Immunolocalization of Full-length NK 1 Tachykinin Receptors in Human Tumors

Abstract

Biological effects of substance P (SP) are mediated by the neurokinin-1 (NK₁) receptor that exists as a full-length and as a carboxy-terminally truncated isoform in humans. Although NK₁ receptor mRNA and binding sites have been detected in certain malignancies, little is known about the cellular and subcellular localization of NK₁ receptor protein in human neoplastic tissues. We developed and characterized a novel anti-peptide antibody to the carboxy-terminal region of the human full-length NK₁ receptor. Specificity of the anti-serum was demonstrated by (1) detection of a broad band migrating at molecular mass 70,000-90,000 Da in Western blots of membranes from NK₁-expressing tissues; (2) cell-surface staining of NK₁-transfected cells; (3) translocation of NK₁ receptor immunostaining after SP exposure; and (4) abolition of tissue immunostaining by preadsorption of the antibody with its immunizing peptide. Distribution of NK₁ receptors was investigated in 72 formalin-fixed, paraffin-embedded human tumors showing that NK₁ receptors were frequently expressed in glioblastomas and breast and pancreatic carcinomas. Immunoreactive NK₁ receptors were clearly confined to the plasma membrane and uniformly present on nearly all tumor cells. Development of this novel NK₁ receptor antibody allows the efficient localization of NK₁ receptor protein in human formalin-fixed, paraffin-embedded tissues. NK₁ receptor visualization with this simple and rapid immunohistochemical method will facilitate identification of tumors with a sufficient receptor overexpression for diagnostic or therapeutic intervention using SP analogs.

Substance P (SP) is an undecapeptide belonging to the tachykinin family of peptides. SP is widely distributed in both central and peripheral nervous systems, where it plays a well-established role in neurotransmission, vasodilatation, and motor responses (Maggi et al. 1993). SP and neurokinin A (NKA) are derived from the preprotachykinin A gene, whereas neurokinin B (NKB) is derived from the preprotachykinin B gene. Biological action of tachykinin peptides is mediated by a family of three G-protein-coupled receptors, namely, NK₁, NK₂, and NK₃, which are preferentially activated by SP, NKA, and NKB, respectively (Maggi et al. 1993). Two NK₁ receptor isoforms have been reported in humans, a full-length receptor (NK₁-Fl) and a truncated receptor (NK₁-Tr) that lacks 100 residues in its cytoplasmic tail (Fong et al. 1992).

NK₁ receptor mRNA and binding sites have been detected in a relatively high percentage in glioblastomas and breast and pancreatic cancers (Johnson et al. 1991; Hennig et al. 1995; Winkler et al. 1995; Ehlers et al. 2000; Singh et al. 2000; Friess et al. 2003). Activation of NK₁ receptors by SP triggers signaling pathways, e.g., extracellular signal-regulated protein kinases (ERK1/2), which are potentially relevant for tumor growth and progression (Luo et al. 1996; Fukuhara et al. 1998; Palma et al. 1999b; Palma and Maggi 2000; Koon et al. 2004). Conversely, administration of NK₁ receptor antagonists has been reported to inhibit tumor cell growth in vitro and in vivo (Palma et al. 1999a,2000; Munoz et al. 2004,2005).

Although expression of NK₁ receptors in human tumors has been well documented, the exact cellular and subcellular sites of NK₁ receptor in human neoplastic tissues still need to be fully elucidated. Expression of NK₁ receptors has previously been detected using binding autoradiography or reverse transcription-polymerase chain reaction (RT-PCR) (Hennig et al. 1995; Ehlers et al. 2000; Singh et al. 2000). However, anatomical resolution of autoradiographic receptor binding is limited, and RT-PCR does not discriminate between receptor transcripts originating from individual target cells.

In the present study we have generated and characterized antibodies directed to the carboxy-terminal tail of the NK₁-Fl receptor. We have also developed an immunohistochemical (IHC) protocol that allows efficient detection of this receptor in formalin-fixed, paraffin-embedded human tissues. Generation of this novel antibody enabled us to determine the cellular distribution of NK₁ receptor proteins in a variety of human tumors.

