The monitoring of circulating tumor cells (CTCs) has recently served as a promising approach for assessing prognosis and evaluating cancer treatment. We have already developed a CTCs enrichment platform by EpCAM recognition peptide-functionalized magnetic nanoparticles (EP@MNPs). However, considering heterogeneous CTCs generated through epithelial-mesenchymal transition (EMT), mesenchymal CTCs would be missed with this method. Notably, N-cadherin, overexpressed on mesenchymal CTCs, can facilitate the migration of cancer cells. Hence, we screened a novel peptide targeting N-cadherin, NP, and developed a new CTCs isolation approach via NP@MNPs to complement EpCAM methods’ deficiencies. NP@MNPs had a high capture efficiency (about 85%) of mesenchymal CTCs from spiked human blood. Subsequently, CTCs were captured and sequenced at the single-cell level via NP@MNPs and EP@MNPs, RNA profiles of which showed that epithelial and mesenchymal subgroups could be distinguished. Here, a novel CTCs isolation platform laid the foundation for mesenchymal CTCs isolation and subsequent molecular analysis.

Single‐cell RNA sequencing on circulating tumor cells (CTCs) proves useful to study mechanisms of tumor heterogeneity, metastasis, and drug resistance. Currently, single‐cell RNA sequencing of CTCs usually takes three prerequisite steps: enrichment of CTCs from whole blood, characterization of captured cells by immunostaining and microscopic imaging, and single‐cell isolation through micromanipulation. However, multiple pipetting and transferring steps can easily cause the loss of rare CTCs. To address this issue, a novel integrated microfluidic chip for sequential enrichment, isolation, and characterization of CTCs at single‐cell level, is developed. And, single CTC lysis is achieved on the same chip. The microfluidic chip includes functions of blood clot filtration, single‐cell isolation, identification, and target single‐cell lysate collection. By spiking tumor cells into whole blood, it is validated that this microfluidic chip can effectively conduct single‐cell CTCs RNA sequencing. The approach lays a solid foundation for the analysis of RNA expression profiling of single‐cell CTCs.

To sequence single circulating tumor cells (CTCs) from whole blood, a microfluidic chip was developed to perform blood filtering/CTC enrichment/CTC sorting and in situ MDA for whole genome sequencing.