P34 Investigating leader cell dynamics in re-epithelialization using a high-throughput scratch assay

Abstract Introduction and aims Epidermal wound re-epithelialization relies on the collective migration of keratinocyte sheets. Leader cells direct keratinocyte migration by sensing the microenvironment and coordinating follower cells. However, the mechanisms underlying leader cell emergence and their defining characteristics remain poorly understood. Notch1 has previously been shown to be expressed in keratinocyte leader cells. This study investigates the role of cell dynamics at the leading edge in leader cell formation during Notch inhibition, hypothesizing that efficient leader cell emergence is Notch-dependent and necessary for effective re-epithelialization. Methods To assess re-epithelialization rates and monitor cellular behaviour, a high-throughput scratch assay was developed using a 3D-printed, autoclavable lid with custom slits for a 96-well plate, ensuring reproducible scratches. Scratch closure was monitored over time using an immortalized keratinocyte cell line (N/TERT). Putative leader cells were identified through a new high-throughput, high-content morphological analysis pipeline with downstream bioinformatic analysis. The Notch pharmacological inhibitor DAPT was used, and scratch closure rates were analysed using one-way Anova. Results The high-throughput morphological analysis identified significant differences in cell morphology at the leading edge (Zone 1) compared with cells further from scratch (Zones 2 and 3). Interestingly, single-cell analysis of cells at the leading edge revealed a unique subset of cells – termed potential leader cells. Notch inhibition significantly decreased the number of these leader cells and delayed scratch closure. Using multiparameter cell tracking over time, keratinocytes with leader cell potential are being further explored to assess novel interventions to influence their potential to orchestrate re-epithelialization. Conclusions Leader cells form during keratinocyte re-epithelialization and can be identified using morphological parameters. Notch inhibition decreases scratch closure rates, correlated with fewer identified leader cells. These findings underscore the critical role of leader cells in wound healing and highlight the therapeutic potential of targeting leader cell pathways to enhance skin repair.