RESUMEN
The clinical diagnosis of a malignant pleural effusion (MPE) is still based on the detection of tumor cells in the pleural effusion. The question of how to improve the efficiency and accuracy of detecting an MPE still remains. This study explores the use of microfluidic technology to concentrate cells in an MPE and achieved the detection of the cell marker TPN in the microarray capture area. TPN is a mitochondria-specific bio-probe that can identify tumor cells on the basis of differences in the mitochondrial potential. First, we designed a microfluidic chip to analyze its performance. The results show that when the total flow rate of the injected chip was 12 mL/h and the volume ratio of cell separation liquid to cell suspension was 1:1, the target cells (A549, MCF-7, and Hela) were enriched and the purity was improved to 98.7-99.3%. Finally, an MPE from cancer patients was used to detect the chip's ability to isolate and enrich tumor cells. Furthermore, the fluorescent identification of the TPN within the tumor cells was simultaneously achieved on the microfluidic chip. In conclusion, the potential to improve the efficiency of the clinical diagnosis of MPEs is provided by the chip structure and analysis conditions explored in this study.
RESUMEN
OBJECTIVE: Ferroptosis is a reactive oxygen species (ROS)- and iron-dependent form of non-apoptotic cell death process. Previous studies have demonstrated that ferroptosis participates in the development of inflammatory arthritis. However, the role of ferroptosis in rheumatoid arthritis (RA) inflammatory hypoxic joints remains unclear. This study sought to explore the underlying mechanism of ferroptosis on lipopolysaccharide (LPS)-induced RA fibroblast-like synoviocytes (FLSs). METHODS: FLSs, isolated from patients with RA, were treated with LPS and ferroptosis inducer (erastin and RSL-3), and ferroptosis inhibitor (Fer-1 and DFO), respectively. The cell viability was measured by CCK-8. The cell death was detected by flow cytometer. The proteins level were tested by Western blot. The cytosolic ROS and lipid peroxidation were determined using DCFH-DA and C11-BODIPY581/591 fluorescence probes, respectively. The small interfering RNA (siRNA) was used to knock down related proteins. The levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), iron, inflammatory cytokines (IL6 and IL8), and LDH were analyzed by commercial kits. RESULTS: Ferroptosis was activated by LPS in RA FLS with increased cellular damage, ROS and lipid peroxidation, intracellular Fe and IL8, which can be further amplified by ferroptosis inducer (erastin and RSL-3) and inhibited by ferroptosis inhibitor (Fer-1 and DFO). Mechanistically, LPS triggered ferroptosis via NCOA4-mediated ferritinophagy in RA FLSs, and knockdown of NCOA4 strikingly prevent the process of ferroptosis. Intriguingly, LPS-induced RA FLSs became insensitive to ferroptosis and NCOA4-mediated ferritinophagy under hypoxia compared with normoxia. Knockdown of HIF-1α reverted ferroptosis and ferritinophagy evoking by LPS-induced RA FLSs inflammation under hypoxia. In addition, low dose of auranofin (AUR) induced re-sensitization of ferroptosis and ferritinophagy through inhibiting the expression of HIF-1α under hypoxia. CONCLUSIONS: NCOA4-mediated ferritinophagy was a key driver of ferroptosis in inflammatory RA FLSs. The suppression of NCOA4-mediated ferritinophagy protected RA FLSs from ferroptosis in LPS-induced inflammation under hypoxia. Targeting HIF-1α/NCOA4 and ferroptosis could be an effective and valuable therapeutic strategy for synovium hyperplasia in the patients with RA.
Asunto(s)
Artritis Reumatoide , Ferroptosis , Sinoviocitos , Humanos , Lipopolisacáridos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Interleucina-8/metabolismo , Artritis Reumatoide/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Hipoxia/metabolismo , Factores de Transcripción/metabolismo , ARN Interferente Pequeño/genética , Fibroblastos/metabolismo , Hierro/metabolismo , Coactivadores de Receptor Nuclear/genética , Coactivadores de Receptor Nuclear/metabolismoRESUMEN
Malignant pleural effusion is a common clinical problem, which often occurs in cases of malignant tumors, especially in lung cancer. In this paper, a pleural effusion detection system based on a microfluidic chip, combined with specific tumor biomarker, hexaminolevulinate (HAL), used to concentrate and identify tumor cells in pleural effusion was reported. The lung adenocarcinoma cell line A549 and mesothelial cell line Met-5A were cultured as the tumor cells and non-tumor cells, respectively. The optimum enrichment effect was achieved in the microfluidic chip when the flow rates of cell suspension and phosphate-buffered saline achieved 2 mL/h and 4 mL/h, respectively. At the optimal flow rate, the proportion of A549 increased from 28.04% to 70.01% due to the concentration effect of the chip, indicating that tumor cells could be enriched by a factor of 2.5 times. In addition, HAL staining results revealed that HAL can be used to identify tumor cells and non-tumor cells in chip and clinical samples. Additionally, the tumor cells obtained from the patients diagnosed with lung cancer were confirmed to be captured in the microfluidic chip, proving the validity of the microfluidic detection system. This study preliminarily demonstrates the microfluidic system is a promising method with which to assist clinical detection in pleural effusion.