Immunocytochemical Identification of VPAC₁, VPAC₂, and PAC₁ Receptors in Normal and Neoplastic Human Tissues with Subtype-Specific Antibodies

Several human tumors overexpress receptors for small regulatory peptides, e.g., somatostatin, bombesin, vasoactive intestinal peptide (VIP), or pituitary adenylate cyclase-activating peptide (PACAP), an observation which has led to a number of clinical applications in the diagnosis and treatment of tumors (1, 2, 3, 4, 5, 6). VIP is a 28-amino acid neuropeptide, which is widely distributed throughout the brain and periphery, and which structurally belongs to the glucagon-growth hormone releasing factor-secretin superfamily (7, 8, 9). PACAP was first isolated from ovine hypothalamic extracts on the basis of its ability to stimulate cAMP formation in anterior pituitary cells (10). PACAP has recently been shown to exert potent gastrointestinal effects (8, 11). The 175-amino acid pro-PACAP precursor molecule is tissue-specifically processed to either PACAP38 or PACAP27 (8, 11).

The biological actions of VIP and PACAP are mediated by a family of three G protein-coupled receptors, which are designated vasoactive intestinal peptide receptor (VPAC)₁, VPAC₂, and pituitary adenylate cyclase-activating peptide receptor (PAC)₁ (5, 12, 13). The PAC₁ receptor exhibits lower affinity for VIP than for PACAP, whereas the VPAC₁ and VPAC₂ receptors exhibit similar affinities for VIP and PACAP (5, 12, 13, 14, 15). Several observations suggest that VIP/PACAP receptors may be potential targets for tumor imaging and treatment with labeled or unlabeled VIP and PACAP analogs. First, in vitro binding studies have shown that VIP/PACAP receptors are expressed in the great majority of most frequently occurring human tumors, including breast, ovarian, colon, insulinoma, carcinoid, pancreas, glioblastoma, meningioma, pituitary adenoma, and pheochromocytoma (16, 17, 18, 19, 20, 21, 22). Second, VIP/PACAP receptor-positive intestinal and endocrine tumors can be visualized by in vivo VIP receptor scintigraphy (1, 4, 6, 23, 24, 25, 26). Third, application of VIP and PACAP analogs modulates tumor growth in animal models (2, 3, 27, 28, 29, 30, 31, 32, 33).

The expression of VIP/PACAP receptors in human tumors has previously been detected with binding autoradiography or reverse transcription-PCR (16, 17, 18, 19, 20, 21, 22). However, the diagnostic value of these methods is limited. The determination of the VIP/PACAP receptor status with in vitro autoradiography in routine tumor diagnostic is costly and time-consuming. Given the limited number of selective ligands, it is also difficult to differentiate between individual VIP/PACAP receptor subtypes. Conversely, with reverse transcription-PCR, it is possible to discriminate between VIP/PACAP receptor subtypes; however, this method is based on total RNA isolation from a fresh tumor sample and therefore would not only detect VIP/PACAP receptor transcripts originating from tumor cells but also from fibrocytes, lymphocytes, or other nonmalignant cells. Progress on this front has been hampered by the lack of specific antibodies for immunohistochemical detection of VIP/PACAP receptor proteins.

In the present study, we have generated and characterized a panel of antibodies directed to the COOH-terminal sequences of VIP/PACAP receptor subtypes VPAC₁, VPAC₂, and PAC₁. We have also developed an immunohistochemical protocol that allows efficient detection of these receptors in formalin-fixed, paraffin-embedded human tissues. The generation of these novel antibodies enabled us to determine the cellular distribution of VIP/PACAP receptor proteins in a variety of human tumors and their tissues of origin.

Ninety-eight tumor specimens were retrieved from the archives of the Departments of Pathology and Neuropathology. All of the tissue specimens had been fixed in formalin and embedded in paraffin. The following tumors were investigated: colorectal adenocarcinoma (n = 5); ductal pancreatic adenocarcinoma (n = 5); diffuse type gastric cancer (n = 9); intestinal type gastric cancer (n = 8); hepatocellular carcinomas (n = 10); breast carcinoma (n = 4); ovarian carcinoma (n = 10); prostate cancer (n = 4); thyroid carcinoma (n = 6); carcinoid (n = 15); panreatic insulinoma (n = 8); pituitary adenoma (n = 4); pheochromocytoma (n = 2); glioblastoma (n = 4); and meningioma (n = 4). Several of the neuroendocrine tumors contained adjacent normal tissue, which was also analyzed. In addition, several fresh tumor specimens were immediately frozen in liquid N₂ and stored at −70°C until Western blot analysis. The following tumors were investigated: colorectal carcinoma (n = 6); breast carcinoma (n = 8); ovarian carcinoma (n = 8); thyroid carcinoma (n = 4); pituitary adenoma (n = 4); glioblastoma (n = 8); and meningioma (n = 4).

