Tissue architecture affects cell state. (a) Schematic of mixtures of fluorescently labeled cells displaying pseudorosette-like formations formed by EwS cells specifically in HBVs. (b and c) Maximum intensity projections (upper panel) or single optical slices (lower panel) of TC32 and TC71 cells expressing F-Tractin-GFP (gray, green) or F-Tractin-mRuby2 (red) in zebrafish larval HBV (3 dpf). Note the pseudorosette-like polarization of cells, with cellular protrusions pointing towards a center of a sphere and cell bodies organized outwards. (d) An H&E-stained histological slice from a human EwS tissue sample from the soft tissue (sacral region) of a 20-yr-old female, evaluated by a pathologist. Pseudorosettes structures outlined with dashed line. (e) Optical slice of TC71 cells expressing nuclear marker H2B-Cherry form ring-like structures (outlined with dashed line) in the HBV that resemble those observed in some human EwS tumors. (f) Cells within a cell cluster of like-cells are classified and analyzed according to percent contact with other cells, as no neighbors (NN, 0–20% contact with other cells), partially exposed (PE, 21–81% contact), or fully embedded (E, 81–100% contact). (g and h) Proportion of contact with other TC32 cells is plotted against two dominant shape features, volume (g) and sphericity (h). Whereas volume was independent of the degree of cell–cell contact, a few embedded cells but not other groups show cells with distinctly low sphericity (<0.8, dashed line). Pink arrows indicate cells from all three regions with distinctive cell morphological states, rendered below and color-coded by k-means clustering in Fig. 2 e. Scale bars, 20 µm.