TECHNICAL NOTE |
Correspondence to: Kim Werther, Dept. of Surgical Gastroenterology 235, Hvidovre Hospital, Univ. of Copenhagen, 2650 Hvidovre, Denmark. E-mail: k.werther@forum.dk
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Summary |
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The presence of disseminated carcinoma cells in bone marrow and peripheral blood has prognostic importance in patients with carcinomas. Much evidence indicates that dissemination of tumor cells may depend on activation of a variety of degradative enzymes. A strong positive correlation has been shown between the expression of tumor cell proteases and tumor invasion. Therefore, phenotypic characterization of disseminated carcinoma cells for expression of protease activators might define the invasive potential of the cells. We present an immunocytochemically enhanced staining method that allows phenotyping of disseminated carcinoma cells in bone marrow and peripheral blood smears. In the first step, the cells were incubated with antibodies against urokinase plasminogen activator receptor (u-PAR) and subsequently with secondary antibodies conjugated to peroxidase-labeled dextran polymers. A brown color reaction was developed with diaminobenzidine as chromogen. In the second step, the cells were incubated with alkaline phosphatase-conjugated murine monoclonal antibodies against a common cytokeratin epitope and a red color reaction was developed with new fuchsin as substrate. This method allows simultaneous and unambiguous immunolabeling of intracellular cytokeratin and of u-PAR intracellularly and on the surface of carcinoma cells. This novel approach can be used for detection and phenotyping of carcinoma cells in blood smears for u-PAR or, presumably, for any other heterogeneously expressed antigen on the surface of the detected cells. (J Histochem Cytochem 47:959963, 1999)
Key Words: carcinoma, cytokeratin, double staining, invasion, smears, u-PAR
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Introduction |
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Metastasis is the major cause of death among patients with carcinoma. Establishment of a secondary tumor is a result of a series of interactions between the carcinoma cell and its environment (
In cancer research, previously described methods for simultaneous demonstration of two different antigens on the same cell have mostly used a combination of immunoenzymatic and immunogoldsilver staining (IGSS) techniques (
The aim of this study was to investigate the applicability of a new, enhanced immunostaining technique to identify and phenotype carcinoma cells for expression of the heterogenously expressed u-PAR.
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Materials and Methods |
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Antibodies and Conjugates
The polyclonal rabbit u-PAR antibody (
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Tumor Cell Line
Cells of the human colon cancer cell line HCC 2998 were used. The cell line was maintained in culture in RPMI-1640 with 10% FCS at 37C with 5% CO2 in a humidified atmosphere. The culture medium was supplemented with L-glutamine 2 mM, penicillin 400 IU/ml, and streptomycin 400 µg/ml. Culture medium and supplements were purchased from GIBCO (Paisley, UK).
Preparation of Tumor Cell Line for Analysis
The adherent growing cells were harvested by detaching the cells from the tissue culture flask by exposure to a 0.01 mol/liter EDTA solution for 10 min at 37C and were subsequently washed three times in RPMI-1640 with 2% FCS. The cells were counted and resuspended at a dilution of 1 x 103 viable cells/ml.
Cytospin Preparation
A volume of 1 ml cell suspension 1000 cells was centrifuged onto a Polysine microslide (CM Laboratories; Stensved, Denmark) using a cytocentrifuge (Hettich Universal 30RF; Hettich, Tuttlingen, Germany). The program used was 1000 rpm, 5 min, 21C, soft. A cytochamber (Hettich; cat. no. 1666) with a capacity of 8 ml was used, giving a cell area on the microslide of approximately 240 mm2. After centrifugation the supernatant was pipetted off and the slide was air-dried overnight and stored at -80C for later immunocytochemical staining.
