproliferative effects of somatostatin analogues require high numbers of somatostatin receptors, whereas the antihormonal effects occur in the presence of a relatively low number of receptors. Thus, in a clinical setting, it may prove useful to determine both the presence and the level of sst2A expression on the tumor cell. However, it is uncertain to what extent sst2A-immunoreactive staining intensity may translate into sst2Aprotein expression on the tumor cells. Therefore, we conducted a prospective study using 16 surgically removed meningiomas. Interestingly, there was an excellent correlation (P <0.001) between the level of sst2A protein expression detected in Western blots and the sst2A-immunoreactive staining seen in tissue sections. This finding highlights yet another advantage of this simple immunohistochemical procedure. Compared with currently available somatostatin receptor detection methods, e.g., binding autoradiography, in situ hybridization, or reverse transcription-PCR, it is less time consuming, suitable for paraffin-embedded tissues, completely subtype selective, and provides information about the level of somatostatin receptor expression on the tumor cell (25, 26, 27, 28).

It has been well documented that many cases of meningioma show a particularly high tracer uptake during somatostatin receptor imaging using[111In-DTPA-d-Phe1]octreotide (10, 11, 12, 13). In fact, somatostatin receptor scintigraphy is often of value in the differentiation of meningiomas from other brain tumors (10, 11, 12, 13). Octreotide, which binds preferentially to sst2 and sst5, has also been implicated in the treatment of meningioma (14, 15, 16). Moreover, at the mRNA level,several studies have detected a particularly high expression of sst2A in the majority of meningiomas (7, 9). Thus, our observation that sst2Aprotein is frequently overexpressed in human meningioma corresponds well to these findings and would thus explain the high rate of true positive somatostatin receptor scintigraphy of this tumor.

What are the implications of immunohistochemical somatostatin receptor determination for the treatment of meningioma? Surgical removal of the tumor is clearly the first option; however, some cases of unresectable tumor or recurrent disease exist that demand further attention. Knowledge of the somatostatin receptor status of these tumors may help in identifying those cases that may possibly respond to therapy with octreotide or other sst2A-selective ligands. It should be noted, however, that the effectiveness of octreotide in the treatment of meningioma is expected to be limited. Although some cases of successful treatment of meningioma with octreotide have been reported (14, 15, 16), somatostatin analogues have also been shown to stimulate the growth of cultured human meningioma cells in vitro(34, 35). Nevertheless, novel nonpeptide agonists for all somatostatin receptors as well as cytotoxic and radiolabeled somatostatin analogues are currently being developed and may provide further options for treatment (36, 37, 38, 39, 40, 41).

In conclusion, we have generated and extensively characterized subtype-selective antibodies for all five somatostatin receptors. We demonstrate that these antibodies are well suited for an immunohistochemical procedure that allowed us to provide precise information about the somatostatin receptor protein expression in a given tumor specimen. With the development of subtype-selective ligands, it will be of particular importance to establish patterns of somatostatin receptor expression for each tumor to select one or more somatostatin analogues for an optimal therapeutic effect.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

Supported by Grant SCHU 924/4-1 (to St. S.)from the Deutsche Forschungsgemeinschaft, Grant QRTL-1999-00908 (to St. S.) from the European Commission, Grant 1908A/0025 (to St. S.)from the Kultusministerium des Landes Sachsen/Anhalt, Grant I/75 172 from the Volkswagen-Stiftung (to St. S.), a grant from Novartis,Germany (to So. S.), and a grant from the Fonds der Chemischen Industrie (to V. H.).

                
3

The abbreviations used are: TPBS, 10 mm Tris, 10 mm phosphate buffer, 137 mm NaCl, and 0.05% thimerosal (pH 7.4); NGS, normal goat serum.

        
4

NIH Image 1.57 is available on the internet at http://rsb.info.nih.gov/nih-image.

Fig. 1.

