6) Notably, the MVDs of HCCLM3-derived and HCCLM3-mock–derived x

6). Notably, the MVDs of HCCLM3-derived and HCCLM3-mock–derived xenografts were 135.2 ± 16.4/0.74 mm2 and 139.2 ± 7.9/0.74 mm2, which were larger

than those of the shRNA-CD151-HCCLM3 (45.2 ± 17.0/0.74 mm2), Hep3B (37.2 ± 12.7/0.74 mm2), and shRNA-MMP9-HCCLM3 CHIR-99021 clinical trial groups (44.8 ± 16.9/0.74 mm2; Fig. 4B,C), and they coincided with the levels of expression of CD151 and MMP9 (Supporting Information Fig. 7A). More importantly, the shRNA-CD151-HCCLM3–derived, shRNA-MMP9-HCCLM3–derived, and Hep3B-derived xenografts also contained masses of necrotic tissues (Fig. 4B), and this suggests that CD151 mediates the expression of MMP9 and has a key role in neoangiogenesis. To explore the role of CD151 in the expression and secretion of VEGF, five HCC cell–derived xenografts were immunostained with antibody to VEGF, and no significant difference was noted (Supporting Information Fig. 8). Fluorescence stereomicroscopy showed that there were more lung metastasis lesions in the HCCLM3-mock group

than in the shRNA-CD151-HCCLM3 group or the shRNA-MMP9-HCCLM3 group (Fig. 4D). Serial sections confirmed that the pulmonary metastasis rates and metastatic tumor clusters per mouse were 100% (5/5) and 132.8 ± 4.0 in the HCCLM3 group and 100% (5/5) and 134.0 ± 8.0 in the HCCLM3-mock group but were 20% (1/5) and 33.6 ± 19.6 in the LDE225 solubility dmso shRNA-CD151-HCCLM3 group, 20% (1/5) and 5.6 ± 12.5 in the Hep3B group, and 20% (1/5) and 24.0 ± 22.8 in the shRNA-MMP9-HCCLM3 group (P < 0.05; Fig. 4E,F and Supporting Information Fig. 7B). The numbers of lung metastatic loci in xenografts were also consistent with their expression of CD151, MMP9, and MVD (Fig. 4C-F and Supporting Information Fig. 7A,B), and this demonstrates that CD151-dependent neoangiogenesis is modulated through modification of MMP9 expression and is involved in the metastasis of HCC. We investigated MCE the expression of CD151, MMP9,

and CD34 by immunohistochemical double staining in a tissue microarray composed of primary tumors of 327 HCC patients. Representative cases of immunohistochemical double staining of all three markers are shown in Fig. 5A-D. Correlation analysis showed that HCC with CD151high expression tended to have high MMP9 expression and MVD and vice versa (rCD151 vs. MMP9 = 0.663, P < 0.001, and rCD151 versus MVD = 0.610, P < 0.001; Fig. 5E). An association between the expression of CD151 and MMP9 was further investigated by RT-PCR and immunoblotting in 60 HCC samples. Semiquantitative analysis of gel bands showed that the expression of MMP9 was tightly associated with the expression of CD151 at the mRNA and protein levels (Fig. 5F). To further assay the role of VEGF-A (VEGF) in CD151-dependent angiogenesis, we also investigated the expression of VEGF by immunohistochemical staining in 327 HCC patients. Representative cases of immunohistochemical staining are shown in Supporting Information Fig. 9A.

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