Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer

Abstract

Cancer-associated carbohydrate antigens are often found on the surface of cancer cells. Understanding their roles in cancer progression will lead to the development of new therapeutics and high-sensitivity diagnostics for cancers. Globo H is a member of this family, which is highly expressed on breast cancer cells. Here, we report the development of a glycan microarray of Globo H and its analogs for measurement of the dissociation constants on surface (K(D,surf)) with three different monoclonal antibodies (VK-9, Mbr1, and anti-SSEA-3), to deduce their binding specificity. The glycan microarray was also used to detect the amount of antibodies present in the plasma of breast cancer patients and normal blood donors. It was shown that the amount of antibodies against Globo H from breast cancer patients were significantly higher than normal blood donors, providing a new tool for possible breast cancer diagnosis. Compared with the traditional ELISA method, this array method required only atto-mole amounts of materials and is more effective and more sensitive (5 orders of magnitude). The glycan microarray thus provides a new platform for use to monitor the immune response to carbohydrate epitopes after vaccine therapy or during the course of cancer progression.

Fig. 1.

Chemical structure of Globo H and abbreviations of Globo H analogs.

Fig. 2.

Binding of monoclonal antibodies to Globo H and its analogues. (A) Slide image obtained from fluorescence scan after antibody incubation assay with VK-9. The grid contain sugars 1–8 printed at an 80 μM concentration. (B and C) Slide images obtained by assay with MBr1 (B) and anti-SSEA-3 monoclonal antibody (C).

Fig. 3.

Binding curves for Globo H printed at different concentrations (100, 80, 50, 40, 20, 10, and 5 μM) are shown. The curves were obtained by using Cy3-labeled goat anti mouse IgG secondary antibodies.

Fig. 4.

Glycan array is a mimic of cell surface expressing glycans for study of multivalent interaction.

Fig. 5.

Ratios of IgG levels against Globo H analogs in sera from breast cancer patients and normal blood donors. The relative fluorescence ratios were obtained from fluorescence intensity of Globo H or Globo H analogs divided by fluorescence intensity of Gb5. The mean of Globo H/Gb5 IgG ratios was significantly higher in sera of breast cancer patients.

Fig. 6.

Ratios of IgM levels against Globo H analogs in sera from breast cancer patients and normal blood donors. The relative fluorescence ratios were obtained from fluorescence intensity of Globo H or Globo H analogs/fluorescence intensity of Gb5. The mean of Globo H/Gb5 IgM ratios was significantly higher in sera of breast cancer patients.

Fig. 7.

The comparison of glycan microarray and ELISA for mornitoring anti-Globo H response. Sera from immunized mice were serially diluted and analyzed for anti-Globo H specific IgG antibody on glycan microarray and ELISA plates. The fold level of signal over background was calculated as (mean of fluorescence of postserum − preserum)/background intensity in glycan micorarray or (OD value of postserum − preserum)/background in ELISA. The antibody levels were detectable up to 1:1,920 dilution method (3.2 ± 1.0-fold of background signal), but only 1:240 dilution in ELISA (1.08 ± 0.48-fold of background OD). Values shown were mean ± SD of four mice.