The Effects Of Activin A On The Migration Of Human Breast Cancer Cells And Neutrophils By Microfluidics

Activin A is an important member of the transforming growth factor beta(TGF?)superfamily,which is highly expressed in breast cancer tissue and inflammation-activated neutrophils.However,the effect and mechanism of activin A on the migration of breast cancer cells and neutrophils is not well characterized.Microfluidics refers to the science and technology involved in the use of microtubules to handle or manipulate tiny fluids.Microfluidic devices are often referred to as microfluidic chips and also known as chip labs(Lab on a Chip).Because the use of biomimetic microstructure and hydrogels and other biological materials,microfluidic chip is very suitable for the physiological function research on the tissue and organ level in vitro,which is also known as "organ chip"(Organs-on-Chips).This can make up for the traditional two-dimensional cell culture and animal experiments,can be dynamically manipulated and real-time observation of important physiological and pathological processes.In this study,a three-channel microfluidic chip was used to analyse the effects of activin A on the migration of human breast cancer cell line MDA-MB-231 cells,neutrophils and their migratory interaction.1.Activin A promotes the migration of human breast cancer cell line MDA-MB-231 cellsIn our study,a three-channel microfluidic device was used to detect the effect of activin A on the migration of MDA-MB-231 cells.At the same time,epidermal growth factor(EGF)was taken as positive contrast.We found that activin A also promoted the migration of MDA-MB-231 cells which is similar to the function of EGF.Furthermore,activin A promoted the migration of MDA-MB-231 cells in a manner of concentration-dependent.In addition,we detected the expression of epidermal growth factor receptor(EGFR)and extracellular regulated kinase 1/2(ERK1/2)using the method of RT-PCR and Western blot.RT-PCR result showed that either activin A or EGF alone could promote the level of EGFR m RNA.Western blot results showed that either activin A or EGF alone could also promote the expression of ERK1/2 and up-regulate the level of p-ERK1/2 in MDA-MB-231 cells.In this study,the function of activin A on the concentration of calcium in MDA-MB-231 cells was further detected by the method of flow cytometry.Our results illustrated that the level of calcium concentration in MDA-MB-231 cells was significantly increased under the stimulation of activin A.2.Activin A inhibits f MLP-induced migration of human neutrophilsThe three-channel microfluidic chip was applied again to detect the function of activin A on the human neutrophils migration while we took the f MLP as a positive control.Cell migration was evaluated by using chemotaxic index(CI)and cell migration distance.The result showed that f MLP gradient could induce the migration of human neutrophils,but activin A itself did not change the migration of neutrophils.Interestingly,activin A could also inhibit f MLP-induced neutrophils migration.In addition,we used human umbilical vein endothelial cells(HUVEC)in the microfluidic device to join and arrange in the docking structure and cultured the HUVEC in the culture medium for 2-4h to better simulate the vascular endothelium.The effect of activin A on the migration of neutrophils across the HUVEC layer was then observed.The results showed that activin A could reduce the migration ability of neutrophils across HUVEC layer.At the same time,flow cytometry result illustrated that activin A was able to reduce the intracellular concentration of calcium in neutrophils stimulated by f MLP.3.Activin A promotes neutrophil migration induced by human breast cancer cell line MDA-MB-231 cellsPrevious studies have shown that neutrophils can promote the transfer of cancer cells,especially the initial process of metastasis.And tumor cells can also recruit neutrophils to tumor microenvironment through the secretion of chemokines.Therefore,in this study,the three-channel microfluidic chip was used to further study the effect of activin A on neutrophil migration induced by MDA-MB-231 cells.Thenormal cell culture medium was taken as a negative control.Activin A was used to stimulate MDA-MB-231 cells for 24 h,then the culture supernatant of the cells was collected and added to the microfluidic device to observe the effect of the culture supernatant on the migration speed and chemotaxis of neutrophils.The results showed that the culture supernatant of MDA-MB-231 cells could significantly induce neutrophil chemotaxic migration compared with the culture medium control group.At the same time,activin A-treated MDA-MB-231 cells culture supernatant could promote the chemotactic migration and migration speed of neutrophils compared with culture supernatant without pretreated of activin A.The results of the above studies showed that activin A could promote the migration of human breast cancer cell line MDA-MB-231 cells,which was related to EGFR-ERK signaling pathway,and also related to the regulation of intracellular calcium concentration.In addition,although the activin A did not affect the motility ability of human neutrophils,it could inhibit f MLP induced chemotactic migration and migration ability across HUVEC layer,which was related to the change of intracellular calcium concentration.It was further confirmed that activin A had different biological effects on different tissue derived cells.However,the related signal transduction mechanism remains to be further studied.In addition,we studied new microfluidic device application with cell docking function which could effectively monitor the dynamic migration of cells and vascular wall barrier penetration process and achieves the quantitative research of mobile cells at single cell level.The study about tumor supernatant function on neutrophil chemotaxis provided a new research foundation for further elucidating the interactions between the tumor microenvironment and neutrophil and also provided new ideas and methods for clinical cancer research and intervention.