Classification of morphologies captures heterogeneity of cell states in EwS cells. (a) Left: Fibroblast (NIH-3T3, n = 52) or EwS cells (TC71 n = 60 cells, and TC32 n = 97) expressing F-Tractin-GFP (green in upper panel, gray in lower panel) or F-Tractin-mRuby2 (red) in zebrafish HBV. Right: TC32 cells expressing a doxycycline-induced cassette, which includes a cytoplasmic GFP (upper panel, green) as well as C shRNA (n = 71) or EF1 shRNA (n = 70). A subset of cells also express F-Tractin-mRuby2 (red in upper panel, gray in lower panel), allowing for sparse labeling of single-cell morphologies. (b) 10 representative 3D cell renderings are displayed within each experimental group, pooled from all three tissue microenvironments. Note the distinction in cell shape and size particularly in NIH-3T3 (left-most panel) and TC32 EF1 shRNA (right-most panel) compared to other experimental groups. (c and d) PCA of all experimental groups shown individually (c) or together (d). (e) K-means based clustering allows visualization of relationships between experimental groups. Note distinctions in all experimental groups, with the exception of TC32 and TC32 + C shRNA, which largely overlap. Scale bars, 20 µm.