Fig. 1: LGR5+ and LGR5- single cells display differences in adhesion, polarity and YAP localization.

a CRC PDOs cultured for 1 week in culture matrix gel stained for CK20 and nuclei. LGR5+ cells are labeled with Tdtomato. Scale bar, 20 μm. Representative images from two independent experiments. b Scheme illustrating the preparation of PDOs for single cell analysis on 2D soft substrates. c Cell roundness measured for sorted cells seeded on collagen-I coated gel substrates of 0.5, 3, 5, 11 kPa in stiffness. d LGR5-, LGR5^med and LGR5+ cells on 0.5 and 11 kPa gel substrates. Representative images of four independent experiments. Scale bar, 50 μm. e Single PDO cells seeded on 0.5 kPa gel substrates stained for Actin, nuclei and YAP. LGR5+ cells are labeled with Tdtomato. Representative images of four independent experiments. Scale bar, 10 μm. f Quantification of YAP nuclear/cytoplasmic ratio of PDO cells seeded on 0.5, 3, 30 kPa gels. In (c) and (f) n ≥ 60 cells/condition from four independent experiments. Statistical significance was determined using two-way analysis of variance, followed by a Šidák multiple-comparison test. Quantification of cell velocity (g) and mean traction (h) exerted by sorted cells on gels of 0.5, 1.5, 5 and 11 kPa in stiffness. In (g) and (h) data are represented as the mean ± s.d. of n ≥ 60 cells/condition from four independent experiments. Statistical significance was determined using Shapiro-Wilk normality test, followed by a Kruskal-Wallis multiple-comparison test. i LGR5-, LGR5^med and LGR5+ cells seeded on 3 kPa gels and their corresponding traction stress field and force dipole. The traction stress vectors and their amplitude are represented by the yellow arrows and colormap, respectively. The two eigenvectors of the dipole matrix are represented by red arrows. Representative images of four independent experiments. Scale bar, 10 μm. j Quantification of the polarization Mδ of LGR5-, LGR5^med and LGR5+ cells. n ≥ 95 cells/condition from four independent experiments. Statistical significance was determined using Shapiro-Wilk normality test, followed by a Kruskal−Wallis multiple-comparison test. All data are represented as the mean ± s.d. Source data are provided as a Source Data file.