CT26 invasion after cutting the collagen gel close to the spheroid.

<p><b>(A)</b> Collagen (z projection, 4x objective): red TAMRA collagen was polymerized and cut with scalpel on one side. Left images show the invasion of GFP-positive cells (green) at 0, 24 and 72 hours post-seeding. The dashed line indicates an edge of the cut collagen and the arrow shows the direction of collagen contraction. Right: Fluorescent images of angular unwinded spheroid where each horizontal line corresponds to the radial fluorescence intensity profile. The y-axis of these images corresponds to the different angles. Arrows show the area of invasion on the side of the cut. The black dotted line makes the left limit of the area used to calculate the average fluorescence as shown in B. <b>(B)</b> Average fluorescent intensity of cells extending over the initial spheroid surface as a function of angle Θ. This is used to estimate the outgrowth of cells from the original spheroid size. The different colors represent the average intensity after 0 (red), 24 (green) and 72 hours (violet) of invasion. The shading of the graph shows the side facing the cut (light blue), the opposing side facing the inside of the gel (light red) and the two sides perpendicular to the cut (white area). The dotted line represents the outgrowth in spheroids embedded in uncut collagen at the corresponding times. <b>(C)</b> Invasion area quantification on the side of the cut (after 0 hours) and average invasion area on uncut side. Invasion area was quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156442#pone.0156442.g003" target="_blank">Fig 3</a> (mean ± standard error, n = 54 of 6 independent experiments). <b>(D)</b> Sketch of tension-dependent invasion model.</p

Invasion of cancer cells CT26 into collagen type I.

<p><b>(A)</b> Image sequence of CT26-GFP (green) cell invasion in TAMRA-labeled collagen type I (red). Cells initiate invasion (white arrow) after approximately 9 hours (onset of invasion). Scale bar: 50 μm. <b>(B)</b> Kymographs of collagen and cells from the image sequence (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156442#pone.0156442.s011" target="_blank">S2 Movie</a>). Images were taken every one hour for 24 hours. Scale 50 μm and 3 hours. <b>(C)</b> Magnification of the boxed region showing movement of the collagen fibers towards the spheroid. The kymograph illustrates the two antagonizing movements of compression due to spheroid growth (close to spheroid, blue arrow and blue line), and the collagen contraction in the invasion zone (stripes toward the spheroid, yellow arrow and yellow line). <b>(D)</b> Zoom in confocal images of CT26-GFP multicellular cancer cell spheroid with cells invading (green) into collagen type I network (red) showing the collagen organization: parallel fibers (white arrow) and radial fibers (yellow arrow). Scale 20 μm. <b>(E)</b> Collagen fiber orientation i) Color-coded orientation map. ii) Quantitative orientation measurement (table—angles) on selected ROIs (circles). Spheroid surface orientation: 45°, orientation normal to surface: -45° <b>(F)</b> Collagen signatures found in a single NICD/p53^-/- mouse intestinal tumor are imaged using intravital two-photon microscopy. i) TACS-1, curly collagen structure; ii) TACS-2, straight and aligned collagen, parallel to the tumor edge and iii) TACS-3, collagen aligned perpendicularly to the tumor edge. Epithelial cancer cells (nuclear GFP, green), collagen (SHG, magenta). Arrowheads point to distinct collagen organization. Scale bars, 50μm.</p

CT26 cell line spheroids characterization.

<p><b>(A)</b> Radial profile of spheroid’s GFP fluorescence over 72 hours of invasion into collagen type I. Different zones of the spheroid can be distinguished: dark area, previously described as necrotic core (dotted line), proliferative rim (dashed line) and invasion (line and shaded area). Insert: representative image of spheroid (GFP) 24 hours post-seeding. Scale bar: 100 μm. <b>(B)</b> Morphology change over time of different-sized spheroids seeded into in collagen type I. Spheroids were imaged in bright field and fluorescence (GFP). Scale bar: 200 μm. <b>(C)</b> Growth kinetics of spheroids for three different initial sizes (<300 μm, 350–400 μm and >400 μm). Values are mean ± standard deviation of n = 6–12 from three independent experiments.</p