Follow-up was terminated on June 30, 2012. Time to recurrence (TTR) was defined as the interval between resection and the diagnosis see more of any type of recurrence,15 with intrahepatic recurrence and extrahepatic metastasis defined as the end points.16 We defined recurrence within 1 year after surgery as early recurrence.17 Cells were enriched from blood samples

within 8 hours after collection using the RosetteSep Human CD45 Depletion Cocktail (StemCell Technologies, Vancouver, Canada) as described.18 The CD45-depleted fraction was subjected to messenger RNA (mRNA) isolation using the RNeasy Micro Kit (QIAGEN, Valencia, CA). Subsequently, reverse transcription was performed using the Quantitect Reverse Transcription Kit (Qiagen). Analysis by qRT-PCR was done with the Light Cycler 480 system (Roche Diagnostics, Basel, Switzerland). All procedures were performed according to the manufacturer’s instructions. Gene expression levels were calculated according to the following equation: 2−ΔCT [ΔCT = Ct(target) − Ct(β-actin)]. PCR conditions were as follows: 10 minutes at

Torin 1 research buy 95°C, followed by 45 cycles of 95°C for 10 seconds and 60°C for 60 seconds. Every sample was measured in triplicate. The primers used are listed in Supporting Table 1. EpCAM+ CTC analysis was performed using CellSearch (Veridex, Raritan, NJ) as described,19 without knowledge of patient clinical characteristics. Results of CTC enumeration were expressed as the number of cells per 7.5 mL of blood (CTC7.5). Blood samples were processed using the CellSearch Profile kit (Veridex) to isolate and collect EpCAM+ cells,20 and cells in the isolated fraction were prepared by cytospin (Thermo Fisher, Waltham, MA) and subjected to immunofluorescence analysis as described.20 The antibodies used in the study are listed in Supporting Table 2. All samples check details were analyzed with a Zeiss confocal microscope (Carl Zeiss, Oberkochen, Germany). To ensure that enough EpCAM+ CTCs were harvested

for tumorigenic assay, we collected 30 mL blood from each of the six patients who had advanced HCC with portal vein thrombosis. Mononuclear cells from whole blood were isolated by density gradient centrifugation using Ficoll-Paque PLUS medium (GE Healthcare,Waukesha, WI) within 1 hour after collection. The isolated cells were then subjected to magnetic-activated cell sorting (Milteny Biotec GmbH, Bergisch Gladbach, Germany), to purify EpCAM+/CD45− CTCs by CD45 depletion and EpCAM selection. All procedures were performed according to the manufacturer’s instructions. Four-week-old nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were purchased from the Shanghai Laboratory Animal Commission of the Chinese Academy of Science, Shanghai, China. Cells to be tested were suspended in 100 μL of Dulbecco’s modified Eagle’s medium and Matrigel (1:1).