Extended Data Fig. 2: ROCK-education of mammary fibroblasts yields a tumor-promoting fibroblast phenotype; Related to Fig. 2.
a, Schematic of the inverse invasion assay assessing fibroblast motility ex vivo. b, Confocal z-stack images (7 µm intervals) through a Matrigel plug following migration by fibroblasts from ROCK-activated R-PyMT and K-PyMT mammary tumors. Repeated more than 3 times with similar results. c, Schematic of ex vivo cell-derived matrix (CDM) establishment to study cancer cell anisotropy. d, Images of picrosirius red-stained CDM formed by fibroblasts from ROCK-activated R-PyMT and K-PyMT mammary tumors. Scales: 100 µm. e, Streaming images of PyMT tumor cells seeded onto CDM generated from ROCK- or KD-educated fibroblasts. Arrows demonstrate streaming direction (insets of boxes shown below). d,e, Repeated 3 times. f, Schematic of co-engraftment strategy to examine educated fibroblast function in vivo. g, ECM proteins fibronectin, periostin and tenascin C (green) in tumors arising in mice that were co-engrafted with PyMT tumor cells and either ROCK- or KD-educated fibroblasts. Counter-label: DAPI (blue). Scales: 100 µm. Dot plots show the area coverage of respective ECM component IF signals. Median±IQR, n = 5 tumors/group, two-sided Mann-Whitney tests. h, Left: Western analysis of pMlc2 in ex vivo-cultured K-PyMT and R-PyMT tumor cells following 4−hydroxy tamoxifen (4HT) treatment. Graphical representations of starved and 4HT-treated densitometries are shown below. Right: pMlc2 (green) in ex vivo-cultured K-PyMT and R-PyMT tumor cells treated with 4HT. Counter-labels: Phalloidin (F-actin, red) and DAPI (blue). Scales: 100 µm. Repeated at least twice with similar results. i, Schematic of co-engraftment strategy to examine the effect of paracrine signaling between ROCK-activated tumor cells and fibroblasts on tumor promotion in vivo. See also Statistics Source Data for Extended Data Fig. 2 and Unprocessed blots for Extended Data Fig. 2.
