Glycan Markers as Potential Immunological Targets in Circulating Tumor Cells

Abstract

We present here an experimental approach for exploring a new class of tumor biomarkers that are overexpressed by circulating tumor cells (CTCs) and are likely targetable in immunotherapy against tumor metastasis. Using carbohydrate microarrays, anti-tumor monoclonal antibodies (mAbs) were scanned against a large panel of carbohydrate antigens to identify potential tumor glycan markers. Subsequently, flow cytometry and fiber-optic array scanning technology (FAST) were applied to determine whether the identified targets are tumor-specific cell-surface markers and are, therefore, likely suitable for targeted immunotherapy. Finally, the tumor glycan-specific antibodies identified were validated using cancer patients' blood samples for their performance in CTC-detection and immunotyping analysis. In this article, identifying breast CTC-specific glycan markers and targeting mAbs serve as examples to illustrate this tumor biomarker discovery strategy.

Keywords: Breast cancer; Breast circulating tumor cells; Carbohydrate microarray; Glycan markers.

Fig. 15.1

Glycan array discovery and FAST-scan validation of a novel glycan marker gp^C1 of bCTCs (Adapted from Wang et al. 2015a)

Fig. 15.2

Carbohydrate microarray analysis of anti-epiglycanin mAb HAE3. Seventy-six glyco-proteins, 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 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 (Adapted from Wang et al. 2015b)

Fig. 15.3

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 (Adapted from Wang et al. 2015b)

Fig. 15.4

Glycan marker gp^C1 is expressed in significant numbers of CTCs in Stage IV breast cancer patients. (a) FAST-scan images of bCTCs. Upper panels: Co-staining of C1 (green) and DAPI (blue); Bottom panels: co-staining of anti-CK (red) and DAPI (blue). (b) Distribution of gp^C1–positive and -negative bCTCs in five subjects. C1-staining of bCTCs was semi-quantitatively measured by the FAST scan as antibody negative (blue), 1+ (purple), 2+ (green), and 3+ (red) as described. A patient with triple-negative BCa (ID# 189370) was measured gp^C1–positive in 37 of 40 CTCs with 50% scored as strong positive (2+ and 3+). (ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2). (c) A summary of patients’ demographics and clinical characteristics (Adapted from Wang et al. 2015a)