Proteomic Differentiation of Colorectal Cancer From Normal Tissue via Ex Vivo E‐Biopsy: A Novel Approach to Molecular Sampling for Diagnostic Precision

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Colorectal cancer (CRC) is one of the most common malignancies worldwide. Early and accurate diagnosis remains a clinical priority, yet current biopsy techniques are invasive, spatially limited, and may not capture the molecular heterogeneity of tumors. We evaluated the feasibility and diagnostic potential of electroporation-based biopsy (e-biopsy) as a minimally invasive technique for proteomic sampling of colorectal cancer tissues. We conducted a multicenter, multinational study involving 19 patients undergoing surgical resection for CRC. Paired tumor and adjacent normal tissues were sampled ex vivo using e-biopsy. Proteins extracted from each sample were analyzed via LC-MS/MS. Bioinformatics pipelines, including differential expression, PCA, and pathway analysis, were used to identify CRC-specific signatures. E-biopsy consistently retrieved more proteins from tumor tissues than from adjacent healthy tissues (mean: 1300 vs. 800). Of the 3246 proteins identified, 54% were significantly upregulated in tumor tissues. Notably, proteins such as DLAT, LETM1, RBBP4, PPIB, and BCAP31 emerged as potential CRC biomarkers. Functional analyses revealed dysregulation in RNA processing, immune response, and metabolic pathways, consistent with known CRC biology. The integrated workflow, spanning tissue collection, electroporation, protein isolation, and mass spectrometry analysis across four institutions in two countries, was successfully executed, demonstrating the feasibility of multi-institutional implementation of the e-biopsy protocol while preserving tissue integrity throughout. E-biopsy enables rapid, reproducible, and minimally invasive molecular sampling of CRC tissue. This study demonstrates its potential to complement standard histopathology, aid in early diagnosis, and support molecularly guided treatment strategies in colorectal oncology.

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