Tissue microarray and liquid biopsy approaches identify EphB3, cMet, and miR-3168 as biomarkers of colorectal cancer

Colorectal cancer (CRC) remains a significant global health concern, and reliable biomarkers are needed to improve early diagnosis, prognostication, and personalized treatment strategies. This study investigated the expression of cell surface proteins and serum exosomal miRNAs in CRC patients. Tissue microarrays (TMAs) constructed from primary and metastatic CRC samples were analyzed for five cell surface proteins: EphB1, EphB3, EphA2, cMet, and EphB4. Immunohistochemistry was performed on the TMAs to validate their expression levels. We found that the distribution of expression for all four receptors, except EphA2, was significantly higher (<i>p</i> < 0.01) in CRC samples compared to non-cancerous tissue. High expression of EphB3 was detected in 37% of patient samples, followed by cMet, which was observed in 35%. Exosomes were isolated from the serum of three CRC patients with tumors exhibiting high expression of LGR5 and/or EphB3, four healthy donors and two CRC cell lines. Serum exosomal miRNA analysis identified miR-3168 as significantly upregulated in CRC patients, showing a 3.8-fold increase compared to healthy controls (<i>p</i> < 0.001) and a 2.6-fold increase in CRC cell lines compared to controls (<i>p</i> = 0.02). Ingenuity Pathway Analysis (IPA) suggested that miR-3168 may regulate cMet, EphB3, and EphB4, along with other CRC-associated molecules and pathways. These findings highlight the potential of EphB3 and cMet as biomarkers in CRC, and miR-3168 as a promising minimally-invasive biomarker for monitoring disease progression and therapeutic response. However, further validation in larger cohorts is needed to establish their clinical utility.