Macrophage-tumour cell interactions: identification of MUC1 on breast cancer cells as a potential counter-receptor for the macrophage-restricted receptor, sialoadhesin

Abstract

In many carcinomas, infiltrating macrophages are commonly found closely associated with tumour cells but little is known concerning the nature or significance of adhesion molecules involved in these cellular interactions. Here we demonstrate in primary human breast cancers that sialoadhesin (Sn), a macrophage-restricted adhesion molecule, is frequently expressed on infiltrating cells that often make close contact with breast carcinoma cells. To determine whether Sn could act as a specific receptor for ligands on breast cancer cell lines, binding assays were performed with a recombinant form of the protein fused to the Fc portion of human immunoglobulin G1 (IgG1) (Sn-Fc). Sn-Fc was found to bind specifically and in a sialic acid-dependent manner to the breast cancer cell lines MCF-7, T47.D and BT-20 both in solid- and solution-phase binding assays. To investigate the nature of the sialoglycoproteins recognized by Sn on breast cancer cells, MCF-7 cells were labelled with [6-3H]glucosamine. Following precipitation with Sn-Fc, a major band of approximately 240000 MW was revealed, which was shown in reprecipitation and Western blotting experiments to be the epithelial mucin, MUC1.

Figure 1

Immunocytochemical double-staining of primary human breast cancer revealing close proximity of tumour cells and infiltrating Sn-positive macrophages. Frozen sections of breast tissue were stained by an indirect immunoperoxidase procedure using the HMFG1 anti-MUC-1 mAb and developed with diaminobenzidine to give a brown colour. The sections were then stained by an indirect alkaline phosphatase method using affinity-purified rabbit anti-Sn IgG and developed with Fast Red. Islands of brown MUC1-positive tumour cells (T) are surrounded by pink Sn-positive macrophages (M) (×250).

Figure 2

Solid-phase binding assays of Sn-Fc to breast cancer cell lines. Fc-proteins were immobilized to plastic wells, cells added and unbound cells washed off after 30 min. (a) Binding of breast cancer cell lines to Sn-Fc compared with CD22-Fc or NCAM-Fc used as a negative control. (b) Sialidase-treated MCF-7 cells bind to Sn-Fc at background levels. Bars show mean±1 SD of triplicate values. Results shown are representative of three experiments performed.

Figure 3

FACS analysis of Sn-Fc binding to MCF-7 cells. Cells were incubated with either Sn-Fc or NCAM-Fc as a control, washed, and binding detected with antihuman IgG-FITC. Sn-Fc shows strong binding which is reduced to background levels after sialidase treatment of the MCF-7 cells.

Figure 4

Precipitation of glycoproteins from 6-[³H]glucosamine-labelled MCF-7 cells. MCF-7 cells were labelled overnight with 6-[³H]glucosamine, lysed and precipitated with the indicated reagents. (a) Sn-Fc specifically precipitates high-molecular-weight material that comigrates with glycoproteins recognized by anti-MUC1 mAb SM3. (b) Lysates from MCF-7 cells labelled with 6-[³H]glucosamine were immunoprecipitated with anti-MUC1 mAb SM3 (left track) and eluates were reprecipitated with either Sn-Fc or NCAM-Fc. Sn-Fc specifically precipitates both allelic forms of MUC1.

Figure 5

Western blot analysis of glycoproteins precipitated by CD22-Fc or Sn-Fc. Lysates prepared from 6-[³H] glucosamine-labelled MCF-7 cells were precipitated with either CD22-Fc or Sn-Fc, proteins separated by SDS-PAGE and transferred to nitrocellulose membranes. The blots were probed with either anti-MUC1 mAb SM3 or anti-CD44 mAb P2A1. Sn-Fc precipitates glycoproteins that are recognized specifically by the anti-MUC1 but not by the anti-CD44 mAb. The nature of the faint band detected with anti-MUC-1 following precipitation with CD22-Fc is unknown.