Structure and Biomechanics of the Endothelial Transcellular Circumferential Invasion Array in Tumor Invasion

<div><p>Cancer cells breach the endothelium not only through cell-cell junctions but also via individual endothelial cells (ECs), or transcellular invasion. The underlying EC forms a circular structure around the transcellular invasion pore that is dependent on myosin light chain kinase (MLCK) and myosin II regulatory light chain (RLC) phosphorylation. Here we offer mechanistic insights into transcellular invasive array formation amid persistent tensile force from activated EC myosin. Fluorescence recovery after photobleaching (FRAP) experiments, sarcomeric distance measurements using super-resolution microscopy and electron microscopy provide details about the nature of the myosin II invasion array. To probe the relationship between biomechanical forces and the tension required to maintain the curvature of contractile filaments, we targeted individual actin-myosin fibers at the invasion site for photoablation. We showed that adjacent filaments rapidly replace the ablat11ed structures. We propose that the transcellular circumferential invasion array (TCIA) provides the necessary constraint within the EC to blunt the radial compression from the invading cancer cell.</p></div

Myosin exchange with the circumferential invasion array.

<p>(A) Representative timelapse images of a FRAP zone (red box) in a transcellular pore. Time  =  mm:ss. (B) A representative fluorescence recovery curve with relative intensity normalized to pre-bleached level. (C–E) Comparison of t_1/2, k value, and mobile fraction between stress fibers and transcellular invasion arrays. n = 8 for each bar. Mean values are displayed on graph.</p

Schematics depicting the postulated balance of mechanical forces during invasion.

<p>At the onset of invasion the EC contractile force is overcome by the compressive force from the cancer cell, and the invasion pore grows in size, characterized by sarcomeric stretching and stress fibers breakage. As the EC recruits myosin for the TCIA assembly, the tensile force begins to counter the compressive force and the size of the invasion pore stays relative constant until the cancer cell exits the microwound, and the force balance reverses.</p

Confocal micrographs of transcellular and paracellular invasion.

<p>(A) Orthogonal view of an early transcellular invasion of MDA-MB231 cell (blue) invading an intact HUVEC monolayer expressing GFP-RLC (green) and stained for VE-Cad (red). (B) Z-sections of invasion pore (white arrowhead). (C) Corresponding Z-sections of cancer cells (arrows) penetrating the monolayer. Inset shows zoomed cancer cell invasive protrusions. Spinning disk confocal images of paracellular (D and E) and transcellular (F and G) invasion. White boxes show zoomed area in panels E and G. (E and G) Intensity-inverted images of GFP-RLC monolayer for ease of visualization. (D) Red lines  =  invading cancer cell, blue  =  uninvaded; red arrowhead highlights stress fiber. (G) Intensity-inverted images of transcellular invasion. Red arrowhead indicates bisecting stress fiber; green arrowheads highlight nascent myosin recruitment. (H) Zoomed area of G showing stretched sarcomeric spacing. Blue arrowheads indicate edges of myosin-denuded bisecting stress fiber. Time is mm:ss. Scale bar, 10 μm.</p

Invasion Array Shape Analysis.

<p>(A–F) Confocal micrographs of gaps between cancer cells (blue) and EC under 3 different conditions. MDA-MB231 are labeled with CellTrace Violet. Maximum intensity projections of VE-Cad delineating cell borders in gray scale. (A and B) Fixed EC and (C and D) live EC expressing GFP-RLC. (E and F) Live EC labeled with CSFE. White arrows show intercellular gaps. Red outlines indicate EC border as determined by VE-Cad stain. (G) Area of gap between EC and invading cancer cell, n = 20 per group. (H) Outline of an invasion pore (gray) fitted to a circle (red) and an ellipse (green). (I) Roundness factor from 11 transcellular arrays (black) and a perfect circle (red). Scale bar, 10 μm.</p

Biomechanics of invasion pore.

<p>(A and B) Laser ablation of GFP-RLC stress fibers (green) in HUVEC during MDA-MB231 (red) invasion. White arrowheads indicate ablation point. (A' and B') GFP-RLC alone is shown. (C) Ablation of a stress fiber in a resting EC. (D–F) Kymographs of ablated zones. Dotted line shows initial positions of ablated fibers. Ablation pulse is indicated by red arrowheads. Time is mm:ss. Scale bar, 5 μm.</p

Ultrastructural studies of invasion pore.

<p>(A and B) SIM images of GFP-RLC (green) and VE-Cad (red) in HUVEC. (C) Example of intensity trace for measuring sarcomeric spacing. (D) Scatter plot of sarcomeric distances (n = 264 for transcellular, 368 for paracellular, and 218 for stress fibers). Blue lines, mean distribution. Red arrows show outliers from myosin-denuded areas of invasion arrays which were not included in the analysis. (E) Platinum replica electron microscopy of transcellular invasion. (F and G) Zoomed areas of purple and cyan boxes. Scale bars, 5 μm (A and B) and 500 nm (E).</p

Tumor Stiffness Is Unrelated to Myosin Light Chain Phosphorylation in Cancer Cells

<div><p>Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors.</p> </div

Quantification of MLC phosphorylation of cells treated with inhibitors.

<p>The phosphorylated and unphosphorylated MLC were separated by urea-glycerol gel electrophoresis, blotted to nitrocellulose and identified using an antibody the 20 kD MLC.</p

Micrographs of cells grown in collagen gels.

<p>HeLa cells were transfected with Lifeact mCherry (red) or Lifeact-GFP and grown in collagen gels. These gels were not released from the walls of the wells to prevent motion artifacts. Panel A shows control (untreated) cells extending filapodia that contact neighboring cells (also see movie in Figure S2). Panels B & C show cells that were treated with 20 μM ML-7 (B) or 6 μM cytochalasin D (C). Cells treated with ML-7 continue to actively extend filopodia. Cells treated with cytochalasin D stop extending filopodia and the actin in these cells appears to collect in large aggregates. The insets are blow ups of the boxed areas. Size bar = 10 μm.</p