Fig. 4. Twist1–thymidine phosphorylase (TP) transcriptional pattern promotes hepatocellular carcinoma cell migration, invasion, and vasculogenic mimicry (VM) formation through enzymatic metabolism of extracellular dT.

PLC-PRF-5 cells were upregulated with Twist1 and/or TP or upregulated with Twist1 and knocked down of TP. Hep3B cells were knocked down of Twist1 and/or TP or knocked down of Twist1 and upregulated with TP. Cells were cultured in glucose-free medium added with the TP substrate of dT. a Wound healing assay showed a significant difference in the speed of cell migration among different treated groups. b Invasion assay showed a significant difference in the speed of cell invasion among different treated groups. c In vitro assay for VM in three-dimensional culture at 24 h. Upregulation of Twist1 and/or TP promoted the tube formation in PLC-PRF-5 cells, and knocking down of TP attenuated the promotion effect of Twist1. Knocking down of Twist1 and/or TP inhibited the tube formation in Hep3B cells, and upregulating TP significantly reduced the inhibitory effect of knocking down of Twist1. d Morphological observation of different treated PLC-PRF-5 cells in pseudopod and cell rounding. e TP relied on the extracellular dT to promote the tube formation of glucose-free cultured PLC-PRF-5 cells. f Transcriptional regulatory activities of Twist1 and TP on VE–Cad, vascular endothelial growth factor receptor 1 (VEGFR1), and VEGFR2. g Protein expression analysis of VE–Cad, VEGFR1, and VEGFR2 with the HCS systems. NG means “No Glucose” (mean ± SD; n = 3 in triplicate; *P < 0.05; **P < 0.01)