Single-cell RNA-sequencing of circulating tumour cells: A practical guide to workflow and translational applications.

The global burden of cancer is rising, with treatment failures often due to the metastatic nature of late-stage malignancies. Circulating tumour cells (CTCs) are metastatic precursors shed from primary tumours, which survive in circulation, extravasate and colonise distant organs. The advent of high-throughput single-cell RNA sequencing (scRNA-seq) has revolutionised the investigation of transcriptomic landscape at single-cell resolution, enabling deep transcriptomic profiling, re-stratifying CTC subtypes and improving the detection of rare new subpopulations. Applications extend to understanding tumour microenvironments, characterising cellular heterogeneity, uncovering metastasis molecular mechanisms and improving prognosis and diagnostic strategies. A timeline of key milestones in CTC scRNA-seq research is also provided. Nevertheless, a knowledge gap remains due to unstandardised protocols and fragmented resources in CTC scRNA-seq research. We address this gap by proposing a 12-step CTC-specific scRNA-seq workflow to overcome methodological inconsistencies. This workflow spans the entire process from enrichment, single-cell sorting and sequencing to data pre-processing and downstream analyses, with a detailed compilation of data analysis tools. An in-depth discussion of the pros and cons of commonly used scRNA-seq tools is also included, specifically evaluating their suitability for CTC research. Additionally, emerging research frontiers, including the discovery of hybrid cells-fusion products of tumour and normal cells-and the integration of machine learning (ML) into scRNA-seq workflows, are explored. Future research should prioritise CTC scRNA-seq workflow standardisation, integrate ML-driven analysis and investigate rare and hybrid populations to advance metastasis research. This review supports these goals by guiding methods, informing tool selection and promoting data sharing for reproducibility.