The prognosis of hepatocellular carcinoma (HCC) is closely associated with recurrence and metastasis which has been proposed to be initiated by circulating tumor cells (CTCs). Yet, the transcriptomic plasticity and immune evasion mechanism of CTCs during circulation are not well defined.
Blood was drawn from 4 different vascular sites, including hepatic vein (HV), peripheral artery (PA), peripheral vein (PV) and portal vein (PoV) of 10 localized HCC patients. Single CTCs were isolated by negative enrichment and robotic micromanipulator, followed by single-cell RNA sequencing (scRNA-seq). After filtering, 113 CTCs with qualified data were subjected to bioinformatics analysis. The scRNA-seq results were further validated in three independent HCC cohorts.
Our scRNA-seq data revealed remarkable intra- and inter-vascular heterogeneity among CTCs from four vascular sites. We determined CTC transcriptional dynamics during transportation through consecutive vascular sites and revealed their adaptation mechanisms under biomechanical stress. We further classified CTCs from different vascular sites into two subsets, namely dormant and activated CTCs. Dormant CTCs were associated with a non-cycling state and upregulation of EMT/angiogenic signatures and showed stronger prognostic ability for early relapse than activated CTCs. Furthermore, we discovered an immune escape mechanism by which CTCs recruited regulatory T cells (Tregs) via expression of CCL5, consequently promoting the formation of an immunosuppressive microenvironment favorable for their survival in bloodstream and distant colonization.We proved that MAX, activated through the p38 pathway, was the key transcriptional factor regulating CCL5 overexpression, which was validated by ChIP, luciferase reporter gene and in vitro/vivo knockdown assays. And we further determined that Tregs-derived TGF-β1 can heighten MAX expression, thus amplifying the CCL5 expression.
Collectively, our results reveal a previously unappreciated spatial heterogeneity of CTCs and a CTC immune-escape mechanism, which may aid in designing new anti-metastasis therapeutic strategies in HCC.
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Legal entity responsible for the study
The State Key Program of National Natural Science of China.
All authors have declared no conflicts of interest.