Analysis of the differential proteome associated with CRC recurrence by in-depth proteomic profiling of FFPE tissue samples and plasma extracellular vesicles

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Colorectal cancer (CRC) is the second leading cause of cancer-related death and the third most common cancer worldwide. Most CRC patients are diagnosed at advanced stages, when the tumour has already metastasized other organs, limiting treatment options and decreasing survival rates. Therefore, the early detection of CRC and monitoring of its progression are crucial steps toward improving patient outcomes.
Here, we aimed to compare the protein expression profiles of tumoral tissues from recurrent and non-recurrent stage II CRC patients during follow-up to identify dysregulated proteins with potential as diagnostic or prognostic biomarkers. We analysed paired protein extracts from FFPE tumour tissue samples and plasma extracellular vesicles (EVs) by Tandem Mass Tag (TMT) 10-plex quantitative proteomics using an Orbitrap Exploris 480 mass spectrometer equipped with FAIMS Pro Duo Interface.
Through two TMT-based proteomics experiments, we identified 25 dysregulated proteins in plasma EVs, and 59 in tumour tissues, with a fold change ≥ 1.5, associated to CRC recurrence. Protein dysregulation was further validated through WB using protein extracts from isogenic CRC cells with different metastatic capabilities, paired healthy and tumoral tissues, and by ELISA using plasma samples from CRC patients and controls. Finally, loss-of-function cell-based assays were performed in CRC metastasis cell models using siRNAs to confirm the relevance of some of these proteins in the disease.

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