Unveiling Novel Tumor-Associated Antigens to Overcome Diagnostic Challenges in Barely Seroreactive Colorectal Cancer Patients Using Mass Spectrometry-Based Proteomics.

The humoral immune response plays a critical role in cancer immune surveillance, producing autoantibodies against tumor-associated antigens (TAAs). These autoantibodies show potential as non-invasive biomarkers for early cancer detection in colorectal cancer (CRC), a leading cause of cancer-related mortality worldwide. Despite their potential, the existence of non-seroreactive CRC patients to common TAAs poses a significant challenge, limiting the effectiveness of autoantibody-based diagnostics. Focusing on previously identified non-seroreactive CRC patients, we have employed high-resolution proteomics to profile their humoral immune response, enabling the discovery of novel CRC-specific TAAs to address this diagnostic gap. These TAAs were expressed in mammalian cells with 6xHisTag and HaloTag to enhance their detection and seroreactivity assessment through ELISA-like tests. Our integrative approach revealed previously undetected TAAs, expanding the panel of CRC-specific biomarkers. By overcoming the limitations of traditional diagnostics, this study provides a deeper understanding of the humoral immune response in CRC and underscores the potential of proteomic-driven strategies to improve early detection. These findings expand the repertoire of CRC-associated serological targets and support the use of immunoproteomic strategies to identify complementary antigens in CRC patients, paving the way for the development of liquid biopsy-based diagnostic approaches for individuals previously classified as non-seroreactive.