Table 1 Comparative summary of migration assay performance in this study. Strengths, limitations, and recommended contexts of use are summarized for each assay type based on our experimental results with five melanoma cell lines under nintedanib treatment.
Assay | Strengths | Limitations | Context of use |
|---|---|---|---|
Scratch wound | Simple, rapid setup; consistently showed faster apparent closure than Z-E in both control and nintedanib-treated conditions; inhibition effects appeared relatively consistent across cell lines | High variability in initial gap size and uneven edges; paracrine stimulation from damaged cells may confound drug-specific inhibition | Suitable for preliminary screening and when throughput is important, but interpretation should account for potential overstimulation artifacts |
Zone-exclusion (Z-E) | Reproducible and uniform gaps; minimized artifacts from cell damage; in some lines (e.g. Mel Pt-4 post) provided the clearest detection of inhibition | Slower closure dynamics; requires longer incubation; inhibitory effects varied by cell line rather than being uniformly strong | More controlled alternative to scratch; useful when minimizing variability and cell damage is critical |
Single-cell tracking | Richest dataset (velocity, MSD, directionality); inhibition by nintedanib was robust across parameters; machine learning achieved 100% accuracy; uniquely allowed quantification of individual cell size and morphology | Labor- and data-intensive; scalability limited; spontaneous migration differs conceptually from collective closure or chemotactic responses | Best suited for precise dissection of drug effects at the single-cell level; the only format that isolates pure migratory behavior, independent of proliferation (gap closure) or deformability (transwell); particularly valuable when cell-intrinsic motility and morphology are central questions |
Transwell | Captured chemotaxis and pore traversal (quasi-3D constraint); revealed strongest inhibitory effects overall, except paradoxical increase in Mel Pt-4 post due to reduced cell size (Fig. S6) | Highly sensitive to morphology and cell size; pore passage may mask true inhibitory effects; results less reproducible when image numbers are low | Valuable for invasion- or chemotaxis-focused studies; important to interpret results together with morphological readouts |
