br Fig AKR B is upregulated
Fig. 5. AKR1B10 is upregulated in Corn oil metastasis. (A-B) Higher levels of AKR1B10 protein was identified in PC9 cells with higher metastatic activity by (A) western blot, and
(B) ELISA assays. (C) Representative immunofluorescence staining images show the expression of AKR1B10 (red) in upstream primary tumor cells and downstream brain
metastasized cells on the chip. Cell nuclei were stained with DAPI (blue). Scale bar, 10 lm. (D) Representative western blot image shows AKR1B10 expression in clinical surgical specimens. (E-F) Serum AKR1B10 levels in clinical groups, as determined by ELISA. Each dot corresponds to one subject (E), and statistical analyses of AKR1B10 levels between indicated groups are shown (F). Data are presented as mean ± SD; ***p < 0.001; ****p < 0.0001. LCBM, lung cancer brain metastasis; PLC, primary lung cancer; PBT, primary brain tumor; HG: healthy group. The bar graphs are summarized results from 3 independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
AKR1B10 level of the LCBM group was significantly higher than that of the other three groups, confirming the diagnostic value of serum AKR1B10 levels in lung cancer brain metastasis (Fig. 5E, F). There-fore, AKR1B10 is upregulated in lung cancer brain metastases and identified as a diagnostic serum marker for lung cancer BM.
3.6. Knockdown of AKR1B10 in lung cancer suppresses BM in vitro and in vivo
To verify the potential association between up-regulated AKR1B10 protein expression and enhanced extravasation ability of brain metastatic cells, we knocked down AKR1B10 with two dif-ferent small interfering RNAs (siR-1 and siR-2) in PC9-BrM3 cells and then evaluated the trans-endothelium ability of these cells using both the Transwell model and our chip. The results showed that silencing AKR1B10 in highly brain metastatic cells markedly diminished their ability to penetrate the endothelial layer in both the Transwell assay and chip extravasation assay (Fig. 6A), while it had minimal influence on the migration capacity and epithelial
mesenchymal phenotype of PC9-BrM3 cells (Fig. S5A, B). In addi-tion, the decrease in trans-endothelium migratory ability of PC9-BrM3 cells upon knocking down AKR1B10 was more obvious on our bionic chip platform. Coincidently, MMP-2 and MMP-9 expres-sion were down-regulated after AKR1B10 knockdown (Fig. 6B). Further silencing of MMP-2 and MMP-9 also reduced the trans-endothelial migration ability of PC9-BrM3 cells in the Transwell assay or on chip (Fig. S6A, B). These data suggest that AKR1B10 promotes BBB extravasation of brain metastatic cells, possibly through an MMP-involved mechanism. r> To further substantiate the association between AKR1B10 expression and trans-endothelium migratory ability of PC9-BrM3 cells, we performed in vivo animal experiments. We inoculated nude mice with either control shRNA-transfected or AKR1B10 shRNA#1-transfected PC9-BrM3 cells via an intracardiac injection. As expected, PC9-BrM3 cells with targeted silencing of AKR1B10 demonstrated significantly diminished in vivo metastatic ability, as evidenced by the bioluminescence images acquired 30 days after inoculation of cancer cells (Fig. 6C). Correspondingly, the mice
Fig. 6. AKR1B10 knockdown suppresses BM in vitro and in vivo. (A) Representative images showing the trans-endothelia on Transwell (upper; scale bar, 20 lm) and penetration of the BBB on the chip (lower; scale bar, 50 lm) of indicated cells. (B) Representative western blot image showing the silence efficiency of AR1B10 and MMP-9 and MMP-2 expression in indicated cells. (C) Representative bioluminescent images of mice at days 30 post intracardiac injection of indicated cells (1x106 cells mouse). (D) Survival curve of nude mice after inoculation of indicated cells. Ctrl, control PC9-BrM3 cells; NC, PC9-BrM3 cells transfected with negative control siRNA oligo; SiR-1, PC9-BrM3 cells transfected with AR1B10 siRNA-1 oligo; SiR-2, PC9-BrM3 cells transfected with AR1B10 siRNA-2 oligo; shAKR1B10, PC9-BrM3 cells transfected with AR1B10-targeted shRNA vector. **p < 0.01; ***p < 0.001. The bar graphs are summarized results from 3 independent experiments.