Abstract
Metastasis accounts for over 90% of colorectal cancer (CRC)-related deaths. Circulating tumour cells (CTCs), seed cells of metastasis, can survive in the bloodstream, adapt to new environments, and form secondary tumours. This plasticity of CTCs generates heterogeneous CTC subpopulations, including CTCs undergoing epithelial-to-mesenchymal transition (EMT-CTCs), which often go undetected by conventional detection methods relying on epithelial markers such as EpCAM (downregulated during EMT). Marker-independent approaches such as single-cell RNA sequencing (scRNA-seq) are needed to capture this diversity. Capturing this diversity may reveal metastatic drivers and improve early detection. However, limited studies have explored this aspect, and this study aimed to characterise CTC diversity across CRC stages to understand their role in disease progression.
We applied scRNA-seq to peripheral blood cells from four people with CRC of different stages, generating transcriptomic profiles for 3,000–9,000 cells per patient. Data were processed using CellRanger and normalised using the SEURAT package. Healthy individuals (public dataset) were included as controls and processed identically. The normalised data were grouped based on patient clinical stage into early-stage (Stage I and II), late-stage (Stage III and IV), and healthy controls. Clustering and differential gene expression analyses were followed by pathway enrichment analysis (PEA) using the CTpathway method to identify pathways that could be linked to CRC-associated CTCs and provide insight into their functional relevance during disease progression.
PEA revealed significant enrichment of Platelet Derived Growth Factor Receptor beta (PDGFR-β) signalling (Log10FDR = 6.03) and Fibroblast Growth Factor (FGF) signalling (Log10FDR = 5.44) in early- and late-stage groups respectively. These pro-survival and migratory pathways were absent in controls, suggesting EMT-CTC involvement.
Our findings show the utility of scRNA-seq for detecting transcriptomically distinct CTC populations in CRC. By revealing EMT-related pathways across stages, this study highlights the value of single-cell approaches in understanding CTC biology and metastasis.