Immunodot-blot analysis of the specificity of anti-sst4 and anti- sst5 antisera. Serial dilutions (0–2000 ng) of the peptides corresponding to the COOH-terminal regions of hsst1, hsst2A,hsst2B, hsst3, msst4,hsst4, or hsst5 were blotted onto nitrocellulose membranes and incubated either with anti-sst4 (6002; top panel) or anti-sst5 (6006; bottom panel) antisera at a dilution of 1:2000. Membranes were developed using the enhanced chemiluminescence method. Note that both the anti-sst4 and the anti-sst5 antisera selectively detected the peptide corresponding to their cognate receptor but did not detect the peptides corresponding to other somatostatin receptors. In addition, the anti-sst4 antibodies detected the sequence corresponding to the mouse as well as the sequence corresponding to the human sst4 receptor.

Fig. 1.

Immunodot-blot analysis of the specificity of anti-sst4 and anti- sst5 antisera. Serial dilutions (0–2000 ng) of the peptides corresponding to the COOH-terminal regions of hsst1, hsst2A,hsst2B, hsst3, msst4,hsst4, or hsst5 were blotted onto nitrocellulose membranes and incubated either with anti-sst4 (6002; top panel) or anti-sst5 (6006; bottom panel) antisera at a dilution of 1:2000. Membranes were developed using the enhanced chemiluminescence method. Note that both the anti-sst4 and the anti-sst5 antisera selectively detected the peptide corresponding to their cognate receptor but did not detect the peptides corresponding to other somatostatin receptors. In addition, the anti-sst4 antibodies detected the sequence corresponding to the mouse as well as the sequence corresponding to the human sst4 receptor.

Close modal
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 μm.

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 μm.

Close modal
Fig. 3.

Western blot analysis of the specificity of anti-sst antisera. Membrane preparations from HEK-293 cells stably transfected to express either sst1, sst2A,sst3, sst4, or sst5 were separated on an 8% SDS-polyacrylamide gel and blotted onto nitrocellulose membranes. Membranes were then incubated with either anti-sst1 (4819), anti-sst2A (6291),anti-sst3 (4823), anti-sst4 (6002), or anti-sst5 (6006) antisera at a dilution of 1:20,000. Blots were developed using enhanced chemiluminescence. Ordinate, migration of protein molecular weight markers(Mr × 10−3).

Fig. 3.

Western blot analysis of the specificity of anti-sst antisera. Membrane preparations from HEK-293 cells stably transfected to express either sst1, sst2A,sst3, sst4, or sst5 were separated on an 8% SDS-polyacrylamide gel and blotted onto nitrocellulose membranes. Membranes were then incubated with either anti-sst1 (4819), anti-sst2A (6291),anti-sst3 (4823), anti-sst4 (6002), or anti-sst5 (6006) antisera at a dilution of 1:20,000. Blots were developed using enhanced chemiluminescence. Ordinate, migration of protein molecular weight markers(Mr × 10−3).

Close modal
Fig. 4.

sst2A immunohistochemical staining in meningioma. Left panels, staining patterns for sst2A in typical meningiomas displaying either strong(+++), moderate (++), weak (+), or negative (−) 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 3,3′-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 μg/ml). Scale bar, 10 μm.

Fig. 4.

sst2A immunohistochemical staining in meningioma. Left panels, staining patterns for sst2A in typical meningiomas displaying either strong(+++), moderate (++), weak (+), or negative (−) 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 3,3′-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 μg/ml). Scale bar, 10 μm.

Close modal
Fig. 5.

Western blot analysis of sst2Aimmunoreactivity 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 (−) or presence (+) of peptide antigen (10 μg/ml). Blots were developed using enhanced chemiluminescence. Note that 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 because it was not completely blocked by preincubation with antigenic peptide. Ordinate, migration of protein molecular weight markers(Mr × 10−3).

Fig. 5.

Western blot analysis of sst2Aimmunoreactivity 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 (−) or presence (+) of peptide antigen (10 μg/ml). Blots were developed using enhanced chemiluminescence. Note that 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 because it was not completely blocked by preincubation with antigenic peptide. Ordinate, migration of protein molecular weight markers(Mr × 10−3).

Close modal
Fig. 6.

Correlation between sst2A-immunoreactive staining and sst2A protein expression in meningioma. Top panel, membrane preparations from 16 surgically removed meningiomas (patients 1–16) 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. Blots were developed using enhanced chemiluminescence. Ordinate, migration of protein molecular weight markers(Mr × 10−3). 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 the immunoreactive score was determined according to the criteria described in “Materials and Methods.”Note that there was an excellent correlation (P <0.001) between sst2A-immunoreactive staining and sst2A protein expression. The correlation coefficient and statistical significance were determined using the Spearman test.

