Urokinase plasminogen activator receptor (CD87) expression of tumor ...

Urokinase plasminogen activator receptor (CD87) expression of tumor-associated macrophages in ductal carcinoma in situ, breast cancer, and resident macrophages of normal breast tissue Ralf Hildenbrand, Georg Wolf, Beatrix Bo¨hme,* Uwe Bleyl, and Andrea Steinborn† Department of Pathology, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim; *Chemotherapeutisches Forschungsinstitut, Frankfurt; and †Department of Gynecology, University of Frankfurt am Main, Germany

Abstract: Macrophages concentrate urokinasetype plasminogen activator (uPA) at the cell surface by expressing urokinase receptors (uPAR) in order to focus the pericellular space plasminogen-dependent proteolysis important in matrix remodeling and cell movement. This study examines the uPAR levels of tumor-associated macrophages (TAM) of invasive breast carcinomas, of TAMs from ductal carcinoma in situ (DCIS) and of macrophages derived from normal (non-tumor) breast tissue. TAMs from invasive breast carcinomas (n 5 30), from DCIS (n 5 12), and macrophages from normal breast tissue (n 5 30) were cultured and immunocytochemically phenotyped by using a panel of antibodies. Urokinase receptor levels were determined by Western blot analysis and in cell-free supernatants by enzyme-linked immunosorbent assay. Urokinase receptor cell surface fluorescence intensity was determined by FACS and by confocal laser scan microscopy. Urokinase-receptor mRNA was detected by in situ hybridization. TAMs of invasive breast carcinomas and of DCIS possess significantly elevated uPAR levels compared with macrophages derived from normal breast tissue. Conclusions: activated macrophages with elevated uPAR levels belong to inflammatory areas in close vicinity of infiltrating and non-infiltrating (DCIS) tumor cells. Blood monocytes that possess elevated uPAR-levels may be selectively recruited from the bloodstream to inflammatory sites close to carcinoma cells, and/or breast cancer and precursor lesions may induce elevated uPAR-levels in TAMs by paracrine interactions. J. Leukoc. Biol. 66: 40–49; 1999. Key Words: urokinase receptor · blood monocytes · inflammation

INTRODUCTION Determination and location of components of the plasminogen activator system in breast cancer is an important issue to 40

Journal of Leukocyte Biology

Volume 66, July 1999

address because there is substantial evidence that high concentrations of proteolytic factors in primary breast cancer tissue (uPA and PAI-1) are conducive to tumor cell spread and metastasis [1, 2]. Tumor cells cross host cellular and extracellular matrix barriers during tumor invasion and metastasis by attachment to and interaction with components of the basement membrane and the extracellular matrix, and by local proteolysis. Tumor cell invasion and metastatic processes require the coordinated and temporal regulation of a series of adhesive, proteolytic, and migratory events. The uPA/uPAR/PAI-1system has been implicated in these processes. Various observations indicate that the uPA system may provide both surfaceassociated protease activity and an adhesion mechanism for cells through interaction with vitronectin [3]. Occupation of uPAR by its ligand uPA and/or interaction with cellular integrins also triggers a signal transduction cascade to cell proliferation, motility, and migration [4]. Macrophages are a major component of the inflammatory foci of various forms of solid tumors [5, 6]. Large numbers of macrophages are present in breast cancer tissues. A positive correlation between the presence of TAMs and tumor progression in breast cancer was reported [7–9]. Macrophages form a heterogeneous cell population because of different developmental and functional stages. Their activities are dependent on the (patho-) physiological situation in their direct environment. It is known that tissue macrophages (resident macrophages) and TAMs are partly different concerning function, phenotype, and cytokine expressions. In all immunohistochemical studies performed so far, there is consent that TAMs display the uPAR antigen, in contrast to breast cancer cells, which express the uPAR antigen in the minority of cases [10–12]. Urokinase receptor facilitates leukocytes to emigrate from the vascular space into sites of inflammation. This process is

Abbreviations: uPA, urokinase-type plasminogen activator; TAM, tumorassociated macrophages; DCIS, ductal carcinoma in situ; PBS, phosphatebuffered saline; SDS, sodium dodecyl sulfate; FITC, fluorescein isothiocyanate. Correspondence: Dr. R. Hildenbrand, Pathologisches Institut der Fakulta¨t fu¨r Klinische Medizin der Universita¨t Heidelberg, Universita¨tsklinikum Mannheim, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. E-mail: [email protected] Received August 21, 1998; revised March 1, 1999; accepted March 2, 1999.

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initiated by a phase of loose adhesion onto the endothelium followed by a firm attachment, mediated in part by complement receptor 3 (CR3; also known as Mac-1, aMb2, CD11b/CD18), a b2 integrin adhesion protein [13]. The physical association between uPAR and CR3 provides a structural basis for coordinating the functions of these two proteins during cellular movement [14, 15]. It was shown by Sitrin et al. [16] that the adhesive functions of CR3 are strongly influenced by its association with uPAR and uPA. They conclude that the uPAR/uPA system exerts an important regulatory control over CR3 function in monocyte adhesion, locomotion, and activation. In this study we compared the uPAR expression of TAMs of invasive breast cancer and of TAMs isolated from ductal carcinoma in situ (DCIS) and of tissue macrophages derived from normal (non-tumor) breast tissue. Our aim was to demonstrate that TAMs (of DCIS and invasive breast carcinomas) possess elevated uPAR levels compared with tissue macrophages and to emphasize that activated macrophages with enhanced uPAR levels belong to inflammatory areas in close vicinity of infiltrating and non-infiltrating (DCIS) tumor cells. Blood monocytes that possess elevated uPAR levels may be selectively recruited from bloodstream to inflammatory sites close to carcinoma cells and/or breast cancer and precursor lesions may induce elevated uPAR levels in TAMs by paracrine interactions.

