Knockdown of PAX3-FOXO1 enhances cell–ECM interaction via TGFβ signaling. (a) Cell spreading areas of PF^KD FPRMS cell lines upon treatment with the TGFβ inhibitor LY2109761 (100 nM, 12 h) (n = 30). (b) Collagen fiber displacement near spheroids of control FPRMS cells (left), PF^KD FPRMS cells (middle), and LY2109761-treated PF^KD FPRMS cells (100 nM, right), monitored by CRM and analyzed by PIV. Top: pseudocolored images showing the displacement degree of collagen fibers caused by spheroids. Bottom: arrows indicate the direction and magnitude of collagen fiber displacement. (c) Quantification of the collagen fiber displacement of PF^KD FPRMS cell lines upon TGFβ inhibition (n = 5). In a and c, each dot represents the value of a corresponding cell or spheroid. Lines are averages of each condition. (d) Collagen fiber alignment 16 h after gelation with control FPRMS cells (left), PF^KD FPRMS cells (middle), and LY2109761-treated PF^KD FPRMS cells (100 nM, right). Dashed lines denote the spheroid edges. Arrows point to the fibers aligned perpendicularly to the spheroid edges, while asterisks (*) highlight the fibers aligned parallel to the edges. (e) Distribution of collagen fiber orientation around the spheroids with and without TGFβ inhibition. Dots represent individual spheroid values (minimum n = 4). Dashed lines indicate a probability of 0.2 indicating an equal distribution of fiber orientations (random orientation). In a, c, and e, error bars represent SE and statistical significance was assessed using the Wilcoxon rank sum test (a) or two-sided Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, and n.s = not significant.