The Mechanism Of Cigarette Smoke Induced Malignant Transformation Of Bronchial Epithelial Cells In A Microfluidic Lung-on-Chip Model

BackgroundChronic obstructive pulmonary disease(COPD)is a severe public health problem commonly associated with prolonged exposure to toxic aeroso.Cigarette smoke(CS)is the principle factor that drives pulmonary disease development and progression.Smoking and aberrant epithelial responses are risk factors for COPD as well as lung cancer.In the last few decades enormous efforts in drug discovery such as the development of novel immunotherapies and molecule-targeting agents had improved prognosis in patients diagnosed with lung cancer,but it remains one of the major causes of death worldwide.The underlying molecular mechanisms that prolonged cigarette smoking induces malignant transformation of bronchial epithelial cells have been widely discussed.It is accepted that airway epithelial barrier dysfunction was the change that came along with inflammation,while micro-vascular endothelium act as an important mediator in this procession and tumor tissue is involed in hyper-angiogenesis.These findings suggested that vascular endothelium is responsible for the pathological process of COPD and lung cancer.Microfluidic 3D culture systems had became a practical tool for in vitro modeling of complex human physiology carcinogenesis in a structurally appropriate context recently.The useful organ surrogate device is also known as organ-on-chip.Lung-on-chip model reconstructed the active air-liguid interface of human lung and studied the inflammation reaction caused by cytokines and toxic nanoparticles.These outcomes provided us a feasible platform to study the continual exposure of cigarette smoke extraction(CSE)in a more comprehensive context.In this article,we fabricated a microfluidic model which contains vascular endothelium and bronchial epithelium to mimic the microenvironment of air-blood interface with a porous membrane for breathing alveolus,and future explored the possible mechanism link between CSE-associated COPD and lung cancer.Methods:1.Design and processing of bionic lung-on-chip(1)Polydimethylsiloxane(PDMS)with favourable biocompatibility and light transmittance was used as the chip substrate.SU-8 lithography was used for chip processing.(2)The lung-on-chip is designed with a multi-layer structure.The size of internal structure simulates the average alveolar volume.The stretchment caused by respiratory movement is simulated by regular suction of the pneumatic chamber.(3)Rat tail collagen type I was used to for the air-liquid dynamic co-culture of the cells,and the viability of the cells was deteced by staining and tracing of viable cells.2.Cigarette smoke exposure experiment was performed based on the lung-on-chip model(1)CCK8 assay was used to detect the sensitivity of epithelial cell BEAS2 B and human umbilical vein endothelial cell HUVEC to CSE.(2)The level of inflammation induced by CSE was detected by ELISA.(3)The m RNA levels of apical junction complexes in epithelial cells and vascular endothelial cells after continuous CSE exposure were detected by RT-PCR.(4)Western blot was used to detect the changes of STAT3 and related protein expression levels in the two types of cells.(5)CCK8 assay was used to detect the sensitivity of BEAS2 B and HUVEC to small-molecule inhibitor HJC0152.(6)Flow cytometry was used to detect the effect of HJC0152 on the epithelial cell cycle induced by continuous CSE exposure and HJC0152 treatment.(7)The effect of HJC0152 on the transcriptional activation of STAT3 in epithelial and endothelial cells induced by CSE was detected by dual-luciferase reporter gene assay.(8)The expression level of related proteins in STAT3 pathway were detected by western blot.Results:1.The lung-on-chip model was able to achieve the status of air-liquid dynamic co-culture,and the cells grew well in the model.2.CSE induced inflammatory responses in the dynamic co-culture system in lung-on-chip model.The level of inflammatory cytokine IL-6 was significantly higher than that in static culture.The IL-6 /STAT3 signaling pathway plays an important role in the pulmonary inflammation induced by cigarette smoke.3.The air-blood barrier was damaged by continuous CSE exposure.This effect is achieved by degradation of several apical-junction complexes.Both epithelial cells and vascular endothelial cells may be involved in this process.Cigarette smoke exposure caused epithelial mesenchymal transformation.This process revolved around E-cadherin and ?-catenin.4.Cigarette smoke exposure increased the expression of c-myc,cyclin D1 and p-erk in lung epithelial cells.The increased level of phosphorylated STAT3 is correlated with the vigorous proliferation of cells.Conclusion:1.The lung-on-chip model could simulate the microenvironment of breathing lung.It confirmed that the co-action of epithelial cells and vascular endothelial cells plays important role in the pulmonary inflammation caused by cigarette smoke.2.In the process of continuous cigarette smoke exposure,the destruction of the air-blood barrier and the occurrence of epithelial-mesenchymal transformation were observed.These results further validated the possible role of STAT3 in the intersection of cigarette smoke induced lung inflammation and malignant transformation.3.It provides a new research site for the prevention and treatment of malignant transformation of COPD,as well as a basis for expanding the application scope of STAT3 pathway blocker HJC0152.In summary,our study proved the correlation between microfluidic chips in simulating the complex pulmonary microenvironment and the prolonged exposure of cigarette smoke.STAT3 may be one of the regulatory factors of the transformation from inflammation to tumorigenesis.The mechanism to be further explored in detail.STAT3 pathway blocker HJC0152 may also play a preventive and therapeutic role in cigarette smoke-related lung diseases and specific mechanism needs to be further studied.