Polyclonal antisera were generated against the COOH-terminal tails of the VIP/PACAP receptor subtypes VPAC₁, VPAC₂, and PAC₁. The identity of the peptides is given in Table 1. Peptides were synthesized, purified, and coupled to keyhole limpet hemocyanin as described previously. The conjugates were mixed 1:1 with Freund’s adjuvant and injected into groups of three rabbits each; 9019 to 9021 for VPAC₁, 9022 to 9024 for VPAC₂, and 9025 to 9027 for PAC₁ antisera production. Animals were given injections at 4-week intervals, and serum was obtained 2 weeks after immunizations beginning with the second injection. The specificity of the antisera as well as possible cross-reactivity with other VIP/PACAP receptor subtypes was initially tested with immuno-dot blot analysis as described previously (34, 35). For subsequent analysis, antibodies were affinity purified against their immunizing peptides with the Sulfo-Link coupling gel according to the manufacturer’s instructions (Pierce, Rockford, IL).

Plasmids encoding VPAC₁ or VPAC₂ and Chinese hamster ovary (CHO) cells stably expressing PAC₁ were kindly provided by Dr. M. Waelbroeck (Brussels, Belgium). Human embryonic kidney 293 (HEK-293) cells were stably transfected with either VPAC₁ or VPAC₂. Cells were grown on coverslips overnight and either not exposed or exposed to 1 μmol/L VIP or 1 μmol/L PACAP27 (Bachem, Weil am Rhein, Germany). Cells were then fixed and incubated with 1 μg/mL anti-VPAC₁ (9020), anti-VPAC₂ (9023), or anti-PAC₁ (9027) antibodies followed by cyanin 3.18-conjugated secondary antibodies (Amersham, Braunschweig, Germany). Specimens were mounted and examined with a Leica TCS-NT laser scanning confocal microscope as described previously (34, 36, 37).

Membranes were prepared from stably transfected HEK-293 or CHO cells as well as fresh tumor specimens. Cells and tissues were lysed in homogenization buffer [5 mmol/L EDTA, 3 mmol/L EGTA, 250 mmol/L sucrose, and 10 mmol/L Tris-HCl (pH 7.6) containing 1 mmol/L phenylmethylsulfonylfluoride, 1 μmol/L pepstatin, 10 μg/mL leupeptin, and 2 μg/mL aprotinin], and membranes were pelleted at 20,000 × g for 30 minutes at 4°C. Membranes were then dissolved in lysis buffer [150 mmol/L NaCl, 5 mmol/L EDTA, 3 mmol/L EGTA, and 20 mmol/L HEPES (pH 7.4) containing 4 mg/mL dodecyl-β-maltoside and proteinase inhibitors as describe above] and incubated with 150 μL wheat germ lectin agarose beads (Amersham) for 90 minutes at 4°C. Beads were washed five times in lysis buffer, and adsorbed glycoproteins were eluted with SDS-sample buffer for 20 minutes at 60°C. When indicated, adsorbed glycoproteins were subjected to enzymatic deglycosylation with peptide N-glycosidase F (PNGase F) according to the manufacturer’s protocol (New England BioLabs, Beverly, MA). Samples were then subjected to 8% SDS polyacrylamide gel electrophoresis and immunoblotted onto nitrocellulose. Blots were incubated with 1 μg/mL anti-VPAC₁ (9020), anti-VPAC₂ (9023), or anti-PAC₁ (9027) antibodies followed by peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection (Amersham). For adsorption controls, antisera were preincubated with 10 μg/mL of their cognate peptides for 2 hours at room temperature.

Seven-μm paraffin sections were cut and floated onto positively charged slides and immunohistochemically stained as described previously (34, 38, 39). Briefly, sections were dewaxed, microwaved in 10 mmol/L citric acid (pH 6.0) for 20 minutes at 600 W, and subsequently incubated with 2 μg/mL anti-VPAC₁ (9020), anti-VPAC₂ (9023), or anti-PAC₁ (9027) antibodies overnight at 4°C. Staining of primary antibody was detected with biotinylated goat antirabbit IgG or biotinylated goat antimouse IgG followed by an incubation with avidin-biotinylated peroxidase solution. Tissue was then rinsed and stained with 3,3′-diaminobenzidine-glucose oxidase for 15 minutes. Cell nuclei were lightly counterstained with hematoxylin. For immunohistochemical controls, the primary antibody was either omitted, replaced by preimmune sera, or adsorbed with several concentrations ranging from 1 to 10 μg/mL of homologous or heterologous peptides for 2 hours at room temperature. A tumor known to stain positively was included in each batch of staining as a positive control.