Materials and Methods

Patients, Tumors, and Tissue Preparation

Seventy two tumor specimens were retrieved from the archives of the Department of Pathology. All 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), ductal invasive breast carcinoma (n=5), ovarian carcinoma (n=10), prostate cancer (n=4), thyroid carcinoma (n=6), carcinoid (n=15), pancreatic insulinoma (n=8), growth-hormone-producing pituitary adenoma (n=4), pheochromocytoma (n=2), glioblastoma (n=4), and meningioma (n=4). In addition, several fresh tumor specimens were immediately frozen in liquid N₂ and stored at −70C until Western blot analysis. The following tumors were investigated: breast carcinoma (n=4) and ovarian carcinoma (n=4).

Generation and Purification of Antipeptide Antibodies

Polyclonal antisera were generated against the carboxy-terminal tail of the NK₁ receptor. Identity of the peptide was SSRSDSKTMTESFSFSSNVLS, which corresponds to amino acids 387-407 of the NK₁-Fl receptor. The peptide was synthesized, purified, and coupled to keyhole limpet hemocyanin as described (Schulz et al. 2000). The conjugate was mixed 1:1 with Freund's adjuvant and injected into groups of three rabbits {9040-9042} for NK₁ antisera production. Animals were injected at 4-week intervals, and serum was obtained 2 weeks after immunizations beginning with the second injection. Specificity of the antisera was initially tested using immunodot-blot analysis as described (Schulz et al. 2000). For subsequent analysis, antibodies were affinity purified against their immunizing peptide using the Sulfo-Link coupling gel according to manufacturer's instructions (Pierce; Rockford, IL).

Immunocytochemistry

Human embryonic kidney 293 (HEK-293) cells were stably transfected with the human NK₁ receptor. Cells were grown on coverslips overnight and either not exposed or exposed to 1 μM SP (Bachem; Weil am Rhein, Germany). Cells were then fixed and incubated with 1 μg/ml anti-NK₁ {9042} antibodies followed by cyanin 3.18-conjugated secondary antibodies (Amersham; Braunschweig, Germany). Specimens were mounted and examined using a Leica TCS-NT laser scanning confocal microscope (Leica; Wetzlar, Germany) as described (Pfeiffer et al. 2001).

Western Blot Analysis

Membranes were prepared from stably transfected HEK-293 cells as well as from fresh tumor specimens. Cells and tissues were lysed in homogenization buffer (5 mM EDTA, 3 mM EGTA, 250 mM sucrose, 10 mM Tris-HCl, pH 7.6, containing 1 mM phenylmethylsulfonylfluoride, 1 μM pepstatin A, 10 μg/ml leupeptin, and 2 μg/ml aprotinin), and membranes were pelleted at 20,000 × g for 30 min at 4C. Membranes were then dissolved in lysis buffer (150 mM NaCl, 5 mM EDTA, 3 mM EGTA, 20 mM Hepes, pH 7.4, containing 4 mg/ ml dodecyl-β-maltoside and proteinase inhibitors as above) and incubated with 150 μl wheat germ lectin agarose beads (Amersham) for 90 min at 4C. Beads were washed five times in lysis buffer, and adsorbed glycoproteins were eluted with SDS sample buffer for 20 min at 60C. Samples were then subjected to 8% SDS polyacrylamide gel electrophoresis and immunoblotted onto nitrocellulose. Blots were incubated with 1 μg/ml anti-NK₁ {9042} antibodies followed by peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection (Amersham). For adsorption controls, antibody was preincubated with 10 μg/ml of its cognate peptide for 2 hr at room temperature.

