24 The vessel area was calculated as follows: All experiments were performed in Napabucasin nmr triplicate. Samples of 8 × 106 HCCLM3, shRNA-CD151-HCCLM3, shRNA-MMP9-HCCLM3, HCCLM3-mock, and Hep3B cells were used for spontaneous metastasis assays, as described

previously.25 Lung metastases of shRNA-CD151-HCCLM3, shRNA-MMP9-HCCLM3, and HCCLM3-mock were visualized with fluorescence stereomicroscopy (Leica Microsystems Imaging Solutions, Ltd., Cambridge, United Kingdom). Serial sections were made for every tissue block from the lung, and the total number of lung metastases was counted under the microscope as described previously.6 There were five animals in each group. Immunohistochemical analysis of subcutaneous xenografts was performed as described elsewhere.6 Antibodies used in this study are listed in the Supporting Information. The construction of tissue microarrays was described in detail in our earlier study.6 Immunohistochemical Selleck Forskolin double staining was performed as described elsewhere.26 Mouse anti-human CD151 antibodies (1:100; 11G5a, Serotec, NK), rat anti-human MMP9 antibodies (1:50; Cell Signal Tec, United States), mouse anti-human CD34 antibodies (1:100; DakoCytomation, Denmark), and rabbit polyclonal VEGF antibodies

(1:100; Neomarkers, Fremont, CA) were used to detect the expression of CD151, MMP9, MVD, and VEGF. The density of positive staining click here of CD151, MMP9, and VEGF was measured as described previously.6 The MVD was evaluated as described elsewhere.27 Statistical analysis was performed with SPSS12.0 software (SPSS, Chicago, IL). Values are expressed as means and standard deviations. The Student t test and one-way analysis of variance were used for comparisons between groups. Correlation analysis was performed. Overall survival (OS) and time to recurrence were defined as described previously.28 OS and the cumulative recurrence rates were calculated by the Kaplan-Meier method and the log-rank test. Cox’s proportional hazards regression model was used to analyze the independent prognostic factors. P < 0.05 was set

as the level of statistical significance. In an earlier study,6 we demonstrated that the bioactivity of MMP9 in the supernatant from HCCLM3 cells with a high level of expression of CD151 was much stronger than that in shRNA-CD151-HCCLM3 cells and HepG2 cells with low CD151 expression by gelatin zymography. We now explore the relationship between the expression of CD151 and MMP9 in HCC cell lines (HCCLM3, MHCC97-L, PLC/PRF/5, Hep3B, and HepG2) with different metastatic potentials by qRT-PCR and immunoblotting. Highly metastatic HCCLM3 cells with CD151high expression showed the highest expression of MMP9 at both the mRNA and protein levels, whereas low-metastatic HCC cell lines (MHCC97-L, PLC/PRF/5, Hep3B, and HepG2) showed low levels of expression of CD151 and MMP9 (Fig. 1A,B).