Adhesion strength and contractility enable metastatic cells to become adurotactic.

Significant changes in cell stiffness, contractility, and adhesion, i.e., mechanotype, are observed during a variety of biological processes. Whether cell mechanics merely change as a side effect of or driver for biological processes is still unclear. Here, we sort genotypically similar metastatic cancer cells into strongly adherent (SA) versus weakly adherent (WA) phenotypes to study how contractility and adhesion differences alter the ability of cells to sense and respond to gradients in material stiffness. We observe that SA cells migrate up a stiffness gradient, or durotax, while WA cells largely ignore the gradient, i.e., adurotax. Biophysical modeling and experimental validation suggest that differences in cell migration and durotaxis between weakly and strongly adherent cells are driven by differences in intra-cellular actomyosin activity. These results provide a direct relationship between cell phenotype and durotaxis and suggest how, unlike other senescent cells, metastatic cancer cells navigate against stiffness gradients.

EGFRvIII uses intrinsic and extrinsic mechanisms to reduce glioma adhesion and increase migration.

A lack of biological markers has limited our ability to identify the invasive cells responsible for glioblastoma multiforme (GBM). To become migratory and invasive, cells must downregulate matrix adhesions, which could be a physical marker of invasive potential. We engineered murine astrocytes with common GBM mutations, e.g. Ink4a (Ink) or PTEN deletion and expressing a constitutively active EGF receptor truncation (EGFRvIII), to elucidate their effect on adhesion. While loss of Ink or PTEN did not affect adhesion, counterparts expressing EGFRvIII were significantly less adhesive. EGFRvIII reduced focal adhesion size and number, and these cells - with more labile adhesions - displayed enhanced migration. Regulation appears to depend not on physical receptor association to integrins but, rather, on the activity of the receptor kinase, resulting in transcriptional integrin repression. Interestingly, EGFRvIII intrinsic signals can be propagated by cytokine crosstalk to cells expressing wild-type EGFR, resulting in reduced adhesion and enhanced migration. These data identify potential intrinsic and extrinsic mechanisms that gliomas use to invade surrounding parenchyma.

Cell Adhesiveness Serves as a Biophysical Marker for Metastatic Potential.

Tumors are heterogeneous and composed of cells with different dissemination abilities. Despite significant effort, there is no universal biological marker that serves as a metric for metastatic potential of solid tumors. Common to disseminating cells from such tumors, however, is the need to modulate their adhesion as they detach from the tumor and migrate through stroma to intravasate. Adhesion strength is heterogeneous even among cancer cells within a given population, and using a parallel plate flow chamber, we separated and sorted these populations into weakly and strongly adherent groups; when cultured under stromal conditions, this adhesion phenotype was stable over multiple days, sorting cycles, and common across all epithelial tumor lines investigated. Weakly adherent cells displayed increased migration in both two-dimensional and three-dimensional migration assays; this was maintained for several days in culture. Subpopulations did not show differences in expression of proteins involved in the focal adhesion complex but did exhibit intrinsic focal adhesion assembly as well as contractile differences that resulted from differential expression of genes involved in microtubules, cytoskeleton linkages, and motor activity. In human breast tumors, expression of genes associated with the weakly adherent population resulted in worse progression-free and disease-free intervals. These data suggest that adhesion strength could potentially serve as a stable marker for migration and metastatic potential within a given tumor population and that the fraction of weakly adherent cells present within a tumor could act as a physical marker for metastatic potential. SIGNIFICANCE: Cancer cells exhibit heterogeneity in adhesivity, which can be used to predict metastatic potential.

Glioblastoma cellular cross-talk converges on NF-κB to attenuate EGFR inhibitor sensitivity.

In glioblastoma (GBM), heterogeneous expression of amplified and mutated epidermal growth factor receptor (EGFR) presents a substantial challenge for the effective use of EGFR-directed therapeutics. Here we demonstrate that heterogeneous expression of the wild-type receptor and its constitutively active mutant form, EGFRvIII, limits sensitivity to these therapies through an interclonal communication mechanism mediated by interleukin-6 (IL-6) cytokine secreted from EGFRvIII-positive tumor cells. IL-6 activates a NF-κB signaling axis in a paracrine and autocrine manner, leading to bromodomain protein 4 (BRD4)-dependent expression of the prosurvival protein survivin (BIRC5) and attenuation of sensitivity to EGFR tyrosine kinase inhibitors (TKIs). NF-κB and survivin are coordinately up-regulated in GBM patient tumors, and functional inhibition of either protein or BRD4 in in vitro and in vivo models restores sensitivity to EGFR TKIs. These results provide a rationale for improving anti-EGFR therapeutic efficacy through pharmacological uncoupling of a convergence point of NF-κB-mediated survival that is leveraged by an interclonal circuitry mechanism established by intratumoral mutational heterogeneity.

Metastatic State of Cancer Cells May Be Indicated by Adhesion Strength.

