Using a 3-step semi-automated image analysis pipeline to quantify differences in VE-cadherin junction morphology in lymphatic endothelial cells in vitro

Lymphatic endothelial cells (LECs) rely on junctions transitioning between button- and zipper-like configurations to regulate fluid flow into vessels. However, existing techniques to measure junction morphology are limited in their ability to analyze LEC monolayers comprehensively. To address this gap, researchers have devised a novel 3-step image analysis pipeline that leverages Cellpose, CellProfiler, and Fiji to assess VE-cadherin expression within lymphatic vessels within a 3D microfluidic chip.

This pipeline enables the quantification of several key metrics, including VE-cadherin radial intensity, object count, object size, branchpoints, end-points, and junction thickness. When exposed to tumor necrosis factor (TNF), LECs exhibit button-like junctions, while unexposed cells display zippered junctions. Notably, radial intensity was found to be elevated at the cell membrane in zippered junctions, whereas perinuclear intensity increased for button-like junctions.

Furthermore, the study revealed that increased branchpoints, endpoints, and objects per cell, as well as increased junction thickness, were associated with buttoning, while larger object size was linked to zippering. Principal component analysis (PCA) supported these findings by identifying metric combinations that distinctly represented each junctional state.

In summary, this semi-automated pipeline provides a powerful tool for quantifying differences in VE-cadherin junction morphology across LEC monolayers. By generating new quantitative metrics, this approach expands the analytical capabilities for studying junction morphology in lymphatic vasculature.