METHODS Cell culture Cell isolation and culture were performed according to previously published methods [17]. Briefly, the tissues of 30 invasive breast carcinomas (2–13 cm in diameter, stage I–III, G1–3) and of 12 DCIS (2.5–5 cm in diameter; 6 high-grade DCIS and 6 non-high-grade DCIS) were minced; collagenase D (200 U/mL; Boehringer-Mannheim, Mannheim, Germany), phosphate-buffered saline (PBS), penicillin (10 U/mL), and streptomycin (100 µg/mL) were added and incubated while being gently stirred at 37°C for 2–6 h. Thereafter the cell suspension was filtered through a 200-µm nylon mesh net. The filtrate was seeded into bags made of hydrophobic Teflon (Biofolie 25, Heraeus, Germany) [18] in RPMI-1640 supplemented with 0.05 mM 2-mercaptoethanol, 0.02 mM L-glutamine, 0.01 mM sodium pyruvate, 10 U/mL penicillin, 100 mg/mL streptomycin, and 5% pooled human AB-group serum. Macrophages adhered to the hydrophobic Teflon. Nonadherent cells were removed and fresh medium was added 5 h after incubation in the Teflon bags (0.5 3 106 cells/mL). Cell-free supernatant was taken for enzyme-linked immunosorbent assay (ELISA) after 10, 14, and 18 h of incubation in the bags. Representative cell samples were carefully detached after 10, 14, and 18 h. Cytospin preparations were performed for immunocytochemistry. The composition of preparations was generally .85% anti-CD68 positive cells. The remaining cells showed a positive staining for leukocyte common antigen, pan-cytokeratin, CD31, or fibroblast antigen. Eighteen hours after isolation cells were detached from the Teflon wall of the bags and incubated with ferritin-labeled monoclonal anti-CD11b antibody [107 cells in 80 µL PBS (5 mmol EDTA, 1% bovine serum albumin) plus 20 µL anti-CD11b antibody (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany); 15 min, 4°C]. Magnetic cell separation (MACS) [19] was performed by using a magnetic column. Magnetic tagging and separation did not affect cell viability. Cells were extensively washed and reseeded into Lab Tek four-chamber slides (Miles Laboratory) and into 500-mL tissue culture flasks (Nunc, Germany). The plastic surface area of the flasks was roughened by cell scrapers (Nunc) to improve the adherence of cells. Viability was .85% throughout experiments, as determined by trypan blue exclusion. The cultures were washed with PBS to

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remove nonadherent cells. Tissue macrophages (n 5 30) of normal (non-tumor) breast tissues were isolated and cultured in the same manner. Representative cell samples from each culture were immunostained using the APAAP method. Cells were phenotyped by using a panel of antibodies: Ki-M2 [20], Ki-M5 [21], Ki-M6 [22], Ki-M7 [23], Ki-M8 [24], RM 3/1 [25], 27E10 [26], 25F9 [27], G16/1 [28], anti-CD11b [29], (all from Dianova, Hamburg, Germany), EMB11 [30], PG-M1 [31], KP1, MAC387, anti-CD45RB (leukocyte common antigen) (lca), anti-CD31, anti-CD18 (MHM23) (all from DAKO, Hamburg, Germany), Leu-M3 [32] (Becton-Dickinson, Heidelberg, Germany), anti-human fibroblast antigen (Dianova, Hamburg, Germany), anti-pan-cytokeratin (ck) (ProGen, Heidelberg, Germany), and anti-uPAR [33, 34] (no. 3936, American Diagnostica, Greenwich, CT). Three representative areas were selected and 100 cells/area were counted. The mean of each case was calculated. The number of positive immunoreactive cells is given in percent. In all experiments the number of cultured TAMs and tissue macrophages per dish (macrophage density) was constant (0.5 3 106 cells/mL). To exclude possible cytotoxic effects of culturing the cells in serum-free medium, we controlled viability by trypan blue staining. Viability was constant .85% throughout experiments. Macrophages (0.5 3 106 cells/mL) were placed in serum-free medium (GIBCO-BRL, Gaithersburg, MD) to assure quiescence and removal of residual factors. We determined uPAR concentration in cell-free supernatants by ELISA kits (American Diagnostica) according to the manufacturer’s instructions 28, 34, and 50 h after cell isolation.