Specificity of the antisera was monitored with Western blot analysis. When membrane preparations from stable transfected cells were electrophoretically separated and blotted onto nitrocellulose, the antisera 9020 (anti-VPAC₁), 9023 (anti-VPAC₂), and 9027 (anti-PAC₁) revealed broad bands in cells transfected with their cognate VIP/PACAP receptor subtype (Fig. 1). In HEK-293 cells stably expressing VPAC₁, the anti-VPAC₁ antibody (9020) detected a broad band migrating at M_r 55,000 to 70,000 (Fig. 1,A). In HEK-293 cells stably expressing VPAC₂, the anti-VPAC₂ antibody (9023) detected a single broad band migrating at M_r 50,000 to 65,000 (Fig. 1,B). In CHO cells stably expressing PAC₁, the anti-PAC₁ antibody detected a broad band migrating at M_r 40,000 to 60,000 (Fig. 1,C). Enzymatic deglycosylation with PNGase F resulted in shift of molecular size and appearance of all three VIP/PACAP receptor proteins (Fig. 1). Antisera were additionally characterized with immunofluorescent staining of transfected cells. When HEK-293 cells stably expressing VPAC₁ or VPAC₂ and CHO cells stably expressing PAC₁ were stained with anti-VPAC₁ (9020), anti-VPAC₂ (9023), or anti-PAC₁ (9027) antibodies, prominent immunofluorescence localized at the level of the plasma membrane was detected (Fig. 2, A, C, and E). After incubation with VIP or PACAP27, VPAC₁-, VPAC₂-, and PAC₁-immunoreactivity was translocated from the plasma membrane into the cytosol, indicating that all of the VIP/PACAP receptor subtypes were rapidly endocytosed in an agonist-dependent manner (Fig. 2, B, D, and F). Whereas VPAC₁ and VPAC₂ were internalized in response to both VIP and PACAP27, PAC₁ was selectively internalized in response to PACAP27 (data not shown). Next, the antisera were tested for possible cross-reactivity with other proteins present in human tissues. When membrane preparations from a variety of human tumors were electrophoretically separated and blotted onto nitrocellulose, the antisera revealed broad receptor-like bands with molecular weights similar to that observed for recombinant VIP/PACAP receptors, e.g., the anti-VPAC₁ antibody (9020) detected a broad band migrating at M_r 55,000 to 75,000 in an ovarian carcinoma (Fig. 3,A), the anti-VPAC₂ antibody (9023) detected a broad band migrating at M_r 60,000 to 75,000 in a breast carcinoma (Fig. 3,B), and the anti-PAC₁ antibody (9027) detected a broad band migrating at M_r 50,000 to 65,000 in a glioblastoma (Fig. 3,C). Immunoreactive bands for each antiserum were completely abolished by preabsorbtion with 10 μg/mL of their immunizing peptides (Fig. 3, A–C).

