Identification of the target protein of the metastatic colorectal cancer-specific aptamer W3 as a biomarker by aptamer-based target cells sorting and functional characterization.

Metastasis is a leading cause of cancer-related deaths. Hence, the discovery of more reliable metastasis-related biomarkers is crucial to improve the survival rate of cancer patients. W3 is an aptamer previously produced by the subtractive cell-SELEX using metastatic colorectal cancer cells as target cells and non-metastatic cells as negative cells. In this study, we aimed to evaluate whether the target molecule of W3 can potentially act as a metastatic biomarker. First, we obtained two cell subpopulations with different expression levels of the target molecule by W3-based cell sorting. Subsequently, we demonstrated that W3high cells have a higher metastatic potential than W3low cells both in vitro and in vivo. Further, immunohistochemical analysis revealed that W3 target expression is positively associated with metastasis and poor prognosis of CRC patients. Using mass spectrometry (MS) combined with pull-down, we identified that Ephrin type-A receptor 2 (EphA2) is the target of W3. EphA2's potential as a metastatic predictor was demonstrated by capturing W3-positive circulating tumor cells from CRC patients using a W3 probe. Based on these results, we put forward a stratagem for cell-SELEX-based biomarker discovery: selecting an aptamer through subtractive cell-SELEX towards the phenotype of interest; evaluating the functional phenotype of the target molecule by aptamer-based target cell sorting and analysis of clinical samples; and identifying the aptamer's target molecule using MS and aptamer-based target enrichment. This stratagem not only shortens the time for the clinical application of aptamers but also enables a more targeted and efficient discovery of biomarkers.