TECHNICAL NOTE

Immunoglobulin and Enzyme-conjugated Dextran Polymers Enhance u-PAR Staining Intensity of Carcinoma Cells in Peripheral Blood Smears

Correspondence to: Kim Werther, Dept. of Surgical Gastroenterology 235, Hvidovre Hospital, Univ. of Copenhagen, 2650 Hvidovre, Denmark. E-mail: k.werther@forum.dk

	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 47:959–963, 1999)

Key Words: carcinoma, cytokeratin, double staining, invasion, smears, u-PAR

	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 (Fidler 1990 ). Invasive carcinoma cells must degrade tissue barriers, such as the basement membrane and the interstitial connective tissue, to reach the circulation for subsequent establishment of a secondary tumor. This invasive behavior is dependent on secretion and activation of a variety of degradative enzymes (Mignatti and Rifkin 1993 ). In particular, plasmin activation has proved to be an important factor in tumor-associated proteolysis and matrix degradation (Dano et al. 1994 ). The urokinase plasminogen activator (u-PA) intensifies the conversion of the inactive plasminogen to the active proteolytic enzyme plasmin. Concomitant binding of u-PA and plasminogen to a receptor for u-PA (u-PAR) on cell surfaces strongly enhances plasmin generation (Cohen et al. 1991 ). The surface of cells expressing u-PAR has therefore been suggested to be the major area of plasmin activation (Reiter et al. 1993 ). In a clinical study, u-PAR expression on disseminated carcinoma cells in the bone marrow of patients with gastric cancer has been shown to reduce survival time (Heiss et al. 1995 ).

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 immunogold–silver staining (IGSS) techniques (Riesenberg et al. 1993 ; Allgayer et al. 1997 ). In the case of simultaneous investigation of a strongly expressed antigen, such as cytokeratin, and a weakly expressed surface antigen, such as u-PAR, this technique has been beneficial because visualization of the weak expressed antigen is performed under epipolarized light and is therefore not masked by the strong cytokeratin staining (Hacker et al. 1985 ; Riesenberg et al. 1993 ). During the past 10 years IGSS has been optimized to provide a sensitive and specific visualization system that is well suited to immunocytochemical detection of low-expressed antigens (Hacker et al. 1996 ; Lackie 1996 ). However, nonspecific background staining is still a risk during the silver enhancement reaction, and introduction of an alternative, reliable, and easy to handle visualization technique for light microscopic studies could be a supplement to IGGS.

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.

	Materials and Methods

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Antibodies and Conjugates
The polyclonal rabbit u-PAR antibody (Ohtani et al. 1995 ) was kindly provided by the Finsen laboratory, (National University Hospital, Copenhagen, Denmark). The u-PAR staining experiments were performed with 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12,800, and 1:25,600 dilutions of the u-PAR antibody stock solution (250 µg/ml). The polyclonal rabbit anti-FITC stock solution (18.5 mg/ml) was from Dako (Glostrup, Denmark) and was used at 1:50 dilution. The EPIMET epithelial cell detection kit was from Baxter (Unterschleissheim, Germany). The Envision was from Dako. The structure of the dextran conjugate is shown in Figure 1. It consists of a high molecular mass polymeric dextran backbone coupled with up to 100 enzyme molecules and up to 20 antibody molecules per backbone. The conjugates are kept water-soluble by using a hydrophilic, noncharged dextran as backbone.

View larger version (98K): [in this window] [in a new window]   Figure 1. The structure of Envision and the staining technique for u-PAR using the antibody–enzyme conjugate. Figure 2. Cytospin preparation of the cell line HCC 2998 mixed with peripheral blood MNCs and immunostained for expression of cytokeratin. Bar = 50 µm. Figure 3. Cytospin preparation of the cell line HCC 2998 mixed with peripheral blood MNCs and immunostained for expression of u-PAR. The small brown-stained cells (arrow) are MNCs. The larger cells (arrowhead) are HCC 2998. Bar = 50 µm. Figure 4. Cytospin preparation of the cell line HCC 2998 mixed with peripheral blood MNCs. Double immunostaining for simultaneous demonstration of intracellular cytokeratin (open arrow) and u-PA (arrows) perinuclearly and on the surface of the cells. Bar = 10 µm.

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 (Kasper et al. 1987 ) with the abundantly expressed cytoskeletal protein cytokeratin in epithelial cells. Fab fragments were used to reduce unspecific Fc-receptor binding of the antibodies. The immunostaining procedure followed the instructions of the kit and was briefly as follows. The cells were permeabilized, fixed, and incubated with a conjugate of MAb A45-B/B3–Fab–AP according to the manufacturer's instructions. Next, an insoluble red reaction product (New Fuchsin ) was developed at the binding site of the Fab–AP conjugate. Negative control staining was performed with AP-conjugated isotype (Fab) antibodies (anti-FITC), and with exclusion of the MAb A45-B/B3. Finally, all slides were mounted in glycerol–gelatin and evaluated by light microscopy.

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 formol–acetone, 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 glycerol–gelatin 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 formol–acetone, 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 formol–acetone 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.

	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.

	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 (Bastholm et al. 1994 ).

Expression of u-PAR on disseminated carcinoma cells in bone marrow appears to be of prognostic significance in patients with gastric cancer (Heiss et al. 1995 ). Expression of the newly discovered transmembrane surface-bound protease activators, such as the membrane-bound matrix metalloproteases (MT-MMP 1–4), appears to be important in invasion of human carcinomas (Ueno et al. 1997 ). Phenotypical characterization of the disseminated carcinoma cells for expression of proteases or protease activators might define the cells' invasive potential. We suggest that this rapid and easy technique could be beneficial in phenotypical characterization of disseminated carcinoma cells in peripheral blood and/or bone marrow aspirations. Furthermore, this double staining technique may very well be beneficially adapted to various other areas of research in which simultaneous demonstration of two differently co-expressed antigens on the same cells is desirable. In conclusion, we believe that the enhanced staining technique presented could be a reliable, fast, and easy to use method to visualize low-expressed antigens on cells and that the method could be an alternative to well-established enhancement techniques such as IGSS.

	Acknowledgments

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 Louis–Hansen 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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