Single circulating tumor cell sequencing based on improved high-porosity membranes and nanoporous microchambers.

Circulating tumor cells (CTCs) are crucial for understanding tumor heterogeneity and progression. Despite extensive research over the years, most studies have focused on CTCs counting, with fewer efforts directed toward single-cell sequencing (SCS) of CTCs. In this study, we developed two novel nanodevices---a high-porosity ultrathin filter membrane and a nanowell chip--- to isolate single CTCs. Automated scanning and single-cell picking systems were employed to locate and isolate individual CTCs, enabling the establishment of an efficient and automated workflow for single CTC sequencing using filter-based systems. We conducted an in-depth comparison to evaluate the effects of different filter membranes and cell adhesion types on genomic integrity, cell viability, sequencing coverage, and depth. The results showed that the high-porosity filter membrane outperformed other photolithographic filters for SCS of CTCs. Validation using NCI-H358 cell lines and patient-derived CTCs demonstrated that this workflow could accurately and comprehensively detect gene mutations, amplifications, and copy number variations (CNVs). CNV profiles of CTCs from patients with the same tumor type were highly consistent, while intra-patient CTCs revealed significant heterogeneity. Furthermore, we identified and overcame challenges related to cell adhesion to the filter membrane and the impact of cell viability on sequencing outcomes during CTC enrichment. This workflow offers new insights into the development of CTC-based approaches for exploring tumor progression, heterogeneity, and mechanisms of drug resistance.