Cancer cells within a tumor are heterogeneous and only a small fraction are able to form secondary tumors. Universal biological markers that clearly identify potentially metastatic cells are limited, which complicates isolation and further study. However, using physical rather than biological characteristics, we have identified Mg2+- and Ca2+-mediated differences in adhesion strength between metastatic and nonmetastatic mammary epithelial cell lines, which occur over concentration ranges similar to those found in tumor stroma. Metastatic cells exhibit remarkable heterogeneity in their adhesion strength under stromal-like conditions, unlike their nonmetastatic counterparts, which exhibit Mg2+- and Ca2+-insensitive adhesion. This heterogeneity is the result of increased sensitivity to Mg2+- and Ca2+-mediated focal adhesion disassembly in metastatic cells, rather than changes in integrin expression or focal adhesion phosphorylation. Strongly adherent metastatic cells exhibit less migratory behavior, similar to nonmetastatic cell lines but contrary to the unselected metastatic cell population. Adhesion strength heterogeneity was observed across multiple cancer cell lines as well as isogenically, suggesting that adhesion strength may serve as a general marker of metastatic cells.

Purification and characterization of lipopolysaccharides from six strains of non-O157 Shiga toxin-producing Escherichia coli.

Certain Shiga toxin-producing Escherichia coli (STEC) are virulent human pathogens that are most often acquired through contaminated food. The United States Department of Agriculture, Food Safety and Inspection Service has declared several serogroups of STEC as adulterants in non-intact raw beef products. Hence, sensitive and specific tests for the detection of these STEC are a necessity for implementation in food safety programs. E. coli serogroups are identified by their respective O-antigen moiety on the lipopolysaccharide (LPS) macromolecule. We propose that the development of O-antigen-specific immunological assays can facilitate simple and rapid discriminatory detection of STEC in beef. However, the resources (antigens and antibodies) required for such development are not readily available. To overcome this, we extracted and characterized LPS and O-antigen from six STEC strains. Using hot phenol extraction, we isolated the LPS component from each strain and purified it using a series of steps to eliminate proteins, nucleic acids, and lipid A antigens. Antigens and crude LPS extracts were characterized using gel electrophoresis, immunoblotting, and modified Western blotting with commercially available antibodies, thus assessing the serogroup specificity and sensitivity of available ligands as well. The results indicate that, while many commercially available antibodies bind LPS, their activities and specificities are highly variable, and often not as specific as those required for serogroup discrimination. This variability could be minimized by the production of antibodies specific for the O-antigen. Additionally, the antigens generated from this study provide a source of characterized LPS and O-antigen standards for six serogroups of STEC.

Binding of nucleoid-associated protein fis to DNA is regulated by DNA breathing dynamics.

Physicochemical properties of DNA, such as shape, affect protein-DNA recognition. However, the properties of DNA that are most relevant for predicting the binding sites of particular transcription factors (TFs) or classes of TFs have yet to be fully understood. Here, using a model that accurately captures the melting behavior and breathing dynamics (spontaneous local openings of the double helix) of double-stranded DNA, we simulated the dynamics of known binding sites of the TF and nucleoid-associated protein Fis in Escherichia coli. Our study involves simulations of breathing dynamics, analysis of large published in vitro and genomic datasets, and targeted experimental tests of our predictions. Our simulation results and available in vitro binding data indicate a strong correlation between DNA breathing dynamics and Fis binding. Indeed, we can define an average DNA breathing profile that is characteristic of Fis binding sites. This profile is significantly enriched among the identified in vivo E. coli Fis binding sites. To test our understanding of how Fis binding is influenced by DNA breathing dynamics, we designed base-pair substitutions, mismatch, and methylation modifications of DNA regions that are known to interact (or not interact) with Fis. The goal in each case was to make the local DNA breathing dynamics either closer to or farther from the breathing profile characteristic of a strong Fis binding site. For the modified DNA segments, we found that Fis-DNA binding, as assessed by gel-shift assay, changed in accordance with our expectations. We conclude that Fis binding is associated with DNA breathing dynamics, which in turn may be regulated by various nucleotide modifications.

Plasmonic photothermal therapy increases the tumor mass penetration of HPMA copolymers.

Effective drug delivery to tumors requires both transport through the vasculature and tumor interstitium. Previously, it was shown that gold nanorod (GNR) mediated plasmonic photothermal therapy (PPTT) is capable of increasing the overall accumulation of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers in prostate tumors. In the present study, it is demonstrated that PPTT is also capable of increasing the distribution of these conjugates in tumors. Gadolinium labeled HPMA copolymers were administered to mice bearing prostate tumors immediately before treatment of the right tumor with PPTT. The left tumor served as internal, untreated control. Magnetic resonance imaging (MRI) of both tumors showed that PPTT was capable of improving the tumor mass penetration of HPMA copolymers. Thermal enhancement of delivery, roughly 1.5-fold, to both the tumor center and periphery was observed. Confocal microscopy of fluorescently labeled copolymers corroborates these findings in that PPTT is capable of delivering more HPMA copolymers to the tumor's center and periphery. These results further demonstrate that PPTT is a useful tool to improve the delivery of polymer-drug conjugates.