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Reassessment of sst₂ Somatostatin Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-1

Department of Pharmacology (T.F., C.D., S.J., S.S.), Julius-Maximilians-University, H 97078 Würzburg, Germany; Department of Pharmacology and Toxicology (C.D., S.S.), Friedrich-Schiller-University, 07743 Jena, Germany; and Department of Pharmacology and Toxicology (A.K., R.S.), Otto-von-Guericke-University, 39120 Magdeburg, Germany

Address all correspondence and requests for reprints to: Stefan Schulz, Department of Pharmacology and Toxicology, Friedrich-Schiller-University, Nonnenplan 4, 07743 Jena, Germany. E-mail: Stefan.Schulz{at}mti.uni-jena.de.

	Abstract

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Objective: The overexpression of somatostatin receptor 2 (sst₂) in neuroendocrine tumors is the molecular basis for diagnostic and therapeutic application of the stable somatostatin analog octreotide. Recent evidence has shown that the immunocytochemical evaluation of sst_2A status is of value for predicting response to octreotide therapy and disease prognosis. However, due to the lack of monoclonal and limited availability of specific polyclonal anti-sst_2A antibodies, only very few patients can currently benefit from in vitro sst₂ evaluation.

Methods: In the present study, we extensively characterized the novel rabbit monoclonal anti-sst_2A antibody (clone UMB-1) using tissues from sst₂-deficient mice and their wild-type littermates. UMB-1 was then subjected to a comparative study of immunohistochemistry on a series of histological specimens from formalin-fixed, paraffin-embedded human tumors and adjacent normal tissues.

Results: Immunoprecipitation experiments unequivocally demonstrated that UMB-1 selectively detected its cognate sst_2A and did not cross-react with other proteins present in crude tissue homogenates. The UMB-1 monoclonal antibody, when compared with currently available polyclonal antisera, yielded several times more effective immunohistochemical staining of fixed-embedded tissues with a predominance of plasma membrane staining and very low cytoplasmic signal even without heat-based antigen retrieval. In addition, dual immunofluorescence revealed for the first time that the sst_2A is present on not only gastrin-containing but also ghrelin-containing cells in human gastric mucosa.

Conclusion: Thus, the rabbit monoclonal antibody UMB-1 may prove of great value in the assessment of sst_2A status in human neuroendocrine tumors during routine histopathological examination.

	Introduction

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The biological actions of somatostatin are mediated by six distinct G protein-coupled receptors encoded by five genes, named sst₁ through sst₅. The somatostatin receptor 2 (sst₂) exists in two variants in rodents: the unspliced sst_2A form and the spliced sst_2B form with a different carboxy terminus. So far, however, only the sst_2A variant has been found in humans. The sst_2A is highly expressed at the plasma membrane of human tumors including pancreatic, gastrointestinal, and pulmonary neuroendocrine tumors, pituitary adenomas, breast carcinomas, meningiomas, neuroblastomas, medulloblastomas, pheochromocytomas, and paragangliomas. This observation has led to the clinical application of stable somatostatin analogs for tumor imaging and tumor therapy. Scintigraphy of sst₂-positive gastroenteropancreatic neuroendocrine tumors with radiolabeled somatostatin analogs is well established. These tumors have also been targeted successfully by sst₂ radiotherapy. Moreover, the somatostatin analogs octreotide and lanreotide are routinely applied to control hormonal hypersecretion of sst₂-expressing neuroendocrine tumors including pituitary adenomas, carcinoids, insulinomas, and glucagonomas.

To evaluate whether a patient is a candidate for in vivo sst₂ targeting, it is of great advantage to know the sst₂ expression in the tumor. It is well established that immunohistochemistry is a fast and reliable method for in vitro sst_2A assessment in human tumors (1, 2, 3, 4). Several recent studies have demonstrated that the clinical response to octreotide in acromegalic patients corresponds well to sst_2A expression (5, 6, 7, 8). Moreover, immunohistochemical sst_2A evaluation in gastroenteropancreatic neuroendocrine tumors has been shown to be of value for predicting the outcome of somatostatin receptor scintigraphy, octreotide response, and disease prognosis (9, 10, 11).

However, the widespread application of immunohistochemistry for in vitro sst_2A evaluation has been hampered by the lack of monoclonal and the limited availability of specific polyclonal anti-sst_2A antibodies, i.e. among many commercially available polyclonal anti-sst_2A antibodies, only one has been found to be useful for routine immunohistochemical assessment of sst_2A in formalin-fixed human tumors (12).

In the present study, we demonstrate that the new rabbit monoclonal anti-sst_2A antibody UMB-1 selectively detects its cognate sst_2A in fixed tissues. Given the numerous advantages of rabbit monoclonal antibodies compared with currently available polyclonal antisera, the development of UMB-1 will facilitate the establishment of routine performance of sst_2A immunohistochemistry in human tumors.

