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Molecular Oncology, Markers, Clinical Correlates |
Departments of Pharmacology and Toxicology [St. S., M. H., V. H.], Neurosurgery [S. U. P., R. F.], Obstetrics and Gynecology [So. S.], and Neuropathology [K. D.], Otto-von-Guericke-University, 39120 Magdeburg, Germany
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 situ hybridization, 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 sst5 and 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.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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.
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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 x 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, 1
µM pepstatin, 10 µg/ml leupeptin, and 2 µg/ml
aprotinin]. The homogenate was spun at 500 x g for 5
min at 4°C to remove unbroken cells and nuclei. Membranes were then
pelleted at 20,000 x g for 30 min at 4°C. Membranes
were then dissolved in lysis buffer [150 mM
NaCl, 5 mM EDTA, 3 mM EGTA,
and 20 mM HEPES (pH 7.4) containing 4 mg/ml
dodecyl-ß-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).4
Extracts 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 
2 test, Fisher’s exact
test, and Spearman test.
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RESULTS |
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, 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.
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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 Mr
52,000–63,000 for sst1,
Mr 62,000–72,000 for
sst2A, Mr
60,000–75,000 for sst3,
Mr 40,000–50,000 for
sst4, and Mr
54,000–63,000 for sst5. These bands were no
longer detected when the antisera were preincubated with their cognate
peptides (data not shown).
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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 sst1
was 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 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 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.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 sst2A protein 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 sst2A protein 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.). 
2 To whom requests for reprints should be
addressed, at Department of Pharmacology and Toxicology,
Otto-von-Guericke-University, Leip-ziger Strasse 44, 39120
Magdeburg, Germany. Fax: 49-391-671-5869; E-mail: Volker.Hoellt{at}medizin.uni-magdeburg.de
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.
Received 11/ 2/99; revised 1/24/00; accepted 1/31/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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