Precision single-cell profiling of Circulating Tumour Cells: novel markers and data-driven characterization by CTCeek

Circulating tumour cells (CTCs) represent a minimally invasive method for monitoring cancer evolution in patients. CTCs are nowadays commonly isolated using antibodies against EPCAM protein. A key limitation regards the extent of EPCAM-negative CTCs, such as those that undergo EMT or whose tumour of origin is EPCAM-low or negative. We studied 3,302 RNA single-cell transcriptomes reported as CTCs in public repositories. Using copy number variation and cell type-specific markers, we discriminated bona fide CTCs from contaminating blood cells, often mislabelled as CTCs. The integration of bona fide CTCs and PBMCs, from multiple datasets, allowed us to identify novel markers, such as CLDN4, CLDN7, EFNA1 and TACSTD2 for epithelial CTCs, KCNK15 and LY6K for epithelial B CTCs, and ITGB4 for both epithelial B and mesenchymal CTCs. We revealed PODXL, AXL, CAV1, and TGM2 as markers of mesenchymal CTCs, which might be undetectable using anti-EPCAM antibodies, and TM4SF1 as universal marker, expressed in all CTC subclasses. Additionally, we found platelets to be physically associated with the epithelial A, but not with the epithelial B or the mesenchymal subtypes. Finally, we developed and implemented CTCeek, the first web-based and public reference tool that automatically annotates bona fide CTCs from scRNA-sequencing profiles.