	Materials and Methods

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Tumor samples

In the majority of tumors selected for this study, sst_2A expression had previously been evaluated with the rabbit polyclonal anti-sst_2A antibody, which is currently considered as the gold standard in sst_2A immunohistochemistry (12). The following tumors were investigated: GH-secreting pituitary adenoma (n = 25), carcinoid (n = 24), pancreatic insulinoma (n = 8), pheochromocytoma (n = 14), breast carcinoma (n = 24), ovarian carcinoma (n = 6), cervical carcinoma (n = 5), colorectal adenocarcinoma (n = 11), pancreatic adenocarcinoma (n = 4), gastric cancer (n = 4), prostate cancer (n = 2), glioblastoma (n = 4), astrocytoma (n = 8), and meningioma (n = 4). Several of the neuroendocrine tumors contained adjacent normal tissue, which was also analyzed. All tissue specimens had been fixed in formalin and were then embedded in paraffin.

Immunohistochemistry

Seven-micrometer paraffin sections were cut and floated onto positively charged slides and immunohistochemically stained as described (7, 13). For antigen retrieval, sections were microwaved in 10 mM citric acid (pH 6.0) for 20 min at 600 W. In selected experiments, antigen retrieval was omitted. Specimens were then incubated with rabbit monoclonal anti-sst_2A antibody UMB-1 at dilutions ranging from 1:10 to 1:100 overnight at 4 C. UMB-1 was obtained from Epitomics (Burlingame, CA). The identity of the immunizing peptide was ETQRTLLNGDLQTSI, which corresponds to residues 335–369 of the human sst_2A. This sequence is identical in mouse, rat, and human sst_2A. Staining of primary antibody was detected using biotinylated goat antirabbit IgG followed by incubation with avidin-biotinylated peroxidase solution. Tissue was then rinsed and stained with 3,3'-diaminobenzidine-glucose oxidase for 15 min. Cell nuclei were lightly counterstained with hematoxylin. For immunohistochemical controls, the primary antibody was either omitted or adsorbed with homologous or heterologous peptides for 2 h at room temperature. For dual immunofluorescence, sections were incubated with a mixture of rabbit monoclonal anti-sst_2A antibody UMB-1 (1:100) and either mouse monoclonal anti-somatostatin-14 antibody (1:10; Biomeda, Foster City, CA), guinea pig antigastrin antibody (1:500; Acris, Hiddenhausen, Germany), goat antighrelin antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal antiglucagon antibody (1:500; Sigma Chemical Co., St. Louis, MO), or guinea pig antiinsulin antibody (1:500; Dako, Glostrup, Denmark). Subsequently, sections were incubated with a mixture of Alexa Fluor 488-conjugated antirabbit antibody and cyanine 3.18-conjugated antimouse, antigoat, or anti-guinea pig antibodies. Specimens were mounted and examined using a Leica TCS SP5 laser scanning confocal microscope as described (14, 15).

Immunoprecipitation and Western blot

Brains from sst₂-deficient mice and their wild-type littermates were lysed in detergent buffer [20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 1 mg/ml pepstatin A, 1 mg/ml aprotinin and 10 mg/ml bacitracin], sst_2A were immunoprecipitated using UMB-1 bound to protein A agarose beads, or glycoproteins were enriched using wheat germ lectin agarose beads as described (16, 17, 18). Beads were washed five times in detergent buffer, and proteins were eluted with SDS-sample buffer for 20 min at 60 C. Samples were then subjected to 10% SDS-PAGE and immunoblotted onto nitrocellulose. Blots were incubated with rabbit monoclonal anti-sst₂ antibody UMB-1 at a dilution of 1:1000 followed by peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection (Amersham, Braunschweig, Germany).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of rabbit monoclonal anti-sst_2A antibody UMB-1