Fig. 6.

Correlation between sst2A-immunoreactive staining and sst2A protein expression in meningioma. Top panel, membrane preparations from 16 surgically removed meningiomas (patients 1–16) 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. Blots were developed using enhanced chemiluminescence. Ordinate, migration of protein molecular weight markers(Mr × 10−3). 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 the immunoreactive score was determined according to the criteria described in “Materials and Methods.”Note that there was an excellent correlation (P <0.001) between sst2A-immunoreactive staining and sst2A protein expression. The correlation coefficient and statistical significance were determined using the Spearman test.

Close modal
Table 1

Somatostatin receptor-like immunoreactivity in meningioma

PatientAge (yrs)GenderDiagnosisaHistological gradesst1sst2Asst2Bsst3sst4sst5
50 G1 b − − − 
49 G1 − ++ − − − 
74 G1 − − − − 
G1 − − − − − − 
72 G1 − − − − − 
61 G1 − − − − − − 
86 G1 − +++ − − − − 
68 G1 − +++ − − 
68 G1 − ++ − − − 
10 67 G1 − +++ − − − 
11 47 G1 +++ 
12 65 G1 − − − − − − 
13 73 G1 − − − − − 
14 64 G1 − +++ − − − 
15 47 G1 − − − − 
16 42 G1 − +++ − − − 
17 62 MC G1 − ++ − − − 
18 80 G1 − ++ − − − − 
19 61 G1 − − − − − 
20 74 G1 − − − − − − 
21 58 G1 − − − − − 
22 61 G1 − ++ − − − − 
23 65 G1 − − − − − − 
24 60 G1 − − − − − 
25 82 G2 +++ − − − 
26 44 G1 − +++ − − − − 
27 46 G1 − ++ − − − 
28 56 G1 − ++ − − − − 
29 51 G1 − − − − 
30 51 G1 − − − − − − 
31 64 G1 − ++ − 
32 64 G1 − ++ − − − − 
33 61 G1 − − − − 
34 75 PS G1 − ++ − − − 
35 62 G1 − +++ − 
36 64 G1 − − − − − 
37 30 G1 − − − − − − 
38 46 G1 − +++ − − − 
39 64 G1 − − − − − − 
40 64 G1 − ++ − − 
PatientAge (yrs)GenderDiagnosisaHistological gradesst1sst2Asst2Bsst3sst4sst5
50 G1 b − − − 
49 G1 − ++ − − − 
74 G1 − − − − 
G1 − − − − − − 
72 G1 − − − − − 
61 G1 − − − − − − 
86 G1 − +++ − − − − 
68 G1 − +++ − − 
68 G1 − ++ − − − 
10 67 G1 − +++ − − − 
11 47 G1 +++ 
12 65 G1 − − − − − − 
13 73 G1 − − − − − 
14 64 G1 − +++ − − − 
15 47 G1 − − − − 
16 42 G1 − +++ − − − 
17 62 MC G1 − ++ − − − 
18 80 G1 − ++ − − − − 
19 61 G1 − − − − − 
20 74 G1 − − − − − − 
21 58 G1 − − − − − 
22 61 G1 − ++ − − − − 
23 65 G1 − − − − − − 
24 60 G1 − − − − − 
25 82 G2 +++ − − − 
26 44 G1 − +++ − − − − 
27 46 G1 − ++ − − − 
28 56 G1 − ++ − − − − 
29 51 G1 − − − − 
30 51 G1 − − − − − − 
31 64 G1 − ++ − 
32 64 G1 − ++ − − − − 
33 61 G1 − − − − 
34 75 PS G1 − ++ − − − 
35 62 G1 − +++ − 
36 64 G1 − − − − − 
37 30 G1 − − − − − − 
38 46 G1 − +++ − − − 
39 64 G1 − − − − − − 
40 64 G1 − ++ − − 
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

+++, strong; ++, moderate; +, weak; −,negative immunostaining.

We thank M. Albrecht, D. Nüβ, and D. Wiborny for skillful technical assistance and Dr. F-W. Röhl for help with statistical analysis.

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