Fig. 1

Epithelial cells exposed to deep hypoxia undertake directional migration towards oxygen. a Schematic diagram of the cell confinement assay. b Z-axis confocal analysis of LifeAct-mCherry-expressing MCF10A cells under confinement (+C) or not (NC), showing a layer of ∼7–10 µm of culture medium supplemented with Oregon Green (green) which separates the cells (red) from the coverslip (black). Right panels are magnified regions from left panels. Scale bars, 100 µm (left), 10 µm (right). c Detection of hypoxia under confinement with the Visisens camera demonstrating the formation of a steep oxygen gradient at the edge of the cell cluster. Oxygen level is shown in pseudocolors (also see Supplementary Movie 1). d Immunoblot showing HIF1A stabilisation after confinement (24 h). Molecular weights (Mw) are indicated in kDa. e Hypoxia gradient at the edge of the cluster revealed by HIF1A stabilisation as soon as 1 h after confinement. HIF1A levels are represented in pseudocolors. Scale bar, 200 µm. f Representative sequential images of H2B-GFP-expressing MCF10A cells confined (+C) or not confined (NC) showing outwards migration of the cells from the edge of the cluster. Scale bars, 1 mm. g Individual tracking of cells (n = 24) at the margin of the cell cluster for 48 h, from experiments depicted in (f). Tracking is representative of more than three different experiments (also see Supplementary Movies 2–3). h Directed migration and redistribution of H2B-GFP-expressing MCF10A cells visualised after 48 h of confinement or unconfined. Left panels: bright field images at 48 h with a red dotted-line indicating the front of the cell cluster at 0 h. Middle panels: tracks of H2B-GFP expressing MCF10A confined or unconfined for 48 h. Right panels: relative distribution of MCF10A cells at the edge of the cluster at 0, 24 and 48 h. Scale bar, 500 µm. i Mean values of directionality and speed calculated from (g) (mean ± SD; n = 3 independent experiments). See Methods for further information about the calculation of motility parameters. ***P < 0.001, *P < 0.05 by two-tailed Student’s t-test. NC not confined, +C confined