Specificity of UMB-1 was monitored in Western blot and immunoprecipitation experiments using tissues from sst₂-deficient mice and their wild-type littermates. In crude extracts from brain tissue of wild-type mice (sst₂^+/+), UMB-1 detected a broad band migrating at M_r 70,000–80,000, which corresponds to the expected size of the sst_2A (14, 16, 17) (Fig. 1A, left lane). In contrast, UMB-1 did not detect any immunoreactive band in brain extracts from sst₂-deficient mice prepared under identical conditions (Fig. 1A, right lane). When UMB-1 was bound to protein A agarose beads and then incubated with crude brain homogenates, immunoreactive sst_2A were detectable as a broad band migrating at the expected size only in extracts from sst₂^+/+ but not in extracts from sst₂^–/– mice (Fig. 1B). Two additional bands were seen in both immunoprecipitates from sst₂^–/– mice as well as in immunoprecipitates that had not been incubated with brain lysates, indicating that these bands result from detection of antibodies eluted from the beads (Fig. 1B). When forebrain sections were immunohistochemically stained, UMB-1 yielded the well-known staining pattern with prominent immunofluorescence in the deep layers of the cortex, claustrum, endopiriform nucleus, und septum in sst₂^+/+ mice but not in sst₂^–/– mice (15) (Fig. 1, C and D). The rabbit monoclonal antibody was further characterized using immunofluorescent staining of transfected cells. When HEK-293 cells stably expressing sst_2A were stained with UMB-1, prominent immunofluorescence localized at the level of the plasma membrane was detected (Fig. 1E). After incubation with somatostatin-14, immunoreactive sst_2A translocated from the plasma membrane into the cytosol, indicating rapid agonist-induced endocytosis (Fig. 1F). UMB-1 did not stain HEK-293 cells stably expressing other somatostatin receptors (not shown).

View larger version (49K): [in this window] [in a new window]   FIG. 1. Characterization of rabbit monoclonal antibody UMB-1. A and B, Western blot and immunoprecipitation analysis of the specificity of UMB-1 using brain tissues from sst2-deficient mice (sst2–/–) and their wild-type littermates (sst2+/+). Note that UMB-1 selectively detects sst2A and does not cross-react with other proteins present in tissue extracts prepared from sst2–/– mice. Ordinate, Migration of protein molecular weight markers (Mr x 10–3). IB, Immunoblot; IP, immunoprecipitation. C and D, characterization of UMB-1 by immunofluorescent staining of brain sections from sst2-deficient mice (sst2–/–) and their wild-type littermates (sst2+/+). Note that UMB-1 selectively detects sst2A and does not cross-react with other proteins present in tissue sections from sst2–/– mice. E and F, HEK-293 cells stably transfected to express sst2A were either not exposed (E) or exposed to 1 µM somatostatin-14 (SS-14) for 30 min (F), subsequently fixed, and immunofluorescently stained with UMB-1. Note that in untreated cells, prominent immunofluorescence was localized at the level of the plasma membrane and that somatostatin-14 exposure induced a rapid translocation of sst2A from the plasma membrane into the cytosol. Representative results from one of three independent experiments are shown. Scale bars, 200 µm (C and D) and 20 µm (E and F).

First, UMB-1 was compared with a polyclonal antiserum drawn at the time of splenectomy from rabbit 2269, the animal from which UMB-1 was derived. Immunohistochemical staining of normal human pancreatic tissue revealed that both rabbit polyclonal antiserum 2269 and rabbit monoclonal antibody selectively detected pancreatic islet cells (Fig. 2, A and B). However, UMB-1 yielded several times more effective immunohistochemical staining of fixed-embedded tissues with a predominance of plasma membrane staining and very low cytoplasmic signal. Initial experiments also demonstrated that in contrast to polyclonal anti-sst_2A antisera, UMB-1 detected sst_2A in fixed-embedded human tissues even without heat-based antigen retrieval (not shown). A series of 143 tissue samples was then immunohistochemically stained with UMB-1. In terms of positive tumor entities and incidence, the results obtained with UMB-1 were in excellent agreement with those obtained using the rabbit polyclonal antibodies currently considered as the gold standard, i.e. prominent staining was observed in the majority of GH-secreting pituitary adenomas, carcinoids, insulinomas, pheochromocytomas, and meningiomas (3, 7, 10, 12, 13). As depicted in Fig. 2 D, E, I, and J, robust plasma membrane staining was homogeneously observed on nearly all tumor cells in many tumors. In contrast, a few examples were observed with heterogeneous distribution of sst_2A immunoreactivity within the tumor (Fig. 2K). UMB-1 also detected sst_2A in a variety of human nonneoplastic tissues, e.g. pancreatic islet cells, a subset of cells in the anterior pituitary, neuroendocrine cells in the gastric and intestinal mucosa, and enteric ganglion cells (Fig. 2, B, C, and F–H). UMB-1 immunostaining was completely abolished by preadsorbtion of the antibody with 10 µg/ml of its immunizing peptide (Fig. 2D). Next, we performed double-labeling experiments using UMB-1 in combination with antibodies directed against somatostatin, gastrin, ghrelin, glucagon, or insulin. Dual-immunofluorescence studies of human fixed-embedded gastrointestinal tissues revealed that the majority of sst_2A-expressing neuroendocrine cells do not contain somatostatin but exist in close proximity to somatostatin-containing cells, suggesting a predominant paracrine mode of regulation (Fig. 3, upper panel). In addition, we found that sst_2A-expressing myenteric neurons were densely innervated by somatostatin-containing fibers and terminals (Fig. 3, upper panel). Within the gastric mucosa, virtually all gastrin-containing as well as all ghrelin-containing cells expressed sst_2A at their plasma membrane, indicating that sst_2A may be involved in the regulation of gastrin and ghrelin release (Fig. 3, middle panel). Finally, sst_2A is present on glucagon- and insulin-secreting cells in human pancreatic islets (Fig. 3, lower panel).

