Identification of ovarian cancer-associated antigens forming complexes with autoantibodies

Tumor-associated antigen (Ag)-autoantibody (AAb) complexes are known to be present in the circulation of patients with multiple types of cancer; however, their significance remains poorly understood. We performed immunoblotting of whole cell extracts of the human ovarian cancer cell lines AMOC2, OVCAR3 and SKOV3 with either free antibodies (Abs) or antigen-dissociated Abs obtained from ovarian cancer ascites and healthy sera. In addition, Ag-AAb complexes isolated from ovarian cancer ascites were analyzed by mass spectrometry to identify tumor-associated antigens. Enzyme-linked immunosorbent assay (ELISA) was then used to evaluate the presence of one extracellular matrix (ECM) candidate antigen, SPON1, in its free versus complexed form in ascites and sera. Immunoblotting revealed that tumor-associated AAbs in ovarian cancer ascites predominantly exist in Ag-AAb complexes rather than as free AAbs. Proteomic profiling identified 72 tumor-associated antigens enriched in ovarian cancer ascites compared with healthy and disease control sera; more than half were ECM- or epithelial–mesenchymal transition (EMT)-related proteins. ELISA demonstrated that SPON1 was preferentially detected as Ag-AAb complexes, but not in its free form, in both ascites and sera of ovarian cancer patients. We uncovered that AAbs predominantly form complexes with tumor-associated antigens in ovarian cancer patients. Since the presence of Ag-AAb complexes has been overlooked in many reports, the identification of tumor-associated antigens and AAbs from Ag-AAb complexes would provide novel insights into their relevance to a variety of cancers and other diseases.