Label-free detection for colorectal circulating tumor cells

Circulating tumor cells (CTCs) in the peripheral blood of colorectal cancer patients can indicate metastatic potential. Immunomagnetic isolation, utilizing interactions between antibodies and epithelial-specific surface antigens, is commonly used but can miss CTCs that have undergone epithelial-mesenchymal transition (EMT) due to suppressed epithelial markers. Alternative methods with high capture sensitivity that rely on physical properties like density gradient and erythrolysis are developed. Immunofluorescence staining characterizes CTCs based on molecular marker expression. Plastin-3 (PLS3) is a protein marker overexpressed in colorectal cancer CTCs undergoing EMT. This study aims to isolate and characterize PLS3-expressing CTCs from colorectal cancer patients, comparing the erythrolysis and density gradient methods for isolation and characterization based on the number of isolated cells. Optimization and validation were performed with spike-in assays using HT-29 cells in healthy donor blood. Results showed erythrolysis isolated more CTCs, with PLS3-expressing CTCs found in all subjects, both as single cells and clusters of varying shapes and sizes. This study suggests that PLS3 is a suitable biomarker for colorectal cancer CTC identification and that both methods are feasible approaches for CTC enrichment.