View larger version (139K): [in this window] [in a new window]   FIG. 2. UMB-1 immunohistochemical staining in human formalin-fixed and paraffin-embedded tissues. A and B, Immunohistochemical staining for sst2A in pancreatic islets using rabbit polyclonal antibody 2269 (A) and rabbit monoclonal antibody UMB-1 (B). Note that UMB-1 yielded several times more effective immunohistochemical staining than the polyclonal antiserum drawn from rabbit 2269, the animal from which UMB-1 was derived. C–K, UMB-1 immunohistochemical staining of a variety of human normal and neoplastic tissues. Sections were dewaxed, microwaved in citric acid, and incubated with UMB-1 at a dilution of 1:100. Sections were then sequentially treated with biotinylated antirabbit IgG and avidin-biotin solution. Sections were then developed in 3,3'-diaminobenzidine-glucose oxidase and lightly counterstained with hematoxylin. Note that UMB-1 detected sst2A in a subset of cells in the anterior pituitary (C), neuroendocrine cells in the gastric and intestinal mucosa (F and G), and enteric ganglion cells (H). UMB-1 also detected sst2A at the plasma membrane of nearly all tumor cells in a variety of tumors including GH-producing pituitary adenoma (D), carcinoid (E), pheochromocytoma (I), and cervical carcinoma (J). In contrast, in a few cases, UMB-1 detected a heterogeneous distribution of sst2A (K) on the tumor cells. Inset in D, Peptide absorption control. Scale bars, 100 µm (A, B, G–K), 20 µm (C and D), and 250 µm (E and F).

View larger version (53K): [in this window] [in a new window]   FIG. 3. Dual-immunofluorescence labeling of sst2A and regulatory peptide hormones. Upper panel, Double-labeling experiments using UMB-1 and an antibody directed against somatostatin-14 (SS-14). Note that the majority of sst2A-expressing neuroendocrine cells in the intestinal mucosa do not contain somatostatin but exist in close proximity to somatostatin-containing cells (upper left panel). Myenteric neurons expressing sst2A are densely innervated by somatostatin-containing fibers and terminals (upper right panel). Middle panel, Double-labeling experiments using UMB-1 and antibodies directed against gastrin or ghrelin. Note that virtually all gastrin-containing and all ghrelin-containing cells expressed sst2A at their plasma membrane. Lower panel, Double-labeling experiments using UMB-1 and antibodies directed against glucagon and insulin. Note that nearly all glucagon-containing and all insulin-containing cells expressed sst2A at their plasma membrane. Scale bar, 100 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Compared with currently available rabbit polyclonal antibodies, the rabbit monoclonal antibody UMB-1 yielded several times more effective immunohistochemical staining of fixed-embedded tissues with a predominance of plasma membrane staining and very low cytoplasmic signal. In addition, UMB-1 immunohistochemistry did not require boiling of samples for antigen retrieval, which resulted in a better preservation of morphology. The use of UMB-1 also permitted us to gain novel insights into sst_2A receptor expression and function; e.g. dual-immunofluorescence studies revealed for the first time that the sst_2A receptor is present on the plasma membrane of not only gastrin-containing but also ghrelin-containing cells in human gastric mucosa. This finding provides a morphological explanation for the observation that somatostatin inhibits ghrelin release in humans (19).

The rabbit monoclonal antibody UMB-1 will also overcome a number of limitations inherent to polyclonal antibodies; e.g. among many commercially available polyclonal anti-sst_2A antibodies, only one has been found to be useful for routine immunohistochemical assessment of sst_2A in formalin-fixed human tumors (12). Naturally, only limited amounts of the gold standard exist, and the quality of this antibody varies from batch to batch. To achieve high-quality labeling, affinity purification is often required, which limits its availability even further. Consequently, only very few patients can currently benefit from in vitro sst_2A evaluation. In contrast, the rabbit monoclonal antibody UMB-1 can be produced in unlimited amounts for unlimited time in identical high quality. Thus, the development of UMB-1 will now allow the establishment of guidelines for the routine performance and interpretation of sst_2A immunohistochemistry in a variety of human tumors.

	Footnotes

 
Disclosure Summary: T.F., C.D., S.J., A.K., R.S., and S.S. have nothing to declare.

First Published Online August 12, 2008

Abbreviation: sst₂, Somatostatin receptor 2.

Received May 16, 2008.

Accepted August 5, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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