A rhabdomyosarcoma cell migration inhibitor screen using phase-contrast microscopy

Rhabdomyosarcoma (RMS) is a malignant soft tissue tumour occurring in young children. Alterations in fibroblast growth factor receptor 4 (FGFR4) signalling pathways are linked to metastasis and poor prognosis in RMS. A critical trait of metastatic disease is the ability of cancer cells to migrate and invade nearby and distant tissues. To study cell migration, we have developed a computational tool that automatically tracks and measures the velocity of migrating cells in phase contrast images. This tool facilitated an inhibitor screen comprising 462 compounds, analysing nearly 2000 videos and tracking approximately 700 000 RMS-derived cells harbouring a constitutively active FGFR4 (RMS559). The highly selective inhibitor screen targeted various signalling pathways, including receptor tyrosine kinases (RTKs), mitogen-activated protein kinases (MAPK), and phosphoinositide 3-kinases (PI3K). Validation of screen hits revealed that inhibiting proteins in the MAPK and PI3K pathway decreased cell migration and, in some cases, cell viability. Inhibiting FGFR4 resulted in decreased RMS559 cell migration, while, surprisingly, inhibitors targeting Rho-associated protein kinase (ROCK) and focal adhesion kinase (FAK) resulted in increased RMS559 cell migration. Mechanistically, the inhibition of ROCK and FAK resulted in fewer focal adhesions, thereby facilitating increased cell velocity. These findings suggest that targeting specific signalling pathways can effectively modulate migration and survival of metastatic RMS559 cells.