Table 1.

Selected quantitative image analysis methods to measure spheroid outgrowth, their advantages and limitations.

Tool	Measurement	Advantages	Limitations
Las X 3D viewer	Length Counting	Visualisation in 3D True length measured	Requires high-quality Z-stack images No automated method available
ImageJ « Sprout morphology » plugin (34)	Nb. of sprouts Total sprout area Total sprout length Average sprout length Branching level	Standardised and automated Information on length and network complexity Reduced experimenter bias	Loss of 3D information Low versatility Requires further optimisation to compute multicellular system
AQuTAS (35)	Sprout area Total sprout length Nb. of associated sprouts and non-associated sprouts	Standardised and automated High throughput analysis Information on length and morphology Reduced experimenter bias	Loss of 3D information Low versatility Requires further optimisation to compute multicellular system
CI Convert/QuPath	Spheroid outgrowth area Core area Additional features: intensity, cell detection, distances	Semi-standardised and semi-automated Compute multiple channels Robust and versatile	Loss of 3D information No information on length, network complexity or morphology