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, gpHAE3, 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.

Exploring Glycan Markers for Immunotyping and Precision-targeting of Breast Circulating Tumor Cells

“…Results of the microarray assay are shown as microarray scores, i.e., means of the log2-transformed fluorescent intensities (MFIs) of multiple detections of a given antigen preparation (Figure 1A and Supplementary Table 1). Glycan-specific ELISA was performed as described in Figure 1 legend following our standard protocol (20). …”

“…This antibody was raised by a murine mammary tumor antigen epiglycanin (EPGN) (21) but was found to cross-react with a number of human epithelial tumors in tissues including the lung, prostate, bladder, esophagus, and ovaries (22–24). In a recent flow-cytometry-based screening for tumor cell surface biomarkers, we found that HAE3 also strongly cross-reacts with human breast tumors (20). …”

“…Glycan markers of bCTCs may have unique value in BCa healthcare, especially in the personalized therapy that targets specific immunotypes of BCa. Thus, our team has worked to identify potential glycan markers of bCTCs (20). …”

“…We therefore explored the potential natural ligands of HAE3 in the repertoire of carbohydrate-based autoantigens. By scanning a large collection of carbohydrate antigens using carbohydrate microarrays, we found that HAE3 is highly specific for a blood group precursor cryptic epitope normally hidden in the cores or internal chains of blood group substances (20). In this study, we further investigated whether this target is applicable for detection and immunotyping analysis of CTCs in patients with metastatic breast cancer.…”

Unraveling Sugar Chain Signatures of the “Seeds” of Tumor Metastasis

“…This microarray finding was further validated by glycan-specific enzyme-linked immunosorbent assay (ELISA) and glyco-conjugate-based epitope competition assays [13,14]. Figure 2 is a schematic of the blood group substance structure with the common blood group precursor core structure highlighted.…”