Immunostaining Tissue blocks were fixed in 4% buffered formalin and embedded in paraffin wax. Immunohistochemical reactions were performed using antibodies against uPAR and CD68. The mouse anti-human uPAR mAb [American Diagnostica no. 3936 (clone 3B10, IgG2a; anti-Mo3f)] [33, 34] was used (50 µg/mL, 60 min, room temperature) after the tissue sections were pretreated by microwave (5 3 3 min in citrate buffer, 700 W). Thereafter the APAAP method [35] was performed. Controls included the following: (1) preabsorption of the primary antibody (uPAR) with an excess of soluble uPAR antigen (CHO-uPAR1–277, a kind gift of Dr. Victor Magdolen, Department of Gynecology, University of Munich, Germany and Dr. Thomas Luther, Department of Pathology, University of Dresden, Germany) and (2) replacement of primary antibody in the initial incubation with non-immune mouse IgG (50 µg/mL).

Immunoblotting Cells were washed twice with ice-cold PBS. The cell pellet was resuspended in 1000 µL of lysis buffer (50 mM HEPES, pH 7.5; 150 mM NaCl; 10% glycerol; 1% Triton X-100; 1.5 mM magnesium chloride; 1 mM EGTA) freshly supplemented with 10 µg leupeptin/mL, 10 µg aprotinin/mL, 1 mM phenylmethylsulfonyl fluoride, and 1 mM natrium orthovanadate. Cell lysates were incubated on ice for 15 min with occasional vortexing and then clarified by centrifugation for 10 min at 12,000 g. The protein concentration was measured using the Pierce BCA protein assay reagents. Protein was fractionated (10 µg/lane) on a sodium dodecyl sulfate (SDS)-10% polyacrylamide gel by electrophoresis. After transfer of the protein onto Immobilon (Millipore Corp.), filters were preincubated for 2 h with 1% bovine serum albumin, 1% gelatin in PBS. The blots were probed with monoclonal anti-uPAR IID7 [36] antibody (a kind gift of Dr. Thomas Luther and Dr. Viktor Magdolen). The blots were washed three times and incubated for 1 h with horseradish peroxidaseconjugated rabbit anti-mouse antibody (Dianova, Hamburg, Germany). Immunoblots were developed using the enhanced chemiluminescence system (DuPont-NEN). To demonstrate that equal amounts of protein were loaded in each lane the anti-uPAR IID7 and the secondary antibodies were completely removed and the same immunoblots were incubated with anti-a-tubulin. Therefore, the membranes were submerged in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incubated at 50°C for 30 min. After washing the blots 2 3 10 min in PBS, the membranes were blocked by immersing in 5% blocking reagent TBS-T for 1 h at room temperature. The immunodetection (as described above) of a-tubulin was performed using a monoclonal anti- a-tubulin antibody (1:2000 dilution; Amersham Pharmacia Biotech Europe GmbH) [37].

uPAR expression in tumor-associated macrophages of the breast

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Flow cytometry TAMs (of DCIS and invasive carcinomas) and tissue macrophages were carefully detached (using 0.02% EDTA in PBS) and harvested by centrifugation. Viability was .85% throughout the experiment, as determined by trypan blue exclusion. Cells (106) were extensively washed and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD18 (clone MHM23, IgG1, DAKO) mAb or with FITC-conjugated anti-CD11b (clone X-5, IgG1, Dianova) for 30 min. After incubation the cells were centrifuged, resuspended in 1 mL PBS, and then cell-associated fluorescence quantified by flow cytometry (FACS) with the FACScan flow cytofluorometer (Becton-Dickinson, low-power argon laser at 488 nm) 14, 28, and 50 h after cell isolation. Binding of monoclonal anti-uPAR antibody to surface receptors on TAMs was determined after acid pretreatment [38]. For this purpose 106 cells were transferred into 0.5 mL of 50 mM glycine-HCl, 0.1 M NaCl, pH 3.0, to dissociate receptor-bound uPA (1 min, 22°C). Subsequently, the buffer was neutralized by the addition of 0.5 M HEPES-0.1 M NaCl, pH 7.5, containing 125 ng of monoclonal anti-uPA antibody (American Diagnostica, no. 3471; mAB no. 3471 reacts with an epitope on GFD, A-chain) to prevent rebinding of dissociated uPA to surface uPAR. After washing in PBS, cells were incubated with anti-uPAR antibody (monoclonal IgG, 1 µg/100 µL, American Diagnostica) for 45 min. After washing, cells were incubated 20 min with FITCimmunoglobulin G (goat anti-mouse IgG, DAKO). After incubation the cells were centrifuged, resuspended in 1 mL PBS, and then cell-associated fluorescence was quantified by flow cytometry (FACS) with the FACScan flow cytofluorometer (Becton-Dickinson, low-power argon laser at 488 nm) 14, 28, and 50 h after cell isolation. Cells incubated with IgG and FITCimmunoglobulins, and cells incubated with anti-uPAR (or anti-CD11b or anti-CD18) antibodies only, served as controls in all experiments.

Confocal microscopy Cultured TAMs and tissue macrophages were immunostained with monoclonal anti-uPAR antibody (no. 3936 American Diagnostica) and Cy3-labeled goat anti-mouse IgG (Amersham Life Sciences, no. PA43002). The macrophages were analyzed with a Sarastro 2000 confocal laser scanning microscope system using a 363/1.4 objective (Zeiss). For Cy3 imaging, a 514-nm laser wavelength filter and a 535-nm primary beamsplitter were used. The confocal aperture was set at 50 µm. A series of optical sections (14–25) was collected at incremental steps of 0.3 µm. Unprocessed optical sections with an image size of 512 3 512 pixels and pixel size of 0.2 µm were obtained. The primary data were Gauss filtered (3 3 3 3 3 kernel size), and a threshold level was set to optimize visualization. Fluorescence images are pseudocolored so that increasing fluorescence intensity is indicated from blue to white. The final image represents the total fluorescence of one cell by overlaying all sections scanned or a transverse section through the middle of the cell.