Immunohistochemistry

Seven-μm paraffin sections were cut and floated onto positively charged slides and IHC stained as described (Schulz et al. 2000,2004,2005; Mundschenk et al. 2003). Briefly, sections were dewaxed, microwaved in 10 mM citric acid (pH 6.0) for 20 min at 600 W, and subsequently incubated with anti-NK₁ {9042} antibodies in concentrations ranging from 0.5 to 2 μg/ml overnight at 4C. Staining of primary antibody was detected using biotinylated goat anti-rabbit 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 IHC controls, primary antibody was omitted, replaced by preimmune sera, or adsorbed with several concentrations ranging from 1 to 10 μg/ml of homologous or heterologous peptides for 2 hr at room temperature. A tumor known to stain positively was included in each batch of staining as positive control.

Assessment of Staining Patterns

All slides were evaluated by two independent investigators. 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. Tumors were only categorized as positive when they exhibited a moderate to strong plasma membrane and/or cytoplasmic staining in the majority of tumor cells, which was easily visible with a low-power objective.

Results

Characterization of NK ₁ Receptor Antibodies

Specificity of the antisera was monitored using Western blot analysis. When membrane preparations from stably transfected cells were electrophoretically separated and blotted onto nitrocellulose, the anti-NK₁ {9042} antiserum detected a broad band migrating at molecular mass 70,000 to 90,000 Da only in cells transfected with its cognate receptor but not in cells transfected with an empty vector (Figure 1). Antisera were further characterized using immunofluorescent staining of transfected cells. When HEK-293 cells stably expressing NK₁ were stained with the anti-NK₁ antibody {9042}, prominent immunofluorescence localized at the level of the plasma membrane was detected (Figure 2A). After incubation with SP, NK₁ immunoreactivity (ir) was translocated from the plasma membrane into the cytosol indicating that the NK₁ receptor was rapidly endocytosed in an agonist-dependent manner (Figure 2B). Next, the NK₁ receptor antisera were tested for possible cross-reactivity with other proteins present in human tissues. When membrane preparations from human breast or ovarian carcinomas were electrophoretically separated and blotted onto nitrocellulose, the anti-NK₁ antibody {9042} detected a broad band migrating at molecular mass 70,000 to 90,000 Da (Figures 3A and 3B). A few cases were observed in which this broad receptor band consisted of several narrower bands, suggesting that the NK₁ receptor may exist in differently glycosylated forms (Figure 3B). All ir bands were completely abolished by preadsorption of the antibody with 10 μg/ml of its immunizing peptide (Figures 3A and 3B).

Figure 1

Western blot analysis of the specificity of anti-NK₁ antibodies. Membrane preparations from HEK-293 cells transfected with NK₁ or empty vector (MOCK) were separated on 8% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were then incubated with affinity-purified anti-NK₁ {9042} antibodies at a concentration of 1 μg/ml. Blots were developed using enhanced chemiluminescence. Two additional experiments gave similar results. MOCK, empty vector; ordinate, migration of protein molecular mass markers (molecular mass × 10^-3).

Figure 2

Characterization of anti-NK₁ antibodies by immunofluorescent staining of transfected cells. (A,B) HEK-293 cells stably transfected to express NK₁ were either not exposed (A) or exposed (B) to 1 μM SP for 30 min, subsequently fixed, and immunofluorescently stained with anti-NK₁ {9042} antibody. Note that in untreated cells prominent immunofluorescence was localized at the level of the plasma membrane and that exposure to SP induced a rapid translocation of NK₁ receptors from the plasma membrane into the cytosol. Representative results from one of three independent experiments are shown. SP, substance P. Bar = 20 μm.

