Isolation and Sequencing of Single Circulating Tumor Cells.

Single-cell sequencing (SCS) technology has become an important tool for researchers to study human diseases, including cancer, providing insights into phenotypic and genetic tumor heterogeneity, targeted therapy resistance, and metastasis. SCS technology has proven to be a remarkable tool for shedding light on the impressive molecular heterogeneity of circulating tumor cells (CTCs). However, the extreme rarity and biological heterogeneity of CTCs have imposed technical challenges for their analyses at the single-cell level, and affected the success of robust processing of complex and costly downstream methodologies. Here, we present the entire workflow we developed to enrich, detect and isolate single non-small-cell lung cancer (NSCLC) CTCs to characterize tumor cell subpopulation resistant to targeted therapy. Once isolated, individual CTCs underwent a molecular workflow including whole-genome amplification (WGA) and qualification, followed by two SCS techniques, including targeted next-generation sequencing (NGS) for somatic variant identification and the low-pass whole genome sequencing (LP-WGS) for the analysis of copy number alterations (CNA).