(H-289) Exosomal Thomsen-Friedenreich Glycoantigen as a New Biomarker for Cancer Liquid Biopsy

Introduction:: Exosomes are cell-secreted, nanosized vesicles that have emerged as potent biomarkers for cancer liquid biopsy. However, most of current exosomal biomarkers are not cancer-specific. We discovered a cancer-specific exosomal marker, Thomsen-Friedenreich glycoantigen (TF-Ag-α; Galβ1-3GalNAc alpha), which is a carbohydrate antigen arising from aberrant glycosylation found in malignant cells but not in normal tissues. We first developed a unique monoclonal antibody, JAA-F11, with high specificity to TF-Ag-α and a surface plasmon resonance (SPR)-based assay to detect exosomal TF-Ag-α in as low as 10μL serum. We demonstrated that cancer cell-derived exosomes carry TF-Ag-α. With both lung cancer and breast cancer as the disease models, we detected significantly higher levels of exosomal TF-Ag-α in sera from cancer patients (n=95) than normal controls (n=26). These results demonstrated that exosomal TF-Ag-α is a specific and potent liquid biopsy biomarker for cancer diagnosis.


Materials and Methods:: To detect exosomal TF-Ag-α using the SPR assay, JAA-F11 antibodies were coated on an Au-coated glass slide through biotin-avidin interaction. Sera (10μL) from lung cancer and breast cancer patients were directly applied onto the biochip. The capture of exosomes expressing TF-Ag-α by JAA-F11 antibodies on the biochip surface changed the local reflective index, allowing optical detection of exosomal TF-Ag-α via SPR. IgG3 control antibodies were used to measure the same samples, and the signals were used to remove background noise caused by nonspecific binding. The analytical performance (limit of detection (LOD), linear range, specificity) of the SPR assay was evaluated using A549 lung cancer and MDA-MB-231 breast cancer cell-derived exosomes. The diagnostic performances (sensitivity, specificity, accuracy) of exosomal TF-Ag-α were investigated using serum samples from normal controls (n=36) and patients with lung cancer (n=40, stage I-IV) and breast cancer (n=65, stage 0-IV).


Results, Conclusions, and Discussions:: We developed the SPR assay utilizing JAA-F11 antibody to allow sensitive detection of exosomal TF-Ag-α (Fig. 1a). The LODs were 5×10⁹ and 1×10⁹ exosomes/mL for exosomes from A549 and MDA-MB-231 cells respectively. Significantly higher levels of TF-Ag-α were observed in exosomes from A549 and MDA-MB-231 cancer cells than those from BEAS-2B and MCF-10a normal cells. The expression of exosomal TF-Ag-α was significantly higher in sera from lung cancer patients (n=40) (Fig. 1b) and breast cancer patients (n=65) (Fig. 1c) than in normal controls (n=20 for lung cancer, n=16 for breast cancer). Results from ROC analysis showed that exosomal TF-Ag-α distinguished normal controls from lung cancer patients with an AUC of 0.92, a sensitivity of 1.0 and a specificity of 0.7, and distinguished normal controls from breast cancer patients with an AUC of 0.95, a sensitivity of 0.89, and a specificity of 0.94. These results demonstrated that exosomal TF-Ag-α is a potent biomarker for cancer diagnosis.


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