Nonradioactive in situ hybridization for human uPAR mRNA applying digoxigenin-labeled oligodeoxynucleotides For in situ hybridization with digoxigenin-labeled oligodeoxynucleotides [10] cells plated into Lab Tek four-chamber slides (Miles Laboratory) were fixed (4% paraformaldehyde in PBS) and incubated with Proteinase K (BoehringerMannheim; 0.01 µg/mL in 50 mM Tris/HCl, pH 7.6, for 15 min at 37°C). After several washes with Tris buffer the slides were prehybridized in 50 µL of hybridization solution [4 3 SSC, 50% formamide, 13 Denhardt’s solution, 10% dextran sulfate, and salmon sperm DNA (150 µg/mL) for 1 h at 37°C]. Subsequently, 50 µL of an equimolar mixture of five different 58- and 38-digoxigenin-labeled antisense or sense oligodeoxynucleotides to uPAR (total concentration of 25 ng/mL hybridization solution) were added. The antisense oligodeoxynucleotides (Biometra, Go¨ttingen, Germany) were complementary to nucleotides 121–150, 321–350, 521–550, 717–746, and 918–947 of uPAR mRNA (according to the nucleotides numbering of accession number X51675 in the EMBL database). Control slides received either the corresponding sense oligodeoxynucleotides or were treated with RNase (Boehringer-Mannheim; 0.1 mg/mL Tris buffer for 1 h at 37°C) before the addition of antisense oligodeoxynucleotides to uPAR. The slides were covered with a coverslip and hybridized in a humidified chamber at 37°C for 16 h. After hybridization, the coverslips were removed and the slides washed twice with 23 SSC (5 min each

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at room temperature) and twice with 13 SSC (5 min each at 50°C). The slides were then rinsed in Tris buffer, followed by an incubation in Tris buffer containing 3% bovine serum albumin (Fraction V; Serva, Heidelberg, Germany) for 30 min at room temperature. One hundred microliters of a 1:600 dilution of an alkaline phosphatase-conjugated antibody directed to digoxigenin (Boehringer-Mannheim) was applied to each slide and incubated for 30 min at room temperature. After washing in TBS the slides were incubated in substrate buffer (10 mM NaCl, 50 mM MgCl2, 100 mM Tris/HCl, pH 9.5) for 5 min at room temperature. Subsequently, the slides were immersed in the color developing solution [0.04% 5-bromo-4-chloro-3-indolyl-phosphate (Boehringer-Mannheim), 0.06% nitro blue tetrazolium (Sigma, Munich, Germany), 0.1 mM levamisole (Sigma), 100 mM NaCl, 50 mM MgCl2, 100 mM Tris/HCl, pH 9.5] for 20 h at room temperature. Finally, the slides were rinsed in TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 7.6).

Statistical analysis Wilcoxon-Mann-Whitney U test (rank sum test) was used for all statistical analysis. Results are expressed as the mean 6 standard error of the mean (SEM) and are considered significant at the P , 0.05 level (two-tailed).

RESULTS Five hours after cells were isolated and seeded into Teflon bags nonadherent cells were removed and fresh medium was added. At this point in time 86 6 3% (range 76–96%) of the cells showed an anti-CD-681 immunoreaction. The remaining cells showed a positive staining for leukocyte common antigen, pan-cytokeratin, CD31, or fibroblast antigen. Ten, fourteen, and eighteen hours after isolation and culturing cells in Teflon bags uPAR concentration was determined in cell culture supernatant by ELISA. Thereafter cells were carefully detached, MACS isolation was performed, and serum-free medium was added. Ten cases of each macrophage group (TAMs of DCIS; TAMs of invasive carcinomas, macrophages of normal breast tissue) were phenotyped by 21 antibodies; the results are listed in Table 1. The numbers of macrophages demonstrating positive immunoreactions by using the antibodies G16/1, RM3/1, 27E10, and 25F9 were significantly lower (P , 0.05) in macrophages derived from normal breast tissue compared with TAMs of DCIS and of invasive breast cancer. Urokinase receptor concentrations in cell culture supernatant were also determined 28, 34, and 50 h after cell isolation. The results are demonstrated in Figure 1. UPAR expression was not significantly different in supernatants of TAMs isolated of DCIS and of invasive breast cancer. UPAR release into the culture medium was significantly different in TAMs (of invasive carcinomas and of DCIS) compared with macrophages derived from normal non-tumor breast tissue (P , 0.05). Similar significant differences of cell-associated uPAR fluorescence of TAMs (of DCIS and of invasive carcinomas) and macrophages of normal breast tissues (P , 0.05) were found by flow cytometry (Table 2). Twenty-eight hours after cell isolation urokinase receptor fluorescence intensity in TAMs of invasive carcinomas (n 5 28) was 2196.3 6 145.6, in macrophages of normal tissue (n 5 28) it was 992.0 6 72.5, and in TAMs of DCIS (n 5 12) it was 1687 6 127.3. The difference of the urokinase receptor fluorescence intensity in TAMs of invasive carcinomas compared with TAMs of DCIS was not significant (P . 0.05). Control values were 89.4 6 7.8, http://www.jleukbio.org

TABLE 1.

