Abstract: We employ a microfluidic chip with three culture chambers to investigate the interactions among lung cancer cells, macrophages and myofibroblasts. By mixing the conditioned media of macrophages and myofibroblasts in this chip, we confirm that these two stromal cells have synergistic effects in accelerating the migration of cancer cells. However, as the myofibroblasts are pretreated with the conditioned medium of macrophages, the myofibroblasts' ability to enhance the migration of cancer cells is lowered. The tumour necrosis factor-α produced by macrophages reduces the expression of α-smooth muscle actin and the secretion of transforming growth factor-β1 in myofibroblasts. Once the tumour necrosis factor-α in the macrophage conditioned medium is neutralized, the macrophage medium-pretreated myofibroblasts can still accelerate the migration of cancer cells.