Figure 4. HOXB9 silencing inhibits brain metastasis of NSCLC and prolongs brain metastasis-free survival in mice. (A) In vivo selection scheme for the isolation of brain metastatic populations (BrM3 cells) derived from H1915 and A549 lung adenocarcinoma cell lines. (B) Representative bioluminescence images (whole body and brain) 60 days after intracardiac inoculation of luciferase-expressing A549 and H1915 cells. (C) Brain metastasis-free survival curves for mice inoculated with H1915 and A549 cells with characteristic high and low HOXB9 expression, respectively (p = 0.0365). (D) Comparison of relative HOXB9 mRNA expression between metastatic NSCLC cell populations (BrM3) and their parental cells. HOXB9 overexpression was confirmed in BrM3 cells (p < 0.001). (E) Western blotting analysis and gray level analysis of HOXB9 expression in BrM3 and parental cells. (F) IHC score-based quantification of HOXB9 expression in primary tumors and matched brain metastasis specimens from NSCLC patients (n = 13; p = 0.0342). (G) Representative images of HOXB9 expression from IHC analysis of 13 primary human NSCLC tumors and their corresponding brain metastases. Scale bars = 50 μm. (H) HOXB9 expression-based analysis of brain metastasis-free survival in 13 NSCLC patients that developed brain metastases. Patients with low HOXB9 expression showed longer brain metastasis-free survival (p = 0.0417). (*p < 0.05, **p < 0.01, ***p < 0.001).