Purpose: The spatial heterogeneity of phenotypic and molecular characteristics of CTCs within the 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 postpulmonary 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 b-catenin related 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. Multivascular measurement of CTCs could help to reveal novel mechanisms of metastasis and facilitate prediction of postoperative relapse or metastasis pattern in HCC.

BackgroundSmall hepatocellular carcinoma (SHCC) is a special type of hepatocellular carcinoma with the maximum tumor diameter ≤ 3 cm and excellent long-term outcomes. However, the prognostic factors for SHCC remain controversial. The purpose of this study is to clarify the predictive factors of SHCC.MethodsThe study population consisted of 458 patients underwent hepatectomy for single SHCC between January 2006 and December 2008. Clinical data (included age, gender, virus infection, serum alfa-fetoprotein level, cirrhosis, capsule, border), histopathologic features (included differentiation, morphology subtype, microvascular invasion, tumor infiltrative lymphocytes (TIL), inflammatory injury grade and fibrosis stage of surrounding liver), were evaluated to identify prognostic factors influencing SHCC patients’ survival and microvascular invasion.ResultsThere were 384 males (83.8%) and 74 (16.2%) females with median ages of 52 years. The median progression-free survival (PFS) and overall survival (OS) durations were 53 and 54 months, respectively. About 91.9% (n = 421) SHCC were infected by Hepatitis B. One hundred forty-seven of the 446 (33.0%) patients with pre-operation serum AFP level record had serum alfa-fetoprotein (AFP) level ≥ 200 ug/ml and 178 of the 280 (63.8%) patients with post-operation serum AFP level record had AFP level ≥ 20 ug/ml. Liver cirrhosis was present in 411 cases (89.7%), while 434 (97.3%) tumors had clear border, and 250 (55.6%) tumors were encapsulated.MVI was identified in 83 patients (18.1%). In univariate analysis, a significant association between the presence of MVI and shortened PFS and OS was found (p = 0.012, 0.028, respectively). Histological differentiation had strong relationship with MVI (p = 0.009), in terms of MVI was more easily presented in patients with worse histological differentiation. In patients with MVI, worse survival was correlated with female patients, patients with G2 or G3 histological differentiation, pre-operation serum AFP level ≥ 200 ug/ml or post-operation serum AFP level ≥ 20 ug/ml, and TIL ≥ 50/HPF.ConclusionsMVI is an independent poorer prognostic factor for PFS and OS of single SHCC patients. Tumor histological differentiation was closely related with MVI.