Cytokeratin Monolabeling Experiments
Staining of the cells for expression of intracellular cytokeratin was performed with the EPIMET epithelial cell detection kit. The identification of epithelial cells with the EPIMET kit is based on the reactivity of alkaline phosphatase (AP)-conjugated Fab fragments of the murine monoclonal antibody A45-B/B3 (
u-PAR Labeling
Monolabeling of the cells for expression of u-PAR was performed with a two-step immunocytochemical method. First, the slides were fixed in formolacetone, pH 6.6, for 10 min and washed twice in TBS, pH 7.6. Second, the slides were incubated with 0.5% H2O2 for 10 min to reduce endogenous peroxidase activity. Then the slides were incubated horizontally for 45 min with the u-PAR antibody diluted in TBS, pH 7.6, containing 1% bovine serum albumin (BSA). Envision peroxidase anti-rabbit (Dako) was applied for 30 min and finally a brown color reaction was developed with diaminobenzidine as chromogen at the binding site of the peroxidase enzymes. Each incubation step was followed by two washings in TBS, pH 7.6, for 2 min to ensure complete removal of unbound antibodies or conjugates. The specificity of the u-PAR staining was tested with irrelevant control antibodies (polyclonal rabbit anti-FITC) and with exclusion of the primary antibody. All slides were mounted in glycerolgelatin and evaluated by light microscopy.
Double Staining Experiments
In the double staining experiments, the two monolabeling experiments were combined. After thawing and before staining the slides were fixed in formolacetone, pH 6.6, for 10 min and washed twice in TBS, pH 7.6. First the staining for u-PAR was performed, followed by cytokeratin staining. The fixation procedure according to the EPIMET kit was omitted because the cells were already fixed in formolacetone before the u-PAR staining. Finally, the cells were counterstained with hematoxylin to visualize nuclear morphology. After the nuclear staining the slides were mounted in glycerol-gelatin and evaluated by light microscopy.
To investigate the optimal u-PAR staining in proportion to the strong cytokeratin staining, the experiments were performed with different dilutions (1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12,800, 1:25,600) of the original u-PAR antibody stock solution. Negative control experiments of the double staining were performed with combinations of irrelevant (anti-FITC) and original antibodies or with exclusion of the primary antibodies.
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Results |
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Our results confirm that monostaining of cytokeratin-expressing cells, in this case the colon carcinoma cell line HCC-2998, using the EPIMET kit is applicable to identification of epithelial cells (Figure 2). Monostaining of the same cell line for expression of the u-PAR using a newly developed two-step immunocytochemically enhanced staining method showed reliable staining properties, although the cells displayed a heterogeneous staining pattern independent of the size or position of the cells (Figure 3). In both the monostaining (Figure 3) and the double staining (Figure 4) experiments, we found that u-PAR was not exclusively localized to the surface of the HCC-2998 cells but was also heterogeneously expressed in the cytoplasm and near the nucleus. The specificity of both monolabeling experiments was supported by the absence of staining with irrelevant antibodies or exclusion of the primary antibodies.
The results of the double staining experiments showed that combination of the two monolabeling techniques allowed simultaneous and unambiguous demonstration of two differently expressed antigens on the same cell (Figure 4).The specificity of the double staining was supported by the consistent absence of unspecific staining in the double staining control experiments that were performed with combinations of irrelevant and original antibodies or with exclusion of the primary antibodies. We found that the optimal u-PAR staining in proportion to the standardized cytokeratin staining with the EPIMET kit was obtained with a 1:800 dilution of the original u-PAR stock solution. At this antibody dilution, unambiguous visualization of the two different antigens was easily performed by light microscopy.
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Discussion |
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We describe a novel enhanced staining technique that allows simultaneous and unambiguous demonstration of two different antigens co-expressed by the same cell. The technique combines a one-step immunoenzymatic staining of cytokeratin with a newly developed two-step enhanced immunoenzymatic staining method based on the reactivity of enzyme- and antibody-conjugated dextran polymers with Fc receptors of antibodies produced in rabbits. Our results show that this method can be used in phenotypical characterization of monolayer cells, such as smears of peripheral blood or bone marrow aspirations. There are conspicuous benefits of this method compared to previously described double staining techniques such as immunoflourescence and the combination of IGSS and immunoenzymatic labeling. Compared to immunoflourescence, this staining does not fade and therefore allows long-term documentation and reexamination of the slides. Compared to IGSS, the technique is easy to use, and although the specificity of IGSS during the past 10 years has been optimized, the risk for false-positive results due to unspecific precipitation of silver granules is still present, which is eliminated with the technique presented. Furthermore, this enhanced staining technique involves fewer steps compared to previously described double staining techniques. The need for several blocking steps to reduce the risk of unspecific staining is therefore minimized, thus making the staining reliable and less time-consuming.
