Interactions between cancer cells and ECs measured with AFM.

<p>A) Photograph of the cantilever with attached fluorescent cancer cell above the HUVEC monolayer. White scale bar corresponds to 20 µm. B) Sketch of the approach-retraction method and typical retraction force curve in terms of the piezo displacement. The cancer cell approaches the EC monolayer at constant speed. Then the cell comes into contact with the EC during 10 seconds (under 1 nN applied force) to create several bond complexes over the adhesion area. The cantilever is retracted at constant velocity in order to detach the adhesive bonds. The retraction curve shows force jumps corresponding to the rupture force (f) of bonds. The adhesive energy (shaded area) represents the detachment work done by the cantilever to completely detach the cell from the substrate. The detachment force is the force necessary to stretch the cancer cell and the EC until bonds start to detach. Note that some force jumps can follow a plateau corresponding to tether formation.</p

Adhesion energies and detachment forces for different cancer cell lines.

<p>Plot of the adhesion energy (A) and detachment force (B) vs. retraction speed after 10s-contact between a TC and an EC on a HUVEC monolayer. Three cancer cell lines: T24 (open circle), J82 (full square) and RT112 (full triangle). Data are plotted as mean ± standard error of the mean. The line is just a guide for the eye.</p

AFM force curves and rupture force histograms for different cancer cell lines.

<p>Typical force curves after 10s-contact between a TC and an EC on a HUVEC monolayer. Probability histograms with collected rupture forces f for J82 (A), T24 (B) and RT112 cells (C) at V = 5 µm/s. Vertical arrows denote examples of force jumps corresponding to breakup of receptor-ligand bonds.</p

Rupture force vs. retraction velocity for different cancer cell lines.

<p>Relationship between rupture force and retraction speed after 10s-contact between a TC and an EC on a HUVEC monolayer. Three cancer cell lines: T24 (full circle), J82 (full square) and RT112 (full triangle) interacting with the endothelium. Data are plotted as mean ± standard error of the mean. The line is just a guide for the eye.</p

Expression of CD43 and MUC1 by the three bladder cell lines used in this study.

<p>Expression levels of CD43 and MUC1 (red line) by FACS analysis in comparison with an irrelevant antibody (black line): (A, D) T24 cells, (B, E) J82 cells and (C, F) RT112 cells.</p

ICAM-1 expression on ECs.

<p><b>A</b>) Confocal microscopy image of an EC monolayer stained for ICAM-1 (green). HUVECs were fixed with PFA. Nuclei are stained in blue using DAPI. <b>B</b>) Quantification of ICAM-1 levels by FACS analysis (dashed line) in comparison with an irrelevant antibody (solid line).</p

Distribution of rupture forces and effect of an anti-ICAM-1 antibody.

<p>Effect of an anti-ICAM-1 antibody on cancer-EC interactions. Rupture force distributions are Gaussian with one or two peaks revealing the presence of receptor/ligand bonds or non specific interactions. Probability histograms of rupture force (V = 5 µm/s) for (A) T24-HUVEC, (B) J82-HUVEC, (C) RT112-HUVEC. Black histograms represent interaction cancer-cell and EC without antibody whereas red ones show the force distribution after using the antibody. Panels D (T24-ICAM-1) and E (T24-BSA) show the rupture force probabilities for T24 cells in contact with coated substrates. The number N of events is indicated on the histograms.</p

Striation patterns in human engineered muscle tissues (A) and in 2D conditions (B).

<p>Immunofluorescence with anti α-actinin (a,d,g,j) and anti-myosin heavy chain (b,e,h,k) 4 and 7 days after the onset of myotube differentiation. Scale bar 10 µm. Panels c,f,i,l: Transmission electron microscopy 4 and 7 days after the onset of myotube differentiation. Single arrow: Z body; Double arrow: thick filament. *: Cytoplasmic membrane; N: nucleus. Scale bar: 1 µm. Insert: close up view of Z bodies; scale bar: 100 nm.</p

Kinetics of differentiation revealed a faster differentiation in 3D cultures.

<p>RT-qPCR of MYOG, ACTN2 and MYH3 during the early time course of differentiation in 3D and 2D cultures. mRNA concentrations of differentiation genes were normalized to 18S expression and expressed as a percentage of maximum. Values are means ±SD, n = 4 independent culture conditions; each qPCR was performed in triplicate, ** p<0.01 <i>versus</i> 2D cultures.</p

Characterization of the 3D fibrin constructs.

<p>A–B: Cell distribution within the fibrin matrix after 1 day (day 1: D1) and 10 days (D10) of 3D culture. Toluidine blue staining of cryostat sections of constructs was analyzed by light microscopy (scale bar = 100 µm). C–D: Cell morphology was analyzed just after gel polymerization (C) (D0) and after 24 hours (D) (D1). Living myoblasts were stained with green fluorescent calcein and visualized by confocal fluorescent microscopy (calcein appeared in green). Fibrin, in red, was visualized by confocal reflectance microscopy. Myoblast alignment was observed as early as day 1. E–F: α-tubulin immunofluorescence visualized by confocal fluorescent microscopy (in red) confirmed the alignment of the myoblasts (E) and myotubes (F) along the gel axis (arrow). Nuclei were stained with DAPI (in blue) (scale bar = 10 µm). G–H: The length of the human engineered muscle tissue greatly reduced overtime due to compaction of the construct. 3D constructs soon after the gel polymerization (G) and 10 days of 3D culture (H). In absence of cells, fibrin gels did not exhibit any compaction.</p