Phenotyping of Macrophages by a Panel of 21 Antibodies

Antibody/clone

Ki-M2 Ki-M5 Ki-M6 (CD68) Ki.M7 (CD68) Ki-M8 G16/1 RM3/1 27E10 25F9 EBM11 (CD68) KP1 (CD68) PG-M1 (CD68) MAC 387 (CD68) Leu-M3 (CD14) CD45RB CD31 Anti-Pan-cytokeratin Anti-fibroblast antigen uPAR (CD87) X-5 (CD11b) MHM23 (CD18)

TAM (DCIS) (n 5 10)

TAM (n 5 10)

TISM (n 5 10)

34.6 6 5.6% 45.8 6 6.8% 83.8 6 6.9% 96.5 6 4.3% 71.8 6 5.6% 67.6 6 5.9% 72.4 6 5.5% 79.6 6 5.1% 82.7 6 3.4% 91.8 6 0.5% 92.9 6 1.3% 87.8 6 1.0% 89.5 6 0.5% 45.7 6 5.1% 1.2 6 0.3% 1.0 6 0.2% 0.2 6 0.01% 0.1 6 0.01% 98.8 6 0.3% 99.5 6 0.2% 98.7 6 0.3%

36.8 6 5.8% 68.9 6 6.8% 84.9 6 6.1% 95.8 6 3.6% 68.9 6 4.5% 78.9 6 6.1% 76.8 6 4.4% 92.4 6 5.6% 86.7 6 5.1% 93.1 6 0.7% 93.9 6 1.0% 91.7 6 1.1% 92.6 6 0.8% 47.9 6 5.3% 1.7 6 0.1% 1.6 6 0.1% 0.4 6 0.01% 0.1 6 0.01% 98.7 6 0.2% 99.7 6 0.3% 98.3 6 0.2%

39.9 6 5.9% 47.8 6 6.5% 84.8 6 3.5% 97.4 6 3.1% 83.9 6 4.0% 55.7 6 6.4% 56.9 6 4.7% 62.7 6 6.5% 59.1 6 2.2% 92.9 6 0.5% 95.2 6 1.5% 90.7 6 1.0% 94.5 6 0.9% 42.8 6 5.9% 1.1 6 0.5% 1.4 6 0.1% 0.2 6 0.01% 0.2 6 0.01% 99.1 6 0.2% 99.0 6 0.2% 98.8 6 0.2%

Values are the percentage of macrophages showing positive immunoreactions (mean 6 SEM).

85.2 6 8.9, and 87.8 6 8.0. One representative case of each macrophage type is demonstrated in Figure 2A. Western blot analysis showed uPAR signals between 45 and 60 kDa in all types of macrophages. Heterogeneity in the bands was observed, probably due to different glycosylation variants of uPAR. To demonstrate that equal amounts of protein (10 µg/lane) were loaded in each lane, a-tubulin (57 kDa) was detected on the same immunoblots (Fig. 2B). TAMs (of DCIS, n 5 12 and of invasive carcinomas, n 5 20) showed stronger uPAR signals in Western blot analysis compared with tissue macrophages (n 5 20). The uPAR bands of DCIS-TAMs were in

Fig. 1. uPAR concentration during cell isolation. After cell isolation from primary tissue, uPAR concentrations were determined in cell culture supernatants by ELISA. After culturing macrophages in Teflon bags for 18 h the MACS procedure was performed by using CD11b-coated magnetic beads. UPAR concentrations of TAMs derived from DCIS (1; n 5 12), from invasive breast cancer (2; n 5 30) and from normal breast tissue (3; n 5 30) are given as mean 6 SEM.