The anti-VPAC₁ (9020), anti-VPAC₂ (9023), and anti-PAC₁ (9027) antibodies were subjected to immunohistochemical staining of a variety of human tissues. Initial experiments showed that heat-induced epitope retrieval is required for efficient immunohistochemical staining of paraffin-embedded tissue (data not shown). Many neuroendocrine tumors contained adjacent noncancerous tissue, which enabled us to analyze the distribution of VIP/PACAP receptors in several parts of the normal gastrointestinal tract. Prominent localizations of VPAC₁, VPAC₂, and PAC₁ receptors in the small intestine are shown in Fig. 4. VPAC₁ receptor immunoreactivity was abundant in the mucosa (Fig. 4,A). The highest densities of immunoreactive VPAC₁ receptors were observed in epithelial cells located in the basal portion of the crypts (Fig. 4,A). In epithelial cells, VPAC₁ receptor immunoreactivity was predominantly confined to the plasma membrane (Fig. 4,B). In contrast, VPAC₂ receptor immunoreactivity was not seen in epithelial cells but selectively localized to neuroendocrine cells (Fig. 4,C). In neuroendocrine cells, immunoreactive VPAC₂ receptors were distributed throughout the cytosol (Fig. 4,C). VPAC₂ receptor immunoreactivity was also seen in smooth muscle of the gastrointestinal tract and blood vessels (Fig. 4,D). VPAC₁ receptor immunoreactivity was observed in myenteric neurons as well as in fibers distributed to the muscle layer (Fig. 4,E). In addition, VPAC₁ receptor immunoreactivity was abundant in neurons of the mucosal plexus (data not shown). PAC₁ receptor immunoreactivity was also observed in many neurons of the myenteric plexus (Fig. 4,F). The anti-VPAC₁ (9020), anti-VPAC₂ (9023), and anti-PAC₁ (9027) antibodies were then subjected to immunohistochemical staining of 98 human tumors. All three antibodies yielded prominent staining predominantly localized to the plasma membrane of the tumor cells (Fig. 5). The staining intensity for each antibody varied greatly between individual tumors giving consistently different sample-specific patterns of VIP/PACAP receptor subtype expression under otherwise identical conditions. Immunostaining for each antiserum was completely abolished by preadsorbtion with 10 μg/mL of the immunizing peptides (Fig. 5). Table 2 summarizes the incidence of VPAC₁, VPAC₂, and PAC₁ receptors in a series of human tumors, including some of the most frequently occurring carcinomas. The VPAC₁, VPAC₂, or PAC₁ receptors were found in every tumor except thyroid cancer, which was completely devoid of any of the VIP/PACAP receptors (Table 2). The VPAC₁ receptor was found most commonly in cancers of the breast, pancreas, and prostate, as well as in pituitary adenomas. The VPAC₂ receptor was commonly expressed in meningiomas and less frequently in carcinoids, insulinomas, gastric cancers, and breast cancers. The VPAC₂ receptor was also found in a single pheochromocytoma. The PAC₁ receptor was most commonly expressed by neuroendocrine tumors, meningiomas, and breast cancer, and was rarely found in cancers of the stomach, liver, colorectum, thyroid, or prostate. A uniform highly abundant expression of all of the VIP/PACAP receptor subtypes was only evident in glioblastomas (Table 2). VPAC₁, VPAC₂, and PAC₁ were also present at the plasma membrane of neuronal cell bodies and processes adjacent to the glioblastomas (data not shown).

In an effort to study the pattern of VIP/PACAP receptor protein expression in normal and neoplastic human tissues, we generated antibodies that exert selective specificity for VPAC₁, VPAC₂, and PAC₁ receptors. We show that the cytoplasmic tails of these receptors can serve as an epitope for the generation of antisera that effectively stain formalin-fixed, paraffin-embedded human tissues. Several lines of evidence indicate that these antibodies specifically detect their targeted VIP/PACAP receptor and do not cross-react. First, in Western blots of membranes from receptor-transfected cells, the anti-VPAC₁, anti-VPAC₂, and anti-PAC₁ antibodies detected broad bands migrating at M_r 50,000 to 70,000. Second, anti-VPAC₁, anti-VPAC₂, and anti-PAC₁ antibodies revealed prominent cell surface staining of VIP/PACAP receptor-transfected cells. This immunostaining translocated from the cell surface into the cytosol after agonist exposure, indicating rapid endocytosis of all three VIP/PACAP receptor subtypes. Third, in Western blots of membranes from receptor-expressing tumors, the anti-VPAC₁, anti-VPAC₂, and anti-PAC₁ antibodies detected single bands migrating at the appropriate molecular weight. Fourth, tissue immunostaining of all three antisera was completely abolished by preadsorbtion with homologous but not heterologous peptides. Finally, it should be noted that two of three VPAC₁, three of three VPAC₂, and two of three PAC₁ antisera gave similar results.

