Further characterisation of immortalised human lymphatic endothelial cells to explore their transcriptomic profile and VEGFC response.

In vitro modelling relies on the availability of suitable cell types that accurately represent the organs under study. In lymphatic research, human dermal lymphatic endothelial cells represent the "gold standard", even though they lose their identity and proliferative capacity over time. A recently established immortalised lymphatic endothelial cell line (imLEC) could become a promising new tool for lymphatic disease modelling. We further characterised this cell line by comparing imLECs and HDLECs in terms of the expression of proteins essential for correct lymphatic function, and the proliferation, migration and sprouting responses to vascular endothelial growth factor C (VEGFC). We show similarities in the expression of lymphatic markers and VEGFC-driven cellular responses, supporting imLECs can retain their VEGFC-driven lymphangiogenic capacity without losing their lymphatic identity. RNA sequencing, however, revealed certain transcriptional differences in genes regulating lymphatic function in health and disease, highlighting the need for further validation at single gene level or specific lymphatic-associated signalling pathways. We acknowledge these limitations should be considered in future applications. Nonetheless, we believe that imLECs represent a useful model for the development of gene editing techniques allowing better modelling of lymphatic disease-associated genetic variants, ensuring long-term culture and providing higher reproducibility in genotype-phenotype validation analyses.