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all cases weaker compared with the signals of TAMs derived from invasive carcinomas. One representative case of each of the three macrophage types is shown in Figure 2B. To examine the effect of isolating macrophages with CD11bcoated magnetic beads on uPAR expression, we extensively washed macrophages (five times, to remove the antibody after MACS) by pelleting cells, removing the supernatant, and resuspending cells in PBS on the one hand and incubated macrophages of the same case with ferritin-conjugated antiCD11b antibody on the other hand. In both groups uPAR expression was determined by ELISA 10, 16, and 32 h after MACS isolation was performed. The uPAR expression in TAMs (n 5 8) incubated in serum-free medium was 0.33 6 0.02 ng/mL (10 h), 0.35 6 0.03 ng/mL (16 h), and 0.32 6 0.02 (32 h). The uPAR expression in TAMs (n 5 8) incubated in serum-free medium plus ferritin-labeled anti-CD11b antibody was 0.35 6 0.03 ng/mL (10 h), 0.35 6 0.03 ng/mL (16 h), and 0.33 6 0.02 ng/mL (32 h). No significant difference was found (P . 0.05). Cell-associated uPAR expression was also determined by flow cytometry 10 h after the MACS procedure. The fluorescence intensity measured by flow cytometry was 2139.8 6 165.5 in TAMs incubated in serum-free medium and 2043.8 6 156.9 in TAMs incubated in serum-free medium supplemented with ferritin-labeled anti-CD11b antibody. No significant difference was found (P . 0.05). TAMs of DCIS and macrophages of normal breast tissues were examined in the same manner; significant differences in uPAR expression were not found (data not shown). Single cell-associated uPAR fluorescence of TAMs (DCIS and invasive carcinomas) and tissue macrophages was determined semiquantitatively by confocal laser-scan microscopy. The fluorescent patches (representing uPA-receptor-bound antibody) in TAMs and tissue macrophages were irregularly located on the outer side of the plasma membrane and not inside the cell. TAMs clearly showed more fluorescent patches compared with macrophages isolated from normal breast tissues. One representative case of each macrophage type is demonstrated in Figure 3. The membrane molecules CD11b and CD18, which are associated with uPAR, were determined in 10 cases by flow cytometry (Table 2). Twenty-eight hours after cell isolation the anti-CD11b (anti-CD18-) -associated fluorescence intensity in TAMs of invasive carcinomas (n 5 10) was 2102.2 6 151.2 (CD18, 2156.9 6 161.9) and of DCIS (n 5 10) was 1752 6 135.8 (CD18, 1789.0 6 167.9, not significant; CD11b, P . 0.05; CD18, P . 0.05). Twenty-eight hours after cell isolation the anti-CD11b (anti-CD18-) -associated fluorescence intensity of macrophages derived from normal breast tissues (n 5 10) was 1161.1 6 112.8 (CD18, 1190.1 6 125.8; Fig. 4). The CD11b (and CD18-) -associated fluorescence intensity in TAMs of invasive carcinomas (and of DCIS) was significantly different compared with macrophages isolated from normal breast tissue (P , 0.05). A strong correlation of the anti-CD11b and the anti-uPAR-associated fluorescence intensity in TAMs of invasive carcinomas (rPearson 5 0.75, P , 0.05; rSpearman 5 0.75, P , 0.05), in TAMs of DCIS (rPearson 5 0.88, P , 0.001; rSpearman 5 0.87, P , 0.01) and of macrophages derived from


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TABLE 2.

Urokinase Receptor, CD11b, and CD18 Fluorescence Intensities Measured by Flow Cytometry 14 h

28 h

50 h

TAMs of invasive breast cancer UPAR (n 5 28) CD11b (n 5 10) CD18 (n 5 10)

2488.1 6 150.6 2508.1 6 140.1 2615.1 6 157.8

2196.3 6 145.6 2102.2 6 151.2 2156.9 6 161.9

2003.1 6 160.6 1999.3 6 145.1 2060.5 6 183.6

TAMs of DCIS UPAR (n 5 12) CD11b (n 5 10) CD18 (n 5 10)

1901.5 6 145.1 1998.6 6 143.6 2050.6 6 149.6

1687.0 6 127.3 1752.0 6 135.8 1789.0 6 167.9

1705.3 6 138.5 1709.8 6 121.1 1647.5 6 131.6

Macrophages of normal breast tissue UPAR (n 5 28) CD11b (n 5 10) CD18 (n 5 10)

1351.5 6 130.8 1390.1 6 128.2 1392.5 6 120.6

992.0 6 72.5 1161.1 6 112.8 1190.1 6 125.8

1089.6 6 101.5 1181.1 6 99.5 1089.1 6 103.1

Values are means 6 SEM at 14, 28, and 50 h after cell isolation.

normal breast tissue (rPearson 5 0.80, Pf45 , 0.01; rSpearman 5 0.76, P , 0.05) exists. TAMs of invasive carcinomas (n 5 30) and of DCIS (n 5 12) and macrophages (n 5 30) of normal breast tissues were probed for the presence of uPAR mRNA by in situ hybridization using digoxigenin-labeled antisense oligodeoxynucleotides. In all cases TAMs and tissue macrophages showed a positive reaction with the antisense probe. In TAMs of invasive carcinomas and of DCIS the staining reaction was always a little stronger compared with macrophages of normal breast tissues. Only a faint reaction was seen substituting the sense probe for the antisense oligodeoxynucleotides (Fig. 5). To visualize the enhanced uPAR expression in TAMs uPAR protein was examined by immunocytochemistry. TAMs of invasive carcinomas (n 5 30) and of DCIS (n 5 12) showed clearly stronger immunoreactions compared with tissue macrophages (n 5 30) when they were incubated with monoclonal anti-uPAR antibody (not shown). The immunohistochemical reactions for uPAR showed a

strong anti-uPAR staining of TAMs (CD68-positive cells) of invasive carcinomas (n 5 30) and of DCIS (n 5 12). A moderate anti-uPAR staining of macrophages in normal breast tissue was found. A moderate immunoreaction of fibroblast-like cells in all breast cancer tissues, DCIS tissues, and normal breast tissues were observed. The carcinoma cells were found to contain uPAR immunoreactivity in 2 of the 30 cases. The tumor cells of DCIS showed a positive immunoreaction in 3 of the 12 cases. No uPAR immunoreaction of endothelial cells was found. Normal epithelial cells showed no anti-uPAR immunoreaction. Negative controls incubated with mouse IgG (or with antiuPAR plus uPAR protein) instead of primary antibody exhibit faint immunoreactions.

DISCUSSION Factors of the plasminogen activator system play a key role in tumor invasion, angiogenesis, and metastasis of breast cancer.

