Lewis x antigen mediates adhesion of human breast carcinoma cells to activated endothelium. Possible involvement of the endothelial scavenger receptor C-type lectin

Abstract

Lewis x (Le(x), CD15), also known as SSEA-1 (stage specific embryonic antigen-1), is a trisaccharide with the structure Galbeta(1-4)Fucalpha(1-3)GlcNAc, which is expressed on glycoconjugates in human polymorphonuclear granulocytes and various tumors such as colon and breast carcinoma. We have investigated the role of Le(x) in the adhesion of MCF-7 human breast cancer cells and PMN to human umbilical endothelial cells (HUVEC) and the effects of two different anti-Le(x) mAbs (FC-2.15 and MCS-1) on this adhesion. We also analyzed the cytolysis of Le(x+)-cells induced by anti-Le(x) mAbs and complement when cells were adhered to the endothelium, and the effect of these antibodies on HUVEC. The results indicate that MCF-7 cells can bind to HUVEC, and that MCS-1 but not FC-2.15 mAb inhibit this interaction. Both mAbs can efficiently lyse MCF-7 cells bound to HUVEC in the presence of complement without damaging endothelial cells. We also found a Le(x)-dependent PMN interaction with HUVEC. Although both anti-Le(x) mAbs lysed PMN in suspension and adhered to HUVEC, PMN aggregation was only induced by mAb FC-2.15. Blotting studies revealed that the endothelial scavenger receptor C-type lectin (SRCL), which binds Le(x)-trisaccharide, interacts with specific glycoproteins of M (r ) approximately 28 kD and 10 kD from MCF-7 cells. The interaction between Le(x+)-cancer cells and vascular endothelium is a potential target for cancer treatment.

Figure 1. HUVEC express von Willebrand factor but not Le^x epitopes

a) Immunofluorescence study of HUVEC endothelial cells immunostained with anti-von Willebrand factor polyclonal antibodies to confirm endothelial phenotype. Cells exhibit a homogeneous staining pattern localized in endothelial-specific organelles called Weibel-Palade bodies. Magnification: × 400. b) Immunofluorescence study performed on PMN adhered to HUVEC monolayers with mAb FC-2.15. A high level of Le^x expression can be seen on PMN, while HUVEC did not stain. Magnification: × 200.

Figure 2. Expression of Le^x epitopes in MCF-7 breast carcinoma cells, PMN, and HUVEC

Le^x expression was analyzed by flow cytometry using two mAbs against nonsialylated-Le^x, FC-2.15 and MCS-1, and a mAb anti-sialylated-Le^x, CSLEX1, followed by incubation with FITC-conjugated goat anti-mouse Ig. Data are taken from one representative experiment. Filled peaks represent staining with the primary anti-Le^x or anti-sLe^x antibodies followed by FITC-secondary antibody. Staining by isotype-matched mouse immunoglobulins is indicated in white (each non-specific isotype control followed by FITC-conjugated goat anti-mouse Ig). Data are represented as histograms of fluorescence intensity for 10,000 light scatter-gated events. Median values of fluorescence intensity (MFI) are indicated in each figure.

Figure 3. Effect of anti-Le^x mAbs on adhesion of MCF-7 cells and PMN to activated and non-activated HUVEC

a) Adhesion assay to HUVEC was performed with different numbers of [³H] labeled MCF-7 cells (5×10⁴ loaded cells corresponded to 20,020 cpm) (upper panel). In the lower panel, HUVEC were activated with 0, 0.1, 1, 5, and 10 μg/ml of LPS, before incubation with [³H] labeled MCF-7 cells (5×10⁴ loaded cells corresponded to 22,619 cpm). Data shown are mean of bound cpm ± SD of triplicate determinations in three independent experiments. b) A PMN adhesion assay was performed by the myeloperoxidase technique as described under Methods. HUVEC were previously activated with 0, 0.1, 1, 5, and 10 μg/ml LPS. Paired Student's t test, P<0.05. Data shown are mean ± SD of triplicate determinations in three independent experiments. c) Adhesion assay was performed with [³H] labeled MCF-7. Left panel: Incubations were performed in the presence of PBS (control), MCS-1 (50 μg/ml), and FC-2.15 (5, 10, 20, and 50 μg/ml) (5×10⁴ loaded cells corresponded to 58,114 cpm). Paired Student's t test, P<0.05. Data shown are mean + SD of triplicate determinations in three independent experiments. Right panel: Adhesion assay was performed with [³H] labeled MCF-7 digested with neuraminidase (NANAse) or not (control) (5×10⁴ loaded cells corresponded to 11,210 cpm). d) PMN adhesion to HUVEC was assayed in the presence of the following mAbs: FC-2.15, MCS-1, and CSLEX1 at 12.5, 25, 50 μg/ml. Paired Student's t test, P<0.05. e) PMN adhesion to activated HUVEC was assayed after PMN treatment with 5 U/ml of neuraminidase from Clostridium perfringens or with PBS (control). X-axis represents the number of PMN/ml. Paired Student's t test, P<0.05. Data shown are mean ± SD of triplicate determinations in three independent .

