Figure 6.

Targeting LCN-2 achieved robust anti-tumor effects. (A) The LCN-2 depleted HCCLM3 cells were cultured with LX2 in the chip for 5 days and the culture medium was gathered from side channels. Then the conditional medium was applied to wide-type HCCLM3 cells combined with sorafenib treatment for 48 h. Flow cytometry analysis was conducted to assess the apoptosis of HCCLM3 cells with indicated treatments. (B) Quantification of HCCLM3 apoptosis in indicated groups from A (n = 3 per group). (C) The clone formation of HCCLM3 cells with indicated treatments. (D) Quantification data from C. (E) The HUVECs were suspended in indicated conditional medium and seeded in the Matrigel for 8 h. Representative bright-field images of HUVEC tubes with indicated treatments. Scale bar: 100 μm. (F) Quantification of HUVEC tube formation from E (n = 3 per group). (G) The HCCLM3 cells (labeled with green) and LX2 cells (no label) were mixed with LCN-2 antibody in the Matrigel and cultured in the chip for 3 days. Then sorafenib was induced from side channel for treatment for 2 days. PI staining was applied to label the dead cells. Scale bar: 1 mm. (H) Quantification of the area occupied by the live HCCLM3 cells (green) from G (n = 3 per group). (I) Fluorescence microscopy images of endothelial cell invasion were shown in indicated groups via chip. Scale bar: 1 mm. (J) Quantification of endothelial cell invasion in indicated groups from I (n = 3 per group). (K) The HCCLM3 (labeled with green) and LX2 (no label) cells were mixed with LCN-2 antibody in the Matrigel and cultured in the chip for 3 days. Then NK-92 cells were induced from side channel for treatment for 2 days. PI staining was applied to label the dead cells. Fluorescence microscopy images showed the live and dead HCCLM3 cells in indicated groups. Scale bar: 1 mm. (L) Quantitative analysis of area occupied by live HCCLM3 cells (green) in the chip from K (n = 3 per group). (M) The xenografts were established by subcutaneous injection of HCCLM3 cells alone or the mixture of HCCLM3 and LX2 cells (at a ratio of 1:1) into the nude mice (n = 6 mice per group). When tumors reached 50–150 mm³ after 7 days, the mice were randomly divided to five groups and administrated with different treatments as below: I) HCCLM3; II) HCCLM3+sorafenib; III) HCCLM3+LX2; IV) HCCLM3+LX2+sorafenib; V) HCCLM3+LX2+sorafenib + LCN-2 antibody. Tumor pictures of indicated groups were shown at the end of the experiments. (N) The tumor growth curves of indicated groups were calculated in the BALB/c nude mice xenograft models. Tumor volumes were quantified. (O) The tumor weights of indicated groups were measured at the end of the experiments. Data are presented as mean ± SEM. ∗P < 0.05; ∗∗P < 0.01.