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  • br Since extravasation through the BBB is


    Since extravasation through the BBB is an important and unique indicator of BM, we compared the ability of PC9-BrM3 cells and parental cells to penetrate the endothelium using both the classic Transwell two-dimensional culture model and our dynamic bionic chip model. Consistently, both models showed that the highly Calcein-AM metastatic PC9-BrM3 cells were more capable of penetrating the endothelial cells compared to the parental cells (Fig. 4C), and the wound healing assays showed that PC9-BrM3 cells were also more migratory compared to PC9 cells with a more obvious mes-enchymal phenotype (Fig. S3A, B). Many studies have shown that matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, play a critical role in promoting BBB leakage by degrading TJs and basement membrane proteins [40,41]. Therefore, we measured the expression of MMP-2 and MMP-9 in brain metastatic subpop-ulations and parental cells, respectively. We found that MMP-2 and MMP-9 protein expression was significantly up-regulated after increasing rounds of BM (Fig. 4D). Taken together, the highly brain
    Fig. 4. Highly-brain metastatic cells more aggressively extravasate the BBB compared to parental cells. (A)(i) In vivo selection scheme for the isolation of metastatic populations (BrM1, BrM2, and BrM3) from the PC9 lung adenocarcinoma line. (ii) Frequency and time circle of BM after inoculation of cells in nude mice. (B) Survive curve of nude mice after inoculation of indicated cells. (C) Representative images showing the GFP-expressing PC9/PC9-BrM3 cells and red-labeled hBMVEC cells in the trans-endothelia on Transwell (upper; scale bar, 20 lm) and after penetrating the BBB on the chip (lower; scale bar, 50 lm). (D) Representative western blot images showing MMP-9 and MMP-2 expression in brain metastatic cells and parental cells. The bar graphs are summarized results from 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    metastatic lung cancer cells recapitulated the salient features of BM and were more capable of extravasating the BBB compared to the parental cells.
    3.5. AKR1B10 is up-regulated in lung cancer brain metastasis
    AKR1B10 is a multi-functional protein expressed in many tumors. To investigate changes in AKR1B10 expression in brain metastatic NSCLC cell lines, we quantified mRNA and protein levels. We found that there was no statistical difference in AKRB10 mRNA levels (Fig. S4A), but the protein levels determined by west-ern blot assay (cellular AKR1B10) and ELISA (secreted AKR1B10 in supernatant) were significantly up-regulated in brain metastatic subpopulations compared to the parent cells (Fig. 5A, B). Since the chip we constructed can mimic the course of in vivo BM, we compared the expression level of AKR1B10 in lung cancers with and without BM on the chip using immunofluorescent staining. Compared with the upstream primary tumor cells, the down-stream brain metastasized lung cancer cells had up-regulated AKR1B10 expression (Fig. 5C), suggesting a good correlation 
    between the brain metastasis cell from chip and that from in vivo BM models.
    To verify AKR1B10 expression in the metastasized tumors from patients, we collected surgical specimens of lung cancer BM (LCBM, n = 6), primary lung cancer (PLC, n = 6), and primary brain tumor (PBT, n = 6). We found that AKR1B10 was expressed in half of lung cancer brain metastases specimens but not in the specimens of two control groups (PLC and PBT), as evidenced by our western blot analyses (Fig. 5D and Fig. S4B). Since AKR1B10 has been recognized as a potential and valuable serum biomarker [42–45], we further determined the diagnostic value of serum AKRIB10 levels for lung cancer brain metastasis. We collected serum samples from lung cancer patients with brain metastasis (LCBM, n = 57), primary lung cancer patients confirmed without lymph node and distant organ metastasis (PLC, n = 46), patients with primary brain tumor (PBT, n = 8) and subjects in the healthy volunteers group (HG, n = 15). The clinical characteristics of the four groups are shown in Table S1, and there were no significant differences among the char-acteristics (age, gender, histology) between the four groups (P > 0.05) (Table S2). ELISA results showed that the average