The availability of subtype-specific VIP/PACAP receptor antibodies will facilitate additional basic morphologic investigation of VPAC₁, VPAC₂, and PAC₁ receptor expression in human tumors and normal human tissues. The immunohistochemical VIP/PACAP receptor evaluation offers several major advantages. This new method can analyze VIP/PACAP receptors in routinely processed archival paraffin-embedded material of any diagnostic pathology center. It requires only an immunopathological laboratory to perform the test, which can be carried out without costly and time-consuming receptor autoradiography. The immunohistochemical evaluation of the complete VIP/PACAP receptor status of a given tumor specimen can be accomplished in <24 hours. Although one could expect the immunohistochemical VIP/PACAP receptor determination to be less sensitive than receptor autoradiography with ¹²⁵I-labeled VIP, the sensitivity of the anti-VPAC₁ (9020), anti-VPAC₂ (9023), and anti-PAC₁ (9027) antibodies generated and characterized in this study was sufficiently high enough to detect these receptors in known human VIP/PACAP target tissues, i.e., detection of VPAC₁ in gastrointestinal mucosa and VPAC₂ in smooth muscle of the gastrointestinal tract. In addition, the immunohistochemical VIP/PACAP receptor determination provided a better histologic quality and cellular resolution compared with receptor autoradiography. Thus, previously unappreciated cellular localizations of VIP/PACAP receptors were uncovered, i.e., presence of VPAC₁ in myenteric nerve fibers and VPAC₂ in neuroendocrine cells.

Although VPAC₁, VPAC₂, and PAC₁ were all highly abundant throughout the gastrointestinal tract, the various VIP/PACAP receptor subtypes exhibited complementary expression patterns. Previous studies have shown that VIP immunoreactivity is present in neuroendocrine cells as well as in submucous and myenteric ganglia of the human gut (40, 41, 42). VIP immunoreactivity is also present in mucosal and myenteric nerve fibers as well as in nerve fibers innervating blood vessels (40, 41, 42). PACAP immunoreactivity exhibits a nearly identical distribution (40, 41, 42). In fact, dual immunofluorescence studies revealed a high degree of coexistence of the two peptides (40, 41, 42). Thus, the present findings suggest distinct roles of individual VIP/PACAP receptor subtypes in gastrointestinal physiology. VPAC₁ in the mucosa is targeted by VIP and PACAP released from mucosal nerve fibers and neuroendocrine cells and is possibly involved in the regulation of secretion. VPAC₂ in the smooth muscle of the gastrointestinal wall is targeted by VIP and PACAP released from myenteric nerve fibers and is probably involved in the regulation of motor activity. Conversely, VPAC₂ in the smooth muscles of blood vessels may regulate gastrointestinal blood flow. VPAC₁ in myenteric nerve fibers is likely to function as autoreceptor, regulating the release of VIP and PACAP. VPAC₂ in neuroendocrine cells may also function as autoreceptor, which may explain the high levels of internalized receptors detected in these cells. Given that PAC₁ has a higher affinity for PACAP than for VIP, it is conceivable that myenteric neurons bearing PAC₁ are selectively responsive to PACAP.

VIP and PACAP can affect the growth of human tumors cells in vivo and in vitro (2, 3, 27, 28, 29, 30, 31, 32, 33). Whereas growth-promoting activities have been reported for VIP, growth-inhibiting properties have been found for VIP antagonists in various tumor models (2, 3, 27, 28, 29, 30, 31, 32, 33). VIP/PACAP receptors are detectable in the great majority of most frequently occurring human tumors, including breast, ovarian, colon, insulinoma, carcinoid, pancreas, glioblastoma, meningioma, pituitary adenoma, and pheochromocytoma. It is therefore very tempting to suggest the use of growth-inhibiting VIP/PACAP analogs for the treatment of human tumors (16, 17, 18, 19, 20, 21, 22). Moreover, a number of clinical and preclinical studies suggest that VIP/PACAP receptors may be promising molecular targets for tumor imaging and targeted radiotherapy (1, 4, 6, 23, 24, 25, 26). However, previous and present evidence indicates a highly abundant expression of VIP/PACAP receptors in normal human target tissues. Thus, for potential clinical application of VIP/PACAP analogs, the ratio of receptor expression in neoplastic versus normal tissues should be determined with subtype-specific VIP/PACAP receptor antibodies.

In conclusion, we have generated and extensively characterized anti-VPAC₁, anti-VPAC₂, and anti-PAC₁ antibodies. Using these antibodies, we provide the first demonstration of all of the VIP/PACAP receptor subtypes in human formalin-fixed, paraffin-embedded tissues. It is now possible to determine the exact cellular and subcellular sites of VIP/PACAP receptor protein expression in normal human tissues, which is needed for a better understanding of VIP/PACAP actions in physiologic target tissues. The rapid immunocytochemical VIP/PACAP receptor visualization may also be helpful to identify those tumors with a sufficient receptor overexpression for diagnostic or therapeutic intervention.

We thank Beate Peter and Dana Mayer for skillful technical assistance and Dr. M. Waelbroeck for plasmids encoding VPAC₁ or VPAC₂ and CHO cells stably expressing PAC₁.