A Dual-Filtration System for Single-Cell Sequencing of Circulating Tumor Cells and Clusters in HCC

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Identification and sequencing of circulating tumor (CT) cells and clusters may allow for noninvasive molecular characterization of HCC, which is an unmet need, as many patients with HCC do not undergo biopsy. We evaluated CT cells and clusters, collected using a dual-filtration system in patients with HCC. We collected and filtered whole blood from patients with HCC and selected individual CT cells and clusters with a micropipette. Reverse transcription, polymerase chain reaction, and library preparation were performed using a SmartSeq2 protocol, followed by single-cell RNA sequencing (scRNAseq) on an Illumina MiSeq V3 platform. Of the 8 patients recruited, 6 had identifiable CT cells or clusters. Median age was 64 years old; 7 of 8 were male; and 7 of 8 had and Barcelona Clinic Liver Cancer stage C. We performed scRNAseq of 38 CT cells and 33 clusters from these patients. These CT cells and clusters formed two distinct groups. Group 1 had significantly higher expression than group 2 of markers associated with epithelial phenotypes (CDH1 [Cadherin 1], EPCAM [epithelial cell adhesion molecule], ASGR2 [asialoglycoprotein receptor 2], and KRT8 [Keratin 8]), epithelial-mesenchymal transition (VIM [Vimentin]), and stemness (PROM1 [CD133], POU5F1 [POU domain, class 5, transcription factor 1], NOTCH1, STAT3 [signal transducer and activator of transcription 3]) (P < 0.05 for all). Patients with identifiable group 1 cells or clusters had poorer prognosis than those without them (median overall survival 39 vs. 384 days; P = 0.048 by log-rank test). Conclusion: A simple dual-filtration system allows for isolation and sequencing of CT cells and clusters in HCC and may identify cells expressing candidate genes known to be involved in cancer biology. Presence of CT cells/clusters expressing candidate genes is associated with poorer prognosis in advanced-stage HCC.