Fig. 2: LGR5+ are stiffer and respond to confinement with a higher survival rate and slower ameboid migration.

a Scheme of the real time-deformability cytometry (RT-DC) setup. Cells suspended in a highly viscous solution are flushed through a microfluidic channel of 20 μm height and width, where they are deformed by shear stress and pressure gradients. Cells are imaged in the region of interest (dashed rectangle) to determine their degree of deformation. b Representative images of LGR5-, LGR5^med and LGR5+ analyzed with RT-DC. Scale bar, 5 μm. c, d Cell deformation and derived elastic modulus of LGR5-, LGR5^med and LGR5+ cells. Data are represented as the mean ± s.d. from one experiment representative of four independent ones. n ≥ 300 cells/subgroup. Statistical significance was determined using Shapiro-Wilk normality test, followed by a Kruskal−Wallis multiple-comparison test. e Scheme of dynamic cell confiner. The central PDMS piston holds a glass slide to which PDMS pillars of fixed height (4.5 μm) are attached. The device acts as a suction cup. When negative pressure is applied, the central piston is pressed down, thereby confining the cells. f Representative examples of the four response categories identified in confined LGR5- and LGR5+ cells. Scale bar, 10 μm. g Percentage of dying/dead, polarized blebs, rounded blebs and no blebs in LGR5- and LGR5+ cells under confinement on a non-adhesive surface. Data are represented as the mean ± s.d. of percentages of n ≥ 112 cells/ subgroup from four independent experiments. Statistical significance was determined using two-way analysis of variance, followed by a Šidák multiple-comparison test. h Representative time lapse images of LGR5- and LGR5+ no blebs, LGR5+ and LGR5- polarized blebs. Scale bar, 10 μm. In (f, h) images are representative images of four independent experiments, with a total of 303 cells. i Migration velocity of tracked nuclei of LGR5- and LGR5+ cells, divided into categories according to the confinement response. Data are represented as the mean ± s.d. of n ≥ 112 cells/ subgroup from four independent experiments. Statistical significance was determined using two-way analysis of variance, followed by a Šidák multiple-comparison test. Source data are provided as a Source Data file.