| Background: Oral squamous cell carcinoma is a common malignant tumor in the head and neck region.In the process of metastasis,tumor cells adhesion to endothelial cells played a key role.Glycocalyx is an important structure on the surface of the endothelial cells.In physical condition,it can inhibit metastasis by preventing the adhesion between tumor cells and endothelial cells.Previous studies reported that glycocalyx could be generated by flow shearing stress.Objectives: The purpose of this study is to construct an endothelial glycocalyx model based on microfluidic technique in vitro.Furthermore,a tumor-induced pathological glycocalyx model will be reproduced on the microfluidic device.Based on the pathological glycocalyx model,we aimed to investigate whether melatonin could suppress tumor-induced glycocalyx damage,thereby preventing tumor cell adhesion to endothelium.Materials and methods: This microfluidic chip was composed of glass and PDMS.PDMS membrane preparation uses photoresist technology processing.Human umbilical vein endothelial cells?HUVEC?were seeded on the bottom of the microchannel to mimic the endothelial barrier of the blood vessel.A syringe pump was used to drive cell culture medium flowing in the microchannels to generate shear stress on endothelial cells.Endothelial glycocalyx was detected by fluorescent labeling of wheat germ agglutinin?WGA-FITC?staining.An oral squamous cell carcinoma cell line,UM-SCC6,was used in this study.Conditioned cell culture medium?CM?was collected after culturing UM-SCC6 for 72 hours.CM without or with melatonin was pumped into the glycocalyx model to assess their effects on endothelial glycocalyx.Results: The microfludic device developed in this study included four parallel microchannels to mimic blood vessels.Different perfusion flow rates of 1 ?L/min,0.75?L/min,0.5?L/min,and 0.25?L/min were applied on the microfluidic device.The averaged bottom wall shear stresses in cell culture channels were estimated to be 0.165 Pa,0.124 Pa,0.082 Pa,and 0.041 Pa,respectively.After 3 days' stimulation with different shear stresses,HUVEC cells were stained with FITC-WGA to quantify changes of the glycocalyx.It was found that glycocalyx significantly increased in both 0.124 Pa and 0.165 Pa groups,compared to both 0.041 Pa and 0.082 Pa groups.Then we assessed the correlation of glycocalyx intensity with the time duration of shear stress stimulation.Quantitative analysis demonstrated that glycocalyx intensity significantly increased from day 3,compared to that at day 0 and day 1 under the shear stress of 0.124 Pa.The spatial distribution of the glycocalyx was analyzed in the reconstructed images recorded by a confocal microscope.It was found that the glycocalyx was weakly stained on the endothelial membrane in the static culture condition,while the intensity of glycocalyx on the HUVEC membrane increased stimulated by shear stress.In addition,HUVEC cells appeared to be flat in static culture condition and the majority of glycocalyx distributed near the edge of HUVEC cell.By contrast,HUVEC cell increased its height in z direction and WGA stained glycocalyx distributed at the apex of the cell membrane above the nucleus after shear stress stimulation.To assess whether cancer cell products could damage glycocalyx,UM-SCC6 CM was introduced into the glycocalyx model established in this study and glycocalyx changes were evaluated after 6,12 and 24 hours,respectively.HUVEC cells with normal culture medium on the glycocalyx model were used as control.As shown in Figure a-h,reduction in glycocalyx staining was observed after 12 and 24 h with CM treatment,compared to the control.Quantitative analysis demonstrated that glycocalyx intensity decreased significantly from 12 h by CM treatment.Melatonin with 1×10-6 and 1×10-3 mol/m L concentrations were tried to antagonize damage of glycocalyx caused by UM-SCC6 CM.We found that melatonin with 1×10-3 mol/m L concentration inhibited tumor CM-induced glycocalyx decrement significantly.Furthermore,we verified the effect of the glycocalyx on UM-SCC6 cell adhesion to endothelium on the physiological and pathological glycocalyx models.It was found that the number of adhesive tumor cells significantly increased on the pathological glycocalyx model with tumor CM pre-treatment than that on the physiological glycocalyx model.Then we assessed the effect of melatonin on tumor cell adhesion to endothelium.It was found that melatonin inhibited UM-SCC6 adhesion to endothelium significantly.Conclusions: A microfluidic-based glycocalyx model was developed in this study.We demonstrated that melatonin suppresses metastasis by maintaining the integrity of glycocalyx.This in vitro glycocalyx model offers a novel platform to study the glycocalyx rapidly and inexpensively screen drugs for their potential to inhibit metastasis. |
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