NK ₁ Receptor IHC Staining in Neoplastic Human Tissues

The anti-NK₁ antibodies were then subjected to IHC staining of human tissues. Initial experiments showed that heat-induced epitope retrieval is required for efficient IHC staining of paraffin-embedded tissues (not shown). All three anti-NK₁ antisera yielded essentially identical IHC staining patterns with prominent ir predominantly localized to the plasma membrane of the tumor cells, although with different staining intensity (Figure 4). Immunostaining for each antiserum was completely abolished by preadsorption with 10 μg/ml of the immunizing peptide (Figure 4B). The anti-NK₁ antibody {9042} produced the most prominent immunostaining and was therefore used throughout the study. Prevalence of NK₁ receptors in human neoplasms is summarized in Table 1. Immunoreactive NK₁ receptors were frequently detected in pancreatic adenocarcinomas (80%), breast carcinomas (75%), and glioblastomas (75%). In most of these tumors, ir NK₁ receptors were homogeneously present on nearly all tumor cells (Figures 4A, 4C, 4F). However, a few tumors were observed with heterogeneous NK₁ expression, i.e., tumor cells with strong to moderate NK₁ receptor staining were seen next to tumor cells lacking ir NK₁ receptors (Figures 4D and 4E). Immunoreactive NK₁ receptors were also found in 4/10 ovarian carcinomas (40%), 2/6 thyroid carcinomas (30%), and 1/4 growth hormone-producing pituitary adenomas (25%). In contrast, ir NK₁ receptors were not detected in neuroendocrine tumors including carcinoid or insulinoma. NK₁ receptors were also not observed in meningiomas, pheochromocytomas, or prostate and colorectal cancer.

Figure 3

Western blot analysis of the specificity of anti-NK₁ antibodies in human tumors. (A) Membrane preparations from a breast carcinoma were separated on 8% SDS-polyacrylamide gels, blotted onto nitrocellulose, and incubated with 1 μg/ml anti-NK₁ antibody {9042} in the absence (-) or presence (+) of 10 μg/ml peptide antigen. (B) Membrane preparations from an ovarian carcinoma were separated on 8% SDS-polyacrylamide gels, blotted onto nitrocellulose, and incubated with 1 μg/ml anti-NK₁ antibody {9042} in the absence (-) or presence (+) of 10 μg/ml peptide antigen. Blots were developed using enhanced chemiluminescence. Representative results from one of three independent experiments are shown. Ordinate, migration of protein molecular mass markers (molecular mass × 10^-3).

Discussion

It is well established that NK₁ receptors are overexpressed in certain malignancies including glioblastomas and breast and pancreatic carcinomas; however, little is known about their cellular and subcellular localization in human neoplastic tissues. We therefore generated antibodies that exert selective specificity for the human NK₁-Fl receptor. We show that the cytoplasmic tail of this receptor 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 this antibody specifically detects its targeted tachykinin receptor and does not crossreact. First, in Western blots of membranes from transfected cells, the anti-NK₁ antibody detected a band migrating at molecular mass 70,000 to 90,000 Da only in NK₁-transfected cells but not in cells transfected with an empty vector. Second, the anti-NK₁ antibody revealed prominent cell surface staining of NK₁-transfected cells. This immunostaining translocated from the cell surface into the cytosol after agonist exposure indicating rapid endocytosis of the NK₁ receptor. Third, in Western blots of membranes from receptor-expressing tumors, the anti-NK₁ antibody detected a broad band migrating at molecular mass 70,000 to 90,000 Da, which corresponds to a glycosylated form of the receptor. Fourth, tissue immunostaining of the anti-NK₁ antiserum was completely abolished by preadsorption with homologous but not heterologous peptides. Finally, it should be noted that three of three NK₁ antisera yielded similar results.

Availability of NK₁ receptor antibodies will facilitate further basic morphological investigation of NK₁ expression in human normal and neoplastic tissues. The IHC NK₁ receptor evaluation offers several major advantages. This method can analyze NK₁ 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 (Hennig et al. 1995). IHC evaluation of the complete NK₁ receptor status of a given tumor specimen can be accomplished in <24 hr. Although one could expect the IHC NK₁ receptor determination to be less sensitive than receptor autoradiography with ¹²⁵I-labeled SP, sensitivity of the anti-NK₁ {9042} antibody generated and characterized in this study was sufficiently high to detect this receptor in human tumors known to express NK₁ receptors in a high percentage, e.g., glioblastomas and breast and pancreatic carcinomas (Hennig et al. 1995; Ehlers et al. 2000; Singh et al. 2000; Friess et al. 2003). In addition, the IHC NK₁ receptor determination provided a better histological quality and cellular resolution compared with receptor autoradiography. Thus, previously unappreciated cellular localizations of NK₁ receptors were uncovered, i.e., heterogeneous expression of NK₁ receptors in a subpopulation of pancreatic carcinomas.