We found that u-PAR staining was not exclusively localized to the surface of the HCC-2998 cells but was also localized in the cytoplasm and near the nucleus. This finding is consistent with an ultrastructural study of the human breast cancer cell line MDA-MB-231 that showed cytoplasmic u-PAR localized inside large vesicles of different morphology and in flat Golgi saccules (
Expression of u-PAR on disseminated carcinoma cells in bone marrow appears to be of prognostic significance in patients with gastric cancer (
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Acknowledgments |
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Supported by grants from the Ingeborg Roikjer Foundation, the M. Kristian and Margrethe Kjaer Foundation, the Gerda and Aage Haensch Foundation, the Kathrine and Vigo Skovgaard Foundation, the Augustinus Foundation, the Gangsted Rasmussen Foundation, the Johanne and Aage LouisHansen Foundation, the Harboe Foundation, the Simon Foùgner Hartman Foundation, and the Danish Cancer Society.
Received for publication September 9, 1998; accepted February 23, 1999.
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Literature Cited |
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Allgayer H, Heiss MM, Riesenberg R, Babic R, Jauch KW, Schildberg FW (1997) Immunocytochemical phenotyping of disseminated tumor cells in bone marrow by u-PA receptor and CK 18: investigation of sensitivity and specificity of an immunogold/alkaline phosphatase double staining protocol. J Histochem Cytochem 45:203-212
Bastholm L, Elling F, Brunner N, Nielsen MH (1994) Immunoelectron microscopy of the receptor for urokinase plasminogen activator and cathepsin D in the human breast cancer cell line MDA-MB-231. APMIS 102:279-286[Medline]
Cohen RL, Xi XP, Crowley CW, Lucas BK, Levinson AD, Shuman MA (1991) Effects of urokinase receptor occupancy on plasmin generation and proteolysis of basement membranes by human tumor cells. Blood 78:479-487[Abstract]
Danø K, Behrendt N, Brünner N, Ellis V, Ploug M, Pyke C (1994) The urokinase receptor. Protein structure and role in plasminogen activation and cancer invasion. Fibrinolysis 8:189-203
Fidler IJ (1990) Critical factors in the biology of human cancer metastasis: Twenty-eighth G.H.A. Clowes Memorial Award Lecture. Cancer Res 50:6130-6138
Hacker GW, Muss WH, HauserKronberger C, Danscher G, Rufner R, Gu J, Su H, Andreasen A, Stoltenberg M, Dietze O (1996) Electron microscopical autometallography: immunogoldsilver staining (IGSS) and heavy-metal histochemistry. Methods 10:257-269[Medline]
Hacker GW, Springall DR, Van Noorden S, Bishop AE, Grimelius L, Polak JM (1985) The immunogold-silver staining method. Virchows Arch [Pathol Anat] 406:449-461
Heiss MM, Allgayer H, Gruetzner KU, Funke I, Babic R, Jauch KW, Schlidberg FW (1995) Individual development and u-PA-receptor expression of disseminated tumor cells in bone marrow: a reference to early systemic disease in solid cancer. Nature Med 1:1035-1039[Medline]
Kasper M, Stosiek P, Typlt H, Karsten U (1987) Histological evaluation of three new monoclonal anti-cytokeratin antibodies. Eur J Cancer Clin Oncol 23:137-147[Medline]
Lackie PM (1996) Immunogold silver staining for light microscopy. Histochem Cell Biol 106:9-17[Medline]
Mignatti P, Rifkin DB (1993) Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 73:161-195
Ohtani H, Pyke C, Danø K, Nagura H (1995) Expression of urokinase receptor in various stromal-cell populations in human colon cancer: immunoelectron microscopical analysis. Int J Cancer 62:691-696[Medline]
Reiter LS, Kruithoff EKO, Cajot JF, Sordat B (1993) The role of urokinase receptor in extracellular matrix degradation by HT29 human colon carcinoma cells. Int J Cancer 53:444-450[Medline]
Riesenberg R, Oberneder R, Kriegmair M, Epp M, Bitzer U, Hofsetter A, Braun S, Riethmüller G, Pantel K (1993) Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells. Histochemistry 99:61-66[Medline]
Ueno H, Nakamura H, Inoue M, Imai K, Noguchi M, Sato H, Seiki M, Okada Y (1997) Expression and tissue localization of membrane-types 1, 2 and 3 matrix metalloproteinases in human invasive breast carcinomas. Cancer Res 57:2055-2060[Abstract]