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

Emmanuel Acheampong, Michael Morici, Afaf Abed, Samantha Bowyer, Du-Bois Asante, Weitao Lin, Michael Millward, Elin S. Gray, Aaron B. Beasley

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)1941-1950
Number of pages10
JournalJournal of Cancer Research and Clinical Oncology
Volume149
Issue number5
Early online date28 Jul 2022
DOIs
Publication statusPublished - May 2023

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