SP can affect the growth of human tumor cells in vivo and in vitro. Whereas growth-promoting activities have been reported for SP, growth-inhibiting properties have been found for NK₁ antagonists in various tumor models (Luo et al. 1996; Fukuhara et al. 1998; Palma et al. 1999a,b,2000; Palma and Maggi 2000; Koon et al. 2004; Munoz et al. 2004,2005). It is therefore very tempting to suggest the use of NK₁ antagonists for treatment of a subset of human malignancies. The present findings also suggest that NK₁ receptors could be promising molecular targets for tumor imaging and targeted radiotherapy using radiolabeled SP analogs (Breeman et al. 1996; Reubi et al. 2005).

Figure 4

NK₁ immunohistochemical (IHC) staining in human neoplastic tissues. NK₁ IHC staining in pancreatic carcinoma (A,B,D,E), glioblastoma (C), and ovarian carcinoma (F). Sections were dewaxed, microwaved in citric acid, and incubated with affinity-purified anti-NK₁ antibody {9042} at a concentration of 1 μg/ml. Sections were then sequentially treated with biotinylated anti-rabbit IgG and AB peroxidase solution. Sections were then developed with 3,3′-diaminobenzidine as chromogen (brown), and cell nuclei were lightly counterstained with hematoxylin (blue). Note that NK₁ receptor immunoreactivity was predominantly localized homogeneously to the plasma membrane of nearly all tumor cells (A,C,F). In contrast, a few tumors were observed with heterogeneous NK₁ expression, i.e., tumor cells with strong to moderate NK₁ receptor staining were seen next to tumor cells lacking immunoreactive NK₁ receptors (D,F). For adsorption controls, primary antibody was incubated with 10 μg/ml of the peptide used for immunizations (B). Representative results from one of three independent experiments are shown. + Peptide, peptide adsorption control. Bars: A,B,C,E,F = 50 μm; D = 100 μm.

It is believed that the human NK ₁ gene could generate NK₁-Fl and NK₁-Tr transcripts (Fong et al. 1992). NK₁-Fl and NK₁-Tr have identical ligand-binding domains but differ in length of their cytoplasmic tails. Specifically, the cytoplasmic tail of NK₁-Tr lacks 100 residues, a region that functions as a substrate for phosphorylation by G-protein-coupled receptor kinases in NK₁-Fl. Consequently, NK₁-Tr shows a reduced capacity to undergo SP-induced internalization and desensitization (Fong et al. 1992). Interestingly, overexpression of NK₁-Tr has recently been shown to promote malignant transformation of breast cells (Patel et al. 2005). Given that the antibody raised and characterized here is directed to the end of the cytoplasmic tail of the NK1-Fl receptor, it was not possible to detect NK₁-Tr in the present study.

Table 1

Prevalence of NK₁ receptors in human tumors

Tumor type (n)	NK1 n (%)
Colorectal adenocarcinoma (5)	0
Pancreatic ductal adenocarcinoma (5)	4 (80)
Breast cancer, ductal invasive (5)	4 (80)
Ovarian carcinoma (10)	4 (40)
Prostate cancer (4)	0
Thyroid cancer (6)	2 (30)
Carcinoid, midgut (7)	0
Carcinoid, hindgut (8)	0
Pancreatic insulinoma (8)	0
Pituitary adenoma, growth-hormone producing (4)	1 (25)
Pheochromocytoma (2)	0
Glioblastoma multiforme (4)	3 (75)
Meningioma (4)	0

In conclusion, we have generated and extensively characterized anti-NK₁ antibodies. Development of this novel antibody enabled us to visualize NK₁-Fl receptors in human formalin-fixed, paraffin-embedded tissues. It is now possible to determine the exact cellular and sub-cellular sites of NK₁ receptor protein in normal human and neoplastic tissues. Rapid immunocytochemical NK₁ receptor visualization may also be helpful to identify those tumors with sufficient receptor overexpression for diagnostic or therapeutic intervention.