br Microfludic chips are usually compared with traditional T
Microfludic chips are usually compared with traditional Tran-swell when it comes to the in vitro research methodology platform. Here we both applied the chip we build and Transwell to investi-gating the effect of AKR1B10 on the trans-BBB capacity of (±)-Baclofen metastasis lung cancer cells. On the one hand, the outcomes from the microfluidic chip followed the same trend as the transwell. On the other hand, whether we knock down the AKR1B10 or MMPs, the inhibitory rates of trans-endothelium ability on chip is more obvious. It seems easier for tumor cells to migrate through the endothelium on Transwell under the same treatment. Following are a few possible reasons contributing to the difference: 1) Studies have shown that the endothelial barrier integrity on microfluidic chip, where endothelial cells were co-cultured with astrocytes under a physiological fluidic shear, was significantly better than Transwell [19,23]; 2) All those cells which crossed the membrane on Transwell were stained with crystal violet and counted, but it was uncertain that the counted cells were all tumor cells because endothelial cells also have the ability to migrate at some degree [52,53]. The number of total counted cells was greater than the actual number of tumor cells due to the penetration of endothelial cells along with tumor cells. It was difficult to differentiate between tumor cells and endothelial cells in uniformly stained cells on Transwell while it was much easy to distinguish cells with-out staining on our chip since the tumor cells stably expressed GFP in green and endothelial cells were labeled in red. To sum up, the chip presented is more convincing than Transwell, indicating that the chip may be an ideal substitute to traditional methodology platform.
AKR1B10 is a member of the AR superfamily and highly expressed in the intestinal system. It has been recognized as a potential serum biomarker for several malignant tumors including lung cancer [42–45,54]. However, little is known about its role in lung cancer-derived BM. To the best of our knowledge, this study demonstrated for the first time that AKR1B10 is up-regulated in lung cancer BM, and validated the value of AKR1B10 as a diagnostic serum biomarker in a cohort including LCBM, PLC, PBT and HG sub-
jects. It was found that the average AKR1B10 level of the PLC group was higher than that of the PBT and HG group, which was consis-tent with previous reports . Further comparison between the LCBM group and PLC group showed that the average AKR1B10 level in the LCBM group was significantly higher than that of the PLC group, indicating the important significance of AKR1B10 in lung cancer that different threshold expression levels can help the diag-nosis of primary lung cancer and secondary brain metastasis and predict the risk of lung cancer brain metastasis. In addition, since AKR1B10 is normally expressed at very low levels in the lung and brain , it can also serve as therapy target for lung cancer BM.
To clarify the role of AKR1B10 in brain metastasis, we proposed a working model for the action mechanism of AKR1B10 in BM that it promotes the extravasation of cancer cells through the BBB, which possibly involves the up-regulation of MMP-2 and MMP-9 expression through a MEK/ERK signaling pathway (Fig. 7B). Here MMPs were proved as important downstream mediators of AKR1B10 in promoting lung cancer brain metastasis. MMPs also play an important role in BBB leakage by degrading TJ proteins , which could lead to an accelerated brain metastasis cascade. Therefore, MMPs could be therapeutic targets, and current new technologies can facilitate the development of specific selective MMPs inhibitors that can be used to prevent and mitigate brain metastasis. Furthermore, microfluidic chip technologies, which allow real-time visualization of the metastasis cascade, offer new insights into how the targeted modulation of MMP activity can be achieved in tumor cells and/or tumor microenvironment.
Moreover, it is worth mentioning that our data showed incon-sistent changes in AKR1B10 expression at the mRNA and protein levels, which indicate there might be an epigenetic modification of AKR1B10, since metastatic microenvironments largely con-tribute to molecular epigenetic changes in tumor cells. Recent studies suggest an undervalued role of post-transcriptional, trans-lational, and degradation regulations in the determination of pro-tein concentrations, and these regulations contribute at least as much as transcription itself [57–60]. In the last decade, it has been reported that S-nitrosylation was a post-translational mechanism that regulated the AR superfamily [61,62]. S-nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), provides a fundamental redox-based cellu-lar signaling mechanism and regulates protein activity, stability, localization, and protein–protein interactions in a wide range of biological processes [63,64]. AR was thiol-modified by NO at the active-site residual Cys-298, which is considered the main site of modification, and this resulted in the changes of AR characteristics like protein stability and catalytic activity . In view of the knowledge that metastatic lung cancer cells face a cascade of oxidative stress during BM and NO is induced by oxidative stress as the ubiquitous second messenger, we hypothesize that AKRIB10 undergoes post-translational S-nitrosylation during BM, which causes an increase in protein stability and a subsequent increase in protein concentrations in metastasized tumors. However, fur-ther well-designed and in-depth studies are warranted.