Study On Invadopodia Formation For Lung Carcinoma Invasion With A Microfluidic3D Culture Device

Objectives: Lung cancer is the leading cause of cancer death worldwide.Recurrence and metastasis are the most major reasons of death in lung cancer patientsdespite advances in the treatment of primary tumors. The initial stage of cancer cellmigration and invasion is the extension of cell protrusions in the direction of cellmovement. The formation of these cell protrusions is usually driven by actinpolymerization at the leading edge. During invasion and intravasation, the invasivecancer cells penetrate basement membranes using subcellular structures calledinvadopodia that localize matrix degrading activity to cell–substrate contact points.Microfluidic chip (lab-on-a-chip), with its miniaturization, integration, high-throughput,short reaction time, less reagent consumption and other advantages is recognized as oneof the main technology platform of system biology research. The micro channel sizeand multidimensional network formed a relative closed cell space which is similar withcharacteristics, especially suitable for cell culture, and can continue to supply nutrientscells, to simulate the body tumor microenvironment. In this work, we built a newinvasion test of lung cancer cells with the mold of3D culture using an integratedmicrofluidic device. We aimed at analyzing the effection of EGF and GM6001forinvadapodia formation in human non-small lung cancer cell line A549using thismicrochip-based system.Methods: In this study, the microfluidic chip system which includes amicrofluidic chip and four MS26injection pumps was built for invasion analysis oflung cancer cell lines. Cells and BME mixture was infused into the3D cell culturechambers via the inlet with the connection of a pump. When the cells attached andspread well, a MS26injection pump was connected to the chip to supply the cells withfresh medium of F12-K continuously at a constant rate of10mm/24h. One hour later,the medium with EGF(200ng/ml) and GM6001(10μM) were injected into the cell chambers of the experiment group for12h. To elucidate invadapodia formation of lungcancer, we detected the expression of F-actin and cortactin by immunofluorescenceassay. And meanwhile, the morphology of invadapodia was also observed.Results: In this study, we have designed and built a microfluidic chip on theinvadapodia formation of lung cancer cells with the mold of3D culture successfully.This device was capable of achieving the assays on one control group and twoexperimental group cells which pretreated with EGF or GM6001simultaneously and inparallel. In control group, there was only23.4%of A549cells with invadopodiaformation and the cells showed an average of1.8invadopodia foci per cell. In EGFgroup, it was observed that the stimulation with EGF remarkably induced the formationof actin dot-like structures while approximately64%of A549cells showed evidence ofinvadopodia formation with an average of5.8invadopodia foci per cell,3-fold greaterthan that of the control group. In GM6001/EGF group, GM6001neutralized theinductive effect of EGF and led to a decrease in the formation of actin dot-likestructures to the level as low as that without EGF stimulation (the control group) oreven lower. Only17%of A549cells formed actin dot-like structures and the cellsdisplayed an average of1.3invadopodia foci per cell with the level near to that of thecontrol group or even lower. From the morphology, invadopodia could be induced byEGF and this induction could be inhibited by GM6001.Conclusion: This study has successfully fabricated a microfluidic3D cultureinvasion platform, which was available for achieving the assays on one control groupand two experimental group cells which pretreated with EGF or GM6001simultaneously and in parallel。MMPs inhibitor might be a powerful candidate againstinvadopodia target anti-invasion therapy. This microfluidic system would be significantin exploring invadopodia formation and investigating therapeutics in a biologicallyrelevant context. The established culture model should be suitable for analysis of theinvasion mechanism and discovering anti-invasion drugs in a well defined3Denvironment.