P44 NOTCH signalling and leader cell morphology in keratinocyte migration: insights from scratch assay models of micro-wound healing
Joel Baby, Metka Gorkic Casey, Cleo BishopAbstract
Introduction and aims
Cutaneous wound healing during the proliferative phase is a major clinical and economic challenge, particularly in older patients and those with comorbidities such as diabetes and peripheral vascular disease. Keratinocytes are central to re-epithelialization, and emerging evidence implicates NOTCH signalling in regulating migration dynamics, especially in leader cells at the wound edge. This study aimed to determine whether NOTCH ligands, namely Jagged1 (JAG1) or Delta-like ligand 4 (DLL4), modulate cell morphology at the leading edge in an in vitro scratch assay. We hypothesized that the addition of NOTCH ligands to an in vitro scratch assay will influence cell morphology at the leading edge.
Methods
Immortalized human basal keratinocytes (NTERT) were seeded in 96-well plates (15 000 cells per cm2) and subjected to scratch assays to simulate micro-wound healing. Experimental conditions included solvent control (phosphate-buffered saline) and ligand treatments (JAG1 or DLL4) at 0.1, 0.3 and 0.5 mg mL−1. Each condition (including control) was tested using three technical replicates (n = 3 wells). Scratches were imaged at baseline and 16–18 h post-scratch using brightfield microscopy. Morphological profiling of leader cells was performed via 4′,6-diamidino-2-phenylindole and CellMask staining, followed by uniform manifold approximation and projection dimensionality reduction and clustering to identify leader subgroups. Analysis was conducted using InCARTA, HALO and R.
Results
High-content image analysis of cellular and nuclear morphology revealed distinct leader cell clusters across different ligand treatments, suggesting that ligand-treated leaders may adopt alternative roles in migration. At least three leader subgroups were identified, suggesting heterogeneity in leader cell behaviour under ligand influence.
Conclusions
NOTCH ligands JAG1 and DLL4 may support the establishment of new morphologies which serve a key point of interest for future investigation. These findings highlight the need for further mechanistic studies involving such ligands, in conjunction with an assessment of cell morphologies to explore their role in the wound healing process.