Carbohydrate Microarrays Identify Blood Group Precursor Cryptic Epitopes as Potential Immunological Targets of Breast Cancer

Abstract

Using carbohydrate microarrays, we explored potential natural ligands of antitumor monoclonal antibody HAE3. This antibody was raised against a murine mammary tumor antigen but was found to cross-react with a number of human epithelial tumors in tissues. Our carbohydrate microarray analysis reveals that HAE3 is specific for an O-glycan cryptic epitope that is normally hidden in the cores of blood group substances. Using HAE3 to screen tumor cell surface markers by flow cytometry, we found that the HAE3 glycoepitope, gp(HAE3), was highly expressed by a number of human breast cancer cell lines, including some triple-negative cancers that lack the estrogen, progesterone, and Her2/neu receptors. Taken together, we demonstrate that HAE3 recognizes a conserved cryptic glycoepitope of blood group precursors, which is nevertheless selectively expressed and surface-exposed in certain breast tumor cells. The potential of this class of O-glycan cryptic antigens in breast cancer subtyping and targeted immunotherapy warrants further investigation.

Figure 1

Carbohydrate microarray analysis of antiepiglycanin mAb HAE3. Seventy-six glycoproteins, glycoconjugates, and polysaccharides were spotted in triplicates in 1 to 2 dilutions to yield the customized microarrays for antibody screening. (a) Microarray detections were shown as the mean fluorescent intensities (MFIs) of each microspot with antigen-binding signal in red and background reading in blue. Each error bar is constructed using one standard deviation from the mean of triplicate detections. The labeled antigens include HCA (ID# 1 and ID# 2), a number of blood group precursors (29^#–32^#), and a microarray spotting marker (80^#). (b) Images of a microarray stained with HAE3 (5 μg/mL). (c) Schematic of a blood group substance structure with the conserved O-glycan core highlighted.

Figure 2

Carbohydrate-specific ELISA and ELISA inhibition assays validate binding specificities of HAE3. (a) An antigen-specific ELISA distinguished HAE3 binding specificity from the T-antigen-specific PNA. ELISA plates were coated with O-core antigen, Tij II (31^#), T (81^#), and Tn (82^#) at 10 μg/mL to react with either HAE3 (5.0 μg/mL) or PNA (1.0 μg/mL). (b) ELISA inhibition assays with a series of carbohydrate antigens as competitors (25.0 μg/mL) to inhibit interaction between EPGN (1.0 μU/mL) and anti-HCA (HAE3, 1.0 μg/mL). These antigens include HCA (1^#), EPGN (85^#), which is the immunogen of HAE3, and blood group substances Tn (83^#), T (84^#), H (4^# or 22^#), A (3^#), B (14^#), Beach P1 (29^#), Tij II (31^#), and OG (32^#). Results are shown as percent inhibition in the presence of an inhibitor.

Figure 3

HAE3 cell surface staining detected selective expression of the HAE3-cryptic glycan markers in human cancer cell lines. (a) Four tumor cell lines, T-47D, A549, PC3, and SKMEL-28, were stained with the C1 preparation of HAE3 (IgM) at 1 : 6 dilution or with an isotype control IgM, 9.14.7 (5.0 μg/mL). (b) Seven breast cancer cell lines were stained with purified mAb HAE3 (5.0 μg/mL) or 9.14.7 (5.0 μg/mL). These cell lines are T-47D, MCF-7, SK-BR-3, BT-549, Hs578T, MDA-MB-231, and MDA-MB-468. An R-PE-conjugated goat anti-mouse IgM antibody was applied to quantify the cell surface-captured IgM antibodies. Blue line: HAE3 stain; Red line: 9.14.7 IgM isotype control.

Figure 4

A Bio-Gel P-10 plot of Tij II antigen. Tij II (20% from 2nd 10%) substance was fractioned in a precalibrated Bio-Gel P-10 column at 0.5 mL per fraction. The neutral sugar content in each fraction was determined by phenol-sulfuric acid color reaction and quantitatively measured at OD490 nm. The sizes of the Tij II substance were measured based on the neutral sugar elution profile with reference to the calibrated saccharide molecular weight standards as indicated in the plot. IM₅ stands for isomaltopentaose.