Figure 6

Activated HSCs possess a migration capacity as a result of increased MMP9 in HCC-MCTS.

(A) HepG2 alone and co-culture spheroids with LX2 cells, which is stained by DiO, WI38 cells stained by DiI, and HUVECs stained by DiD were cultured for 3 days. After spheroid formation, each spheroid was transferred to a collagen I coated plate and cultivated for 4 days. The cells were stained with Hoechst33342 after fixation and the images were obtained (left). The distance from surface of spheroid to boundary of stretched cells was measured (right). (B,C) HepG2 and PLC/PRF/5 spheroids were cultured alone or with LX2, WI38, and HUVEC stromal cells at a 1:1 ratio for 3 days. After 3 days, mRNA expression in each spheroid was evaluated by (B) RT-PCR and (C) real-time PCR using MMP and cadherin primers. Values were normalized to GAPDH. (D) LX2 cells were transfected with siCont and siMMP9 and its efficacy was examined by real-time PCR. (E) HepG2 alone and co-culture spheroids with LX2-siCont or LX2-siMMP9, which were stained by DiO, were cultured for 3 days. After 3 days, each spheroid was transferred to a collagen I coated plate and cultivated for 4 days. The cells were fixed and stained with Hoechst33342. The images were obtained using the same method (left). The distance from surface of spheroid to boundary of stretched cells was measured (right). Data represent the mean values ± SD from three independent experiments. **P < 0.005. Scale bar = 200 μm.