Background

Cell migration is an individual or collective cell movement that plays an important role in embryonic development (Weijer, 2009), immune response (Luster et al., 2005), and cancer metastasis (Friedl and Glimour, 2009). To investigate these essential physiological phenomena in vitro, wound healing assay or random migration assay are often applied (Jin et al., 2016).

Compared with traditional analyses of wound healing assay focusing on general migration speed, our script mainly focuses on precisely tracking each cell in each timeframe image, leading to speed, directionality, and coordination being analyzed simultaneously. Moreover, we can conquer the difficulties in analyzing the wound healing assay even when the edge of the wound is not smooth.

Since wound healing assay provides cell information like migration speed and directionality, and random migration provides cell coordination information, we applied both assays to elucidate the mechanisms of cell migration thoroughly. We found several pairs of genes and pathways that affect migration speed and cell coordination through these assays after performing two-hit inhibition: the knockdown of a migration-related gene using shRNA combined with the addition of an inhibitor of migration-related pathways at the same time. One of our best candidates, STK40 and MAPK, showed a synergistic effect on migration speed. By applying shSTK40 and MAPK inhibitor PD98059, we found that the absence of either of them has a mild effect on cell migration speed, while the speed significantly dropped when both of them were inhibited at the same time.

Our method is a half-automatic way to trace single cells in the time-lapse immunofluorescence images, which provides multiple parameters for a comprehensive analysis of cell migration, including speed, density, directionality, and cell–cell coordination. In sum, our method not only solves the aforementioned concern but provides a fast, cheap, and comprehensive analysis of cell migration.