Fig. 4: LGR5+ adhere better to endothelial monolayers and form transendothelial gaps.

a Schematic of the preparation of PDOs for adhesion analysis on an endothelial monolayer. b PDO clusters seeded on HUVEC monolayers formed on collagen I coated 3 kPa gel. Representative time lapse of LGR5^low, LGR5^med and LGR5^high clusters on a HUVEC monolayer. The last two columns show the fluorescence image of the HUVEC monolayers at 8 h including a zoom at the contact point between clusters and monolayers. Scale bars, 50 μm. Representative images from four independent experiments. c Mean Tdtomato fluorescence labeling LGR5+ cells in CRC clusters that either remained attached to the monolayer during the whole time-lapse or detached. Data are represented as the mean ± s.d. of n ≥ 31 clusters/ subgroup clusters from four independent experiments. Statistical significance was determined using Shapiro-Wilk normality test, followed by a Kruskal−Wallis multiple-comparison test. d Percentage of clusters that formed a gap in the endothelial monolayer for LGR5^low, LGR5^med and LGR5^high cell clusters. Data are represented as the mean ± s.d. of percentages from four independent experiments. Statistical significance was determined using two-way analysis of variance, followed by a Šidák multiple-comparison test. e Time for gap formation for LGR5^low, LGR5^med and LGR5^high cell clusters. f Gap area measured 8 h after beginning of time lapse acquisition. In (e) and (f) data are represented as the mean ± s.d. of n = 77 clusters that remained attached during time lapse acquisition from four independent experiments. Statistical significance was determined using Shapiro-Wilk normality test, followed by a Kruskal−Wallis multiple-comparison test. Source data are provided as a Source Data file.