Circulating Tumors Cells from Different Vascular Sites Exhibit Spatial Heterogeneity in Epithelial and Mesenchymal Composition and Distinct Clinical Significance in Hepatocellular Carcinoma

Purpose: The spatial heterogeneity of phenotypic and molecular characteristics of CTCs within circulatory system remains unclear. Herein, we mapped the distribution and characterized biological features of CTCs along the transportation route in hepatocellular carcinoma (HCC).  Experimental Design: In 73 localized HCC patients, blood was drawn from peripheral vein (PV), peripheral artery (PA), hepatic veins (HV), infrahepatic inferior vena cava (IHIVC) and portal vein (PoV) before tumor resection. Epithelial and mesenchymal transition (EMT) phenotype in CTCs were analyzed by a 4-channel immunofluorescence CellSearch assay and microfluidic quantitative RT-PCR. The clinical significance of CTCs from different vascular sites was evaluated. Results: The CTC number and size gradient between tumor efferent vessels and post-pulmonary peripheral vessels was marked. Tracking the fate of CTC clusters revealed that CTCs displayed an aggregated-singular-aggregated manner of spreading. Single-cell characterization demonstrated that EMT status of CTCs was heterogeneous across different vascular compartments. CTCs were predominantly epithelial at release, but switched to EMT-activated phenotype during hematogeneous transit via Smad2 and β-catenin signaling pathways. EMT activation in primary tumor correlated with total CTC number at HV, rather than epithelial or EMT-activated subsets of CTCs. Follow-up analysis suggested that CTC and circulating tumor microemboli burden in hepatic veins and peripheral circulation prognosticated postoperative lung metastasis and intrahepatic recurrence, respectively. Conclusions: The current data suggested that a profound spatial heterogeneity in cellular distribution and biological features existed among CTCs during circulation. Multi-vascular measurement of CTCs could help to reveal novel mechanisms of metastasis and facilitate prediction of postoperative relapse pattern in HCC.

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