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

Effect of inhibitors on 3D gel contractions.

<p>HUF (left) and HeLa (right) cells were grown in 3D cultures and treated with inhibitors as described. The gels were photographed 24 hrs later and the surface area of the individual gels was quantified. The data represent the mean +SEM. One way ANOVA * = p value < 0.05, *** = p value < 0.001.</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

Analysis of MLCK expression in normal and cancer tissues.

<p>Quantitative PCR (A) and western blot (B) analyses of normal and cancer tissues. RNA was isolated from various normal and cancer tissues and total MLCK (A), including all splice variants of nmMLCK and smMLCK, was detected using primers targeting exons 26 and 27. The gene for H3F3A was used as an internal control in all experiments. The data in Panel A depict the averages of qPCR analyses performed in triplicate and the error bars show the standard deviation. Panel B shows a western blot analysis using affinity purified antibodies to MLCK. GAPDH was used as a loading control.</p

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

Contraction of 3D collagen gels.

<p>HeLa, LNCaP, MCF7, MCF10A, HUF and primary prostate cells were seeded in 24 well dishes in liquid collagen. After the collagen hardened, the gels were released from the sides of the wells and allowed to contract for 24 hrs. The wells were photographed from above at the times shown. Note that the HUF and primary prostate cells contract the gels to a greater extent than HeLa and LNCaP cells and that MCF 10A cells contract more than MCF-7 cells. Scale bar = 2 mm.</p

A. Bias compared to FASCount as the reference technology. B. Bias compared to FASCalibur as the reference technology.

<p>A. Bias compared to FASCount as the reference technology. B. Bias compared to FASCalibur as the reference technology.</p

Operating characteristics of dedicated single platform CD4+ T-cell enumeration systems.

<p>PCA = personal cell analysis, SSC = side scatter.</p><p>Operating characteristics of dedicated single platform CD4+ T-cell enumeration systems.</p

PRISMA flow diagram of study selection.

<p>EQA: external quality assurance, FC: flow cytometry.</p

Operating characteristics of Point of Care (POC) technologies for CD4+ T-cell enumeration.

<p>Operating characteristics of Point of Care (POC) technologies for CD4+ T-cell enumeration.</p