Unraveling CTC Heterogeneity: DLD-Assisted Immunomagnetic Sorting and ICP-MS for the Isolation and Profiling of Epithelial–Mesenchymal Transition-Related States

Accurate analysis of circulating tumor cell (CTC) heterogeneity at the single-cell level, in addition to circulating tumor cell (CTC) enumeration, holds great promise for advancing our understanding of cancer progression and therapeutic response. Magnetic-activated cell sorting integrated with microfluidics has emerged as a powerful tool for CTC isolation and phenotyping. However, this method is still plagued by inherent trade-offs among sample throughput, magnetic separation resolution, and recovery of CTC subpopulations for downstream analysis. To address these limitations, we report a DLD@MACS chip engineered to generate a magnetic field gradient within a deterministic lateral displacement (DLD) channel, which facilitates the simultaneous size-based enrichment of CTCs and magnetic separation of their epithelial cell adhesion molecule (EpCAM) expression-related phenotypes into distinct streams, including the separation and recovery of EpCAM-negative CTCs. The DLD@MACS chip enabled significantly higher sample throughput (200 μL/min) compared to reported MACS-based phenotyping methods. Furthermore, a synergistic effect between DLD and the magnetic field gradient yields an enhanced separation resolution even at low magnetization. Through single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) detection, this method was successfully applied to enumeration and phenotyping of CTCs in the blood samples from more than 30 breast cancer patients. The detected CTC number and relative EpCAM expression were correlated to the breast cancer stage and clinical treatment, demonstrating the promising clinical applicability of this method.