Figure 4. Complement-dependent cytotoxicity (CDC) of ⁵¹Cr-MCF-7 and ⁵¹Cr-PMN

a) ⁵¹Cr-release assay was used to test the ability of mAb FC-2.15 (0, 2.5, 10, 20 μg/ml) to mediate CDC on ⁵¹Cr-MCF-7 and ⁵¹Cr-PMN in suspension. b) ⁵¹Cr-release assay was used to test the ability of mAb FC-2.15 (0, 2.5, 10, 20 μg/ml) to mediate CDC on ⁵¹Cr-MCF-7 and ⁵¹Cr-PMN adhered to HUVEC. c) Evaluation of the lytic effect on ⁵¹Cr-HUVEC of bound cells undergoing FC-2.15-mediated CDC. ⁵¹Cr-release assay was used to test the ability of complement alone (C') or FC-2.15 (20 μg/ml) plus complement (FC-2.15 + C') to mediate CDC when unlabeled PMN, unlabeled fMLP-activated PMN, and unlabeled MCF-7 are adhered to ⁵¹Cr-HUVEC. Data shown are mean ± SD of triplicate determinations in three independent experiments. Paired Student's t test, P<0.05. d) MCS-1 mediates CDC: MTT assay was performed on MCF-7 in suspension after treatment with mAbs FC-2.15 or MCS-1 (2.5, 10, 20 μg/ml) in the presence of C'. Control isotype-matched immunoglobulins used were IgM and IgG₃. Paired Student's t test, P<0.05. Data shown are mean ± SD of triplicate determinations in three independent experiments. e) MCS-1 mediates CDC: MTT assay was performed on PMN in suspension after treatment with mAbs FC-2.15 or MCS-1 (2.5, 10, 20 μg/ml) in the presence of C'. Control isotype-matched immunoglobulins used were IgM and IgG₃. Paired Student's t test, P<0.05. Data shown are mean ± SD of triplicate determinations in three independent experiments.

Figure 5. In vitro FC-2.15-induced PMN aggregation exhibits dose- and time-dependence

Suspensions of PMN were incubated in different conditions: a) with different FC-2.15 concentrations for 1 h at 37°C; b) with 20 μg/ml FC-2.15 during different incubation times at 37°C. c) PMN aggregation was evaluated in the presence of different concentrations of FC-2.15, control IgM, MCS-1 or control IgG₃. The percentage of aggregated cells was calculated for each condition as described under Methods. Determinations were performed by duplicate (bars: mean ± SD).

Figure 6. In vitro aggregation of PMN by FC-2.15 appears to be partially mediated by β₂-integrins, which express Le^x

a) Isolated PMN were preincubated in the absence (control) or the presence of mAb TS1/18, a β₂-integrin blocking antibody. Then, the agglutination assay was performed in the presence of FC-2.15 (20 μg/ml), KIM185 (20 μg/ml) or PBS (control). Each determination was performed in duplicates in two independent experiments (bars: mean ± SD). b) PMN membrane extracts were immunoprecipitated with either MEM-170 mAb, MEM 48 mAb or isotype-matched immunoglobulins (not shown), and immunoprecipitates were subjected to SDS-PAGE and immunoblotting with either MEM-170, MEM-48 or FC-2.15 mAbs as indicated.

Figure 7. Glycoproteins of MCF-7 cells are bound by the scavenger receptor C-type lectin

a) Immunoblotting analysis of MCF-7 cells glycoproteins containing Le^x epitopes. Membrane extracts of MCF-7 cells were submitted to SDS-PAGE (15%) and immunoblotted with FC-2.15 and MCS-1 mAbs. b) Membrane extracts of MCF-7 cells were subjected to SDS-PAGE (17.5%) and parallel lanes were stained with Coomassie blue or blotted onto a nitrocellulose membrane that was probed with ¹²⁵I-labeled extracellular domain of SRCL as described under Methods.