Powering single-cell genomics to unravel circulating tumour cell subpopulations in non-small cell lung cancer patients

BACKGROUND: Circulating tumour cells (CTCs) are attractive “liquid biopsy” candidates that could provide insights into the different phenotypes of tumours present within a patient. The epithelial-to-mesenchymal transition (EMT) of CTCs is considered a critical step in tumour metastasis; however, it may confound traditional epithelial feature-based CTC isolation and detection. We applied single-cell copy number alteration (CNA) analysis for the identification of genomic alterations to confirm the neoplastic nature of circulating cells with only mesenchymal phenotypes. METHODS: We isolated CTCs from blood samples collected from 46 NSCLC patients using the Parsortix system. Enriched cells were subjected to immunofluorescent staining for CTC identification using a multi-marker panel comprising both epithelial and mesenchymal markers. A subset of isolated CTCs was subjected to whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) for the analysis of copy number alterations (CNAs). RESULTS: CTCs were detected in 16/46 (34.8%) patients, inclusive of CK(+)/EpCAM(+) CTCs (3/46, 6.5%) and Vim(+) CTCs (13/46, 28.3%). Clusters of Vim(+) cells were detected in 8 samples, which constitutes 50% of the total number of NSCLC patients with CTCs. No patients had detectable hybrid CK(+)/EpCAM(+)/Vim(+) cells. All of the tested CK(+)/EpCAM(+) CTCs and 7/8 Vim(+) CTCs or CTC clusters carried CNAs confirming their neoplastic nature. Notably, the Vim(+) cluster with no CNAs was characterised by spindle morphology and, therefore, defined as normal mesenchymal circulating cells. CONCLUSION: Our results revealed that CK-negative, vimentin-expressing cells represent a large proportion of CTCs detected in NSCLC patients, which are likely missed by standard epithelial-marker-dependent CTC categorisation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00432-022-04202-y.