Exosomal Thomsen-Friedenreich Glycoantigen: A New Liquid Biopsy Biomarker for Lung and Breast Cancer

Introduction: Tumor-derived exosomes (TEX) have become promising liquid biopsy biomarkers for cancer screening and diagnosis. The clinical applications of several exosome components including nucleic acids, proteins and lipids in cancer diagnosis have been extensively investigated. However, carbohydrates, an important type of biomolecule, have not yet been studied. In this study, we reported a new exosomal marker that is carbohydrate-based, specifically the alpha-linked Thomsen-Friedenreich glycoantigen (TF-Ag-α).

TF-Ag is a typically cryptic antigen that is aberrantly expressed in cancer cells and closely related to tumor growth, immune evasion, and metastasis. TF-Ag-α is specifically expressed in tumor tissues but not in normal issues. Whether exosomes carry TF-Ag-α and whether exosomal TF-Ag-α can be used as a blood-based biomarker for cancer diagnosis need to be investigated. We demonstrated, for the first time, that exosomes do carry TF-Ag-α. To transform our discovery for clinical applications, we developed a surface plasmon resonance (SPR)-based liquid biopsy assay to detect exosomal TF-Ag-α in serum samples. The clinical potential of exosomal TF-Ag-α in cancer diagnosis was demonstrated using both lung cancer and breast cancer as disease models (Fig. 1a). Significantly higher levels of exosomal TF-Ag-α were observed in sera from cancer patients (n=155) than normal controls (n=78), demonstrating it is a potent liquid biopsy biomarker for cancer diagnosis.

Materials and

Methods: A SPR assay was developed to detect exosomal TF-Ag-α in sera. Briefly, mouse JAA-F11 antibodies (a monoclonal antibody with high specificity to TF-Ag-α) were attached on a 49nm Au-coated glass slide through biotin-avidin interaction. Serum (10uL) was directly applied on the biochip. The binding between exosomes and JAA-F11 antibodies changed the local reflective index, allowing optical detection of exosomal TF-Ag-α.

SEM was used to visualize captured exosomes on the biochip surface. The levels of TF-Ag-α in sera, exosomes isolated from sera and exosome-depleted sera (n=10) were measured to confirm that exosomes carry TF-Ag-α. The analytical performances (limit of detection, linear range, and repeatability) of the SPR assay were determined using a normal control serum sample spiked with exosomes derived from A549 lung cancer cells and MDA-MB-231 breast cancer cells (0 to 1011 exosomes/mL). For lung cancer, the diagnostic performances of exosomal TF-Ag-α were evaluated using exosomes derived from BEAS-2B normal lung bronchial epithelial cells, A549 and DMS-114 lung cancer cells, and sera from normal controls (n=47, including both low risk and high risk patients) and lung cancer patients (n=60, stage I-IV). For breast cancer, the diagnostic performances of exosomal TF-Ag-α were evaluated using exosomes derived from MCF-10A normal cells, MDA-MB-231 and MCF-7 breast cancer cells, sera from normal controls (n=34 including women with benign breast conditions) and breast cancer patients (n=95, stages 0-IV). Receiver operating characteristics analysis was performed to determine the sensitivity, specificity, and AUC of exosomal TF-Ag-α in detecting lung and breast cancer.

Results, Conclusions, and Discussions: SEM successfully observed exosomes expressing TF-Ag-α captured on the biochip surface. Significantly higher levels of TF-Ag-α were detected in sera and exosomes isolated from sera than in exosomes-depleted sera, demonstrating that TF-Ag-α was mainly carried by exosomes. The SPR assay may detect as low as 0.01% exosomes that express TF-Ag-α in total exosomes. It also showed excellent intra-day, inter-day and inter-operator reproducibility.

For lung cancer diagnosis, at an exosome concentration of 4×1010 exosomes/mL, 2.1-fold and 2.2-fold higher levels of TF-Ag-α were detected in exosomes from A549 and DMS-114 lung cancer cells, respectively, than in those from normal BEAS-2B cells. Significantly higher levels of exosomal TF-Ag-α were observed in sera from lung cancer patients (n=40, stage I-IV) than normal controls (n=41, 20 low risk and 21 high risk patients) (Fig. 1b). Exosomal TF-Ag-α detected lung cancer with AUC of 0.95, sensitivity of 0.98 and specificity of 0.85. This diagnostic performance was further validated in a blinded study including 20 lung cancer patients (stage I-IV) and 6 normal controls.

For breast cancer diagnosis, at an exosome concentration of 1011 exosomes/mL, 13.5-fold and 7.5-fold higher levels of TF-Ag-α were observed in exosomes from MDA-MB-231 and MCF-7 breast cancer cells than in those from normal MCF-10A cells. The levels of exosomal TF-Ag-α in sera from breast cancer patients (n=70) were significantly higher than those in normal controls (n=29, 14 without breast conditions and 15 with benign breast conditions) (Fig. 1c). Exosomal TF-Ag-α detected breast cancer with AUC of 0.97, sensitivity of 0.93 and specificity of 0.90. This diagnostic performance was further validated in a blinded study including 25 breast cancer patients (stage 0-IV) and 5 normal controls.

With 233 patients and controls in training and validation sets, exosomal TF-Ag-α distinguished lung and breast cancer patients from normal controls with AUC ≥0.95 and ≥0.97, respectively. These results collectively demonstrated the great potential of exosomal TF-Ag-α in lung and breast cancer liquid biopsy. In the future, we will further validate the diagnostic potential of exosomal TF-Ag-α in a larger cohort of patients and controls and expand the cancer type to include more cancers.

Acknowledgements (Optional):