FNRMS exhibits higher TGFβ signaling activity than FPRMS. (a) GO analysis of significantly low-expressed genes in Subgroup 3, reflecting FPRMS (top); highly expressed genes in Subgroup 4, reflecting FNRMS (middle); and genes highly expressed in FNRMS compared with FPRMS (bottom), using KEGG gene sets as the reference gene sets. We highlight TGFβ signaling as a suppressed signaling pathway in Subgroup 3, an activated signaling pathway in Subgroup 4, and an enhanced signaling pathway in FNRMS compared with FPRMS. Color represents the count of inquired genes overlapping with each specific reference gene set. Circle size indicates P value, and strength means the ratio of inquired to reference gene counts, implying the enhanced expression of the corresponding reference. Gene expression data of FPRMS and FNRMS are from GSE114621. (b) Dynamic SMAD3 nuclear localization upon TGFβ treatment (4 ng/ml) in FNRMS and FPRMS cell lines, monitored using NG-Smad3 live-cell sensor. (c) TGFβ signaling activity, quantified by the ratio of intensity in nuclear SMAD3 after TGFβ treatment to before the treatment, of FNRMS and FPRMS cell lines. Each dot represents the value of a corresponding cell, and lines are averages of each cell line (n = 14). (d) Higher expression of collagen type I (Col 1) and CTGF (TGFβ signaling downstream molecules) in FNRMS compared with FPRMS, analyzed by immunoblotting. (e) IF images of RD and SMS-CTR (FNRMS), with and without the treatment of a TGFβ inhibitor (LY2109761, 100 nM, 12 h), stained for F-actin (with phalloidin) and vinculin to show focal adhesion. (f) Cell spreading areas for FNRMS and FPRMS cell lines after treatment with TGFβ inhibitor in e. Each dot represents the value of a corresponding cell, and lines are averages of each cell line (n = 30). (g) Collagen fiber displacement near FNRMS spheroids with and without the treatment of LY2109761 (100 nM), monitored by CRM and analyzed by PIV. Top: pseudocolored images showing the displacement degree of collagen fibers caused by FNRMS spheroids. Bottom: arrows indicate the direction and magnitude of collagen fiber displacement. (h) Quantification of the collagen fiber displacements surrounding FNRMS spheroids with and without TGFβ inhibition (n = 5). (i) Collagen fiber alignment 16 h after gelation with FNRMS spheroids, with and without the treatment of LY2109761. Dashed lines denote FNRMS spheroid edges. Arrows point to the fibers aligned perpendicularly to the spheroid edges, while asterisks highlight the fibers aligned parallel to the spheroid edges. (j) Distribution of collagen fiber orientation around FNRMS 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). Asterisks (*) denote statistical differences of values between spheroids with and without LY2109761. In c, f, h, and j, error bars represent SE and statistical significance was assessed using two-sided Student’s t test or Wilcoxon’s rank sum test (h): *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, and n.s. = not significant. Source data are available for this figure: SourceData F3.