Fig. 2: Progressive changes in composition and architecture of complex VTSs during cultivation.

a Image analysis routine: After pre-processing, 3D images underwent a four-pronged analysis routine of generating surface renderings of the (i) individual cellular compartments, (ii) localization of individual cells, (iii) tracing of the PV network, and (iv) evaluation of distal distribution from the pseudovasculature within the VTS. This resulted in a multiparametric data set for each VTS allowing for the side-by-side comparison of effects from cultivation conditions, VTS composition, or treatment. b Changes in VTS size over 12 days of cultivation. Volumes calculated from LSFM-acquired images in fixed VTSs. c Time-resolved changes of MCF7-based VTSs over a cultivation period of 12 days. 3D-rendering of surfaces of the three imaged cellular compartments and 3D representation of traced pseudovessels, colored according to segment mean diameter. d Changes in VTS composition over 12 days of cultivation. Relative volume ratios of ECs, TCs, and fibroblasts over time. e Time-resolved distribution of PV-segment diameters over 12 days of cultivation in MDA-MB-231-, MDA-MB-435s- and MCF7-based VTSs. Displayed are the mean values of three VTSs/cell lines. f Time-resolved distribution of PV-segment branch levels over 12 days of cultivation in MDA-MB-231-, MDA-MB-435s- and MCF7-based VTSs. Branch levels give the hierarchical distance of a multi-segment PV branch from the longest (main-) branch of a PV network. Displayed are the mean values of three VTSs/cell lines. g Time-resolved distribution of voxel distances from the nearest PV over 12 days of cultivation in MDA-MB-231-, MDA-MB-435s-, and MCF7-based VTSs. Displayed are the mean values of three VTSs/cell lines. 3D grid spacing: 50 µm, error bars: ± SEM, n = 3 individual biological samples.