B

Fig. 2. (A) Cell surface uPAR expression measured by flow cytometry. TAMs of DCIS (1), of invasive breast cancer (2), and macrophages derived from normal breast tissue (3) were incubated with anti-uPAR and FITC-conjugated immunoglobulins. Fluorescence intensities were measured by FACScan. One representative case of each macrophage type is demonstrated. (B) UPAR expression in macrophages analyzed by immunoblotting. Western blot analysis in macrophages of DCIS (1), of invasive breast carcinomas (2), and of normal breast tissue (3) demonstrates heterogeneous uPAR signals between 45 and 60 kDa (same cases as in panel A). To demonstrate that equal amounts of protein were loaded in each lane, a-tubulin (57 kDa) was detected on the same blot.

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Fig. 3. Identification of uPAR in TAMs of DCIS (A, B), of invasive breast cancer (C, D), and in macrophages derived from normal breast tissue (E, F) by confocal laser scan microscopy. Cells were incubated with anti-uPAR antibody no. 3936 and Cy3-labeled goat anti-mouse IgG. The images represent the total fluorescence of the cell (A, C, E) by overlaying 20 sections (0.3 µm each) scanned. In B, D, and F a transverse section through the middle of the cell is depicted. The fluorescent spots seen represent patches of anti-uPAR antibody bound to uPAR on the surface of the cell. Fluorescence images are pseudocolored so that increasing fluorescence intensity is indicated from blue to white. Scale bar 2 µm.

The fact that in addition to the strong prognostic impact of uPA and PAI-1 elevated uPAR are also associated with poor prognosis adds to the importance of the plasminogen activator system in tumor spread [4, 39]. In all immunohistochemical studies performed so far, there is consent that TAMs do display the uPAR antigen in all cases, in contrast to breast cancer cells, which express the uPAR protein in the minority of cases (5–8%) [11, 12]. In the majority of our examined tumors, uPAR immunoreactivity was observed in macrophages located in inflammatory sites in close vicinity to infiltrating carcinoma cells or in inflammatory areas surrounding the ducts of DCIS. After isolation from primary tissue, macrophages were phenotyped by a panel of antibodies (Table 1). No significant differences were found by using different anti-CD68 antibodies or other macrophage markers. Striking differences were found by using antibodies that detect macrophage-associated inflammatory antigens. The antibodies 27E10 (early), RM3/1 (intermediate), 25F9 (late), and G16/1 (chronic) characterize acute, intermediate, late, and chronic stage inflammatory macrophages. The numbers of macrophages demonstrating positive immunoreactions by using these inflammation markers were significantly lower (P , 0.05) in macrophages derived from normal breast tissue compared with TAMs of DCIS and of invasive breast cancer. This suggests that TAMs of DCIS and of carcinomas are activated and involved in inflammatory proHildenbrand et al.

cesses in close vicinity to infiltrating or non-infiltrating (DCIS) carcinoma cells. Our findings, that TAMs (of breast cancer and DCIS) exhibit more uPAR compared with macrophages derived from normal breast tissue, may suggest two important hypotheses. (1) Monocytes that possess elevated uPAR-levels may be selectively recruited from the bloodstream to inflammatory sites in close vicinity of infiltrating carcinoma cells or carcinoma precursor lesions. This hypothesis is supported by the findings of Gyetko et al. [40] who have found that uPAR and CR3 collaborate in polymorphonuclear leukocytes (PMN) and monocytes and that CD87 facilitates CR3 functions like adhesion and directional migration to a chemotactic gradient. They demonstrated that uPAR is required for PMN chemotaxis because chemotaxis was substantially reduced by treatment of cells with anti-uPAR mAb, and that PMN chemotaxis was selectively inhibited by special saccharides that disrupt the CD87/CR3 association. They conclude that CR3-uPAR interaction affects monocyte trafficking. In our study the elevated levels of uPAR in TAMs may be a characteristic of monocytes/ macrophages that are able to infiltrate easily from the bloodstream into inflammatory sites surrounding malignancies. (2) Another explanation for the higher uPAR levels in TAMs compared with normal breast macrophages is that TAMs may acquire the high uPAR levels after extravasation into inflamma-


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Fig. 4. Expression of uPAR and CR3 (CD11b/CD18) in macrophages derived from DCIS (TAM/DCIS), from invasive breast cancer (TAM), and from normal breast tissue (tissue macrophages, TISM) measured by flow cytometry. One representative case of each macrophage type is demonstrated.

