Proteomic Analysis of Paired FFPE Tissue and Extracellular Vesicles Reveals Proteins Associated with Recurrence in Stage II Colorectal Cancer

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide. Stage II CRC poses a clinical challenge due to its heterogeneous outcomes, with 15-20% of patients experiencing recurrence. Current prognostic models based on clinicopathologic features lack sufficient precision, highlighting the need for new molecular markers. In this study, we employed an integrative TMT-based proteomics strategy to identify biomarkers of recurrence in stage II CRC. We analyzed paired formalin-fixed paraffin-embedded (FFPE) tissues and small extracellular vesicles (sEVs) from stage II recurrent and nonrecurrent CRC patients. Validation of proteomics data was performed in silico and by WB, immunohistochemistry, ELISA, and in vitro functional cell-based assays. This multifaceted approach identified several dysregulated proteins associated with CRC recurrence, including MANF, TLN1, TALDO1, and CDCA2, among others. CDCA2 knockdown altered the tumorigenic properties of CRC cells in vitro, correlating findings with its association with prognosis. Conversely, higher plasma levels of MANF were found in nonrecurrent CRC patients, aligning results with its favorable prognosis profile. Collectively, our findings support the value of combining paired FFPE tissue and sEVs proteomics analyses to uncover recurrence-associated biomarkers, offering the potential for risk stratification and management of stage II CRC patients.