FNRMS shows higher cell–ECM interaction than FPRMS. (a) IF images of FNRMS (RD and SMS-CTR) and FPRMS (Rh30 and Rh41) cell lines, stained for F-actin (with phalloidin) and pFAK, displaying focal adhesion. (b) Cell spreading areas for each RMS subtype cell line. Each dot represents the value of a corresponding cell, and lines are averages of each cell line (minimum n = 36). (c) Initial 3 h collagen fiber displacement near spheroids, 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. (d) Quantification of collagen fiber displacement (n = 5). (e) Collagen fiber alignment 16 h after gelation with spheroids. Dashed lines denote spheroid edges. Arrows point to the fibers aligned perpendicularly to FNRMS spheroid edges, while asterisks highlight the fibers aligned parallel to FPRMS spheroid edges. (f) Distribution of collagen fiber orientation around RMS cell spheroids. Angles at 90° represent perpendicular alignment to spheroid edges; 0° represents parallel alignment. Dashed lines indicate a probability of 0.2 indicating an equal distribution of fiber orientations (random orientation). Dots represent individual spheroid values (minimum n = 4). Asterisks (*) denote statistical differences from RD spheroid values, and pounds (#) indicate differences from SMS-CTR spheroid values. In b, d, and f, error bars represent SE and statistical significance was assessed using Wilcoxon’s rank sum test (b) or two-sided Student’s t test: *P < 0.05, **P < 0.01, ^###P < 0.005, ***P < 0.005, and ****P < 0.001.