tory sites in close vicinity of infiltrating or non-infiltrating carcinoma cells (DCIS). It is possible that cancer cells may activate TAMs by paracrine and juxtacrine interactions, and that this could result in elevated uPAR levels. Previously we have demonstrated that transforming growth factor b derived from breast cancer cells is able to induce elevated levels of uPA and uPAR in TAMs [17]. A further explanation for high uPAR levels in TAMs is a combination of these two hypotheses. It was reported by Ying Wei et al. [3] that uPAR is also a high-affinity receptor for vitronectin and that uPA is a physiological activator of this vitronectin receptor, which means that uPA stabilizes the vitronectin-uPAR binding and thereby the cellmatrix contact. The PAI-1-mediated release of cells attached to vitronectin seems to occur independently of the ability of PAI-1 to function as a protease inhibitor and results from its direct interaction with vitronectin rather than with uPA [41]. Recently Bajou et al. have provided evidence that PAI-1 is essential for invasiveness [42] by demonstrating that PAI-1 can impair cellular adhesion and promote tumor cell detachment. In this context uPA, uPAR, and PAI-1 are multifunctional proteins involved not only in extracellular matrix proteolysis, but also in cellular adhesion and migration through their binding sites for vitronectin. The elevated uPAR levels in TAMs of inflammatory areas in close vicinity of infiltrating carcinoma cells demonstrate that TAMs are activated and particularly participate in all these functions. However, the high uPAR expression in TAMs 46



does not address uPAR function concerning tumor invasion in this context, which means it is not possible to imply that elevated uPAR levels in TAMs are associated with a degradative macrophage phenotype promoting tumor invasion. We also have no evidence that released uPAR competes with cellassociated uPAR for binding of uPA, thereby influencing cell-associated local proteolysis. It has been shown that the uPAR/uPA/PAI-1 system is involved in regulating tumor angiogenesis [42] and that macrophages play a key role in neovascularization [43]. Previously we have demonstrated that the uPAR/uPA/PAI-1 system is associated with tumor angiogenesis in breast cancer and that elevated uPA- and PAI-1 levels are strongly correlated with tumor angiogenesis and with numbers of macrophages within inflammatory sites of breast cancer tissue [44]. CD87 has been shown to colocalize with the b2 integrin CR3 (CD11b/CD18) [16] on human monocytes. It was demonstrated by Sitrin et al. [16] that CR3 and CD87 are associated via a carbohydrate-lectin interaction in human blood monocytes and that this physical association is necessary for collaboration. We have examined cell-associated expression of CD11b and CD18 by flow cytometry. In Figure 4 exemplary cases of uPAR-, CD11b-, and CD18 expressions of TAMs and normal macrophages are demonstrated. The strong correlations (see Results) between uPAR and CR3 expressions in macrophages (stress imposed, activated, and recovered cells) derived from DCIS, http://www.jleukbio.org

Fig. 5. In situ hybridization for uPAR mRNA in TAMs of DCIS (A), of invasive breast cancer (B, D), and in macrophages derived from normal breast tissue (C) with the use of digoxigenin-labeled antisense (A, B, C) or sense (D) oligodeoxynucleotides specific for uPAR mRNA.

invasive carcinomas, or normal breast tissue may suggest that the uPAR and CR3 are also associated in TAMs and tissue macrophages of the breast. We have examined possible influencing effects of the culturing procedure on macrophage uPAR expression. Figure 1 demonstrates the uPAR release into culture medium over 8 h within the Teflon bags and over 32 h within the culture flasks after the MACS procedure. Mincing and digesting the tissue by collagenase D may activate the uPAR expression in cells and increase the uPAR release into the culture medium. UPAR levels decreased in all types of macrophages until 28 h after beginning the culturing procedure. In the time following, constant uPAR levels were found. This suggests that the stress-imposed macrophages recover during 28 h after cell isolation from primary tissue. This experiment demonstrates that the same culturing procedure, used in all types of macrophages, does not change uPAR expression in just one type of macrophage but may change the uPAR expression either in all types or not at all. Therefore the differences of uPAR levels found in macrophages are reliable. Macrophage uPAR expression was not influenced by isolating the cells through CD11b-coated magnetic beads. The differences of uPAR basal levels in macrophages isolated from DCIS, invasive carcinomas, and normal breast tissues may be due to the conditions within the primary tissue. Corresponding significant differences of the cell-associated uPAR expression were found by flow cytometry and Western Hildenbrand et al.

blot analysis. The fluorescent patches found by confocal laser-scan microscopy were irregularly located on the outer side of the plasma membrane and TAMs (of DCIS and invasive carcinomas) showed clearly more fluorescent patches (representing uPAR-bound antibody) compared with tissue macrophages. Schmitt et al. [45] have shown similar irregular uPAR distributions on macrophage cell line U937. We have compared the uPAR expression of TAMs and tissue macrophages that adhered to a roughed (by a cell scraper) surface with macrophages that adhered to a smooth surface. No significant differences in uPAR expression were found by flow cytometry and by ELISA. TAMs (n 5 30) and tissue macrophages (n 5 30) were probed for the presence of uPAR mRNA by in situ hybridization using digoxigenin-labeled antisense oligodeoxynucleotides. In all cases TAMs and tissue macrophages showed a positive reaction with the antisense probe. In TAMs the staining reaction was always a little stronger compared with tissue macrophages. These uPAR mRNA detections may support our results concerning the different uPAR protein levels in TAMs and tissue macrophages.

ACKNOWLEDGMENTS This work was supported by grants from the Faculty of Clinical Medicine Mannheim, University of Heidelberg. The authors


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gratefully appreciate the excellent technical assistance of Mr. Walter Hofer (Max-Planck-Institut fu¨r Hirnforschung, Frankfurt, Germany), Mrs. S. Trochimczyk, Mrs. R. Hanagarth, and Mrs. T. Gunst. The generous supply of reagents by E. H. Nacih, Diagnostic International, Dossenheim/Heidelberg, Germany is gratefully acknowledged.

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