Morphologies and cytoskeletal structure for HT-1080 fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) in synthetic extracellular matrix (ECM).

<div><p>(<b>A</b>) Schematic representation of synthetic extracellular matrix (synthetic ECM) formed through “thiol-ene” photopolymerization chemistry to couple norbornene C=C bonds on 4-arm poly(ethylene glycol) (PEG) molecules with thiol (-SH) bonds of cysteine-containing peptides. Crosslinks were formed using matrix metalloproteinase (MMP)-degradable peptides with cysteine groups on each end while adhesion was promoted using pendant RGD-containing peptides (C<b>RGDS</b>) with a single cysteine (2.5 or 3 wt% by mass PEG-NB + MMP-degradable crosslinking peptide, shear moduli = 140 Pa or 220 Pa respectively). Constant total pendant peptide was maintained using non-bioactive C<i><b>RDGS</b></i> (1500 μM active C<b>RGDS</b> + non-active C<i><b>RDGS</b></i>). </p> <p>(<b>B</b>-<b>I</b>) Projected z-stack immunofluorescence (IF) images illustrating hDFs and HT-1080s seeded in synthetic ECM (220 Pa, 1000 μM CRGDS). All overlay images are counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nuclei, blue). (<b>B</b>,<b>C</b>) Overview to illustrate morphological differences (F-actin, red; Nuclei, blue) for (<b>B</b>) hDFs and (<b>C</b>) HT-1080s. (<b>D</b>-<b>F</b>) IF images illustrating myosin IIb expression for hDFs: (<b>D</b>) Overlay image (Myosin IIb, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>E</b>) F-actin and (<b>F</b>) Myosin IIb. White arrows point to actomyosin filaments. (<b>G</b>-<b>I</b>) IF images illustrating myosin IIb expression for <b><i>HT-1080s</i></b>: (<b>G</b>) Overlay (Myosin IIb, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>H</b>) F-actin and (<b>I</b>) Myosin IIb. </p> <p>(<b>J</b>-<b>L</b>) Comparison of quantified mean (<b>J</b>) circularity and (<b>K</b>) elongation for hDFs and HT-1080s calculated using Nikon NIS Elements software (n > 150 cells, ≥ 6 hydrogels, at least two separate experiments; *** = p<0.001). (<b>L</b>) Fraction of elongated (Elongation ≥ 3.0), middle (2.0 ≤ Elongation < 3.0), and rounded (Elongation < 2.0) cells. Differences in fraction of elongated and rounded hDFs compared to HT-1080s were each statistically significant (N ≥ 6 total gels, at least two separate experiments; *** = p<0.001). </p></div

Matrix influences on migration and morphologies for HT-1080s in synthetic ECM.

<p>(<b>A</b>) Cell speed and (<b>B</b>) directionality (DTO/TD) for HT-1080s as a function of matrix conditions (≥ 6 gels, ≥ 40 cells, at least two separate experiments; * = p<0.05; ** = p<0.01). X-axis: Modulus in Pa / RGD concentration in μM. <i>Box</i> and <i>whisker </i><i>plot </i><i>for </i><i>cell </i><i>speed</i>: White diamond = mean, white line = median, boxes = middle upper (top) and middle lower (bottom) quartile of the cell population, whiskers = highest (above) and lowest (below) migration speeds. Error bars for DTO/TD represent standard error of the mean for individual cells. There is also a statistical difference in cell speed for the 140 Pa (1500 μM RGD) and 220 Pa (250 μM RGD) conditions (p<0.05, not shown on graph for clarity). (<b>C</b>) A comparison of the fraction of rounded HT-1080s (Elongation Index < 2.0) as a function of synthetic ECM conditions (x-axis: Modulus in Pa / RGD concentration in μM; white bar = 140 Pa; black bars = 220 Pa). Error bars represent standard deviation for fraction of rounded cells per gel (≥ 6 gels, at least two separate experiments; * = p<0.05; ** = p<0.01; *** = p<0.001). </p

Comparison of adhesion properties for HT-1080s and hDFs.

<div><p>(<b>A</b>-<b>M</b>) Projected z-stack immunofluorescence (IF) images for hDFs and HT-1080s in synthetic ECM (220 Pa, 1000 μM CRGDS). All overlay images are counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nuclei, blue). </p> <p>(<b>A</b>-<b>D</b>) IF images illustrating vinculin expression for <i><b>hDFs</b></i> (boxed region from A shown in B-D): (<b>A</b>) Overlay image (Vinculin, green; F-actin, red; Nuclei, blue). White arrows point to regions enriched with vinculin. (<b>B</b>) Overlay (Vinculin, green; F-actin, red); Single channel images (grayscale) illustrate (<b>C</b>) F-actin and (<b>D</b>) Vinculin. (<b>E</b>-<b>G</b>) IF images illustrating vinculin expression for <b><i>HT-1080s</i></b>: (<b>E</b>) Overlay image (Vinculin, green; F-actin, red; Nuclei, blue). Single channel images (grayscale) illustrate (<b>F</b>) F-actin and (<b>G</b>) Vinculin. </p> <p>(<b>H</b>-<b>J</b>) IF images illustrating β1-integrin expression for <i><b>hDFs</b></i>: (<b>H</b>) Overlay (β1-integrin, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>I</b>) F-actin and (<b>J</b>) β1-integrin (White arrows point to punctate β1-integrin features; Also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s003" target="_blank">Fig. S3</a>). (<b>K</b>-<b>M</b>) IF images illustrating β1-integrin expression for <b><i>HT-1080s</i></b>: (<b>K</b>) Overlay (β1-integrin, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>L</b>) F-actin and (<b>M</b>) β1-integrin. </p> <p>(<b>N</b>) Comparison of attachment for hDFs and HT-1080s on RGD-SAMs as a function of RGD density. Attachment was statistically significant (cell number > 0 RGD control spots) for HT-1080s on surfaces with ≥ 0.19% mol fraction RGD and for hDFs on surfaces ≥ 0.007% mol fraction RGD. Error bars represent standard error of the mean (SEM) for array spots at given RGD density. Significance for cell attachment on individual spots was calculated for hDFs relative to HT-1080s (* = p<0.05; ** = p<0.01; *** = p<0.001). </p></div

A Quantitative Comparison of Human HT-1080 Fibrosarcoma Cells and Primary Human Dermal Fibroblasts Identifies a 3D Migration Mechanism with Properties Unique to the Transformed Phenotype

<div><p>Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels (“synthetic extracellular matrix” or “synthetic ECM”). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced β1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with β1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results, we propose that HT-1080s migrate in synthetic ECM with functional properties that are a direct consequence of their transformed phenotype. </p> </div