Kandice R. Levental

Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling

Tumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM remodeling to cancer is appreciated; the relevance of stiffening is less clear. We found that breast tumorigenesis is accompanied by collagen crosslinking, ECM stiffening, and increased focal adhesions. Induction of collagen crosslinking stiffened the ECM, promoted focal adhesions, enhanced PI3 kinase (PI3K) activity, and induced the invasion of an oncogene-initiated epithelium. Inhibition of integrin signaling repressed the invasion of a premalignant epithelium into a stiffened, crosslinked ECM and forced integrin clustering promoted focal adhesions, enhanced PI3K signaling, and induced the invasion of a premalignant epithelium. Consistently, reduction of lysyl oxidase-mediated collagen crosslinking prevented MMTV-Neu-induced fibrosis, decreased focal adhesions and PI3K activity, impeded malignancy, and lowered tumor incidence. These data show how collagen crosslinking can modulate tissue fibrosis and stiffness to force focal adhesions, growth factor signaling and breast malignancy.

Tissue mechanics modulate microRNA-dependent PTEN expression to regulate malignant progression.

Tissue mechanics regulate development and homeostasis and are consistently modified in tumor progression. Nevertheless, the fundamental molecular mechanisms through which altered mechanics regulate tissue behavior and the clinical relevance of these changes remain unclear. We demonstrate that increased matrix stiffness modulates microRNA expression to drive tumor progression through integrin activation of β-catenin and MYC. Specifically, in human and mouse tissue, increased matrix stiffness induced miR-18a to reduce levels of the tumor suppressor phosphatase and tensin homolog (PTEN), both directly and indirectly by decreasing levels of homeobox A9 (HOXA9). Clinically, extracellular matrix stiffness correlated directly and significantly with miR-18a expression in human breast tumor biopsies. miR-18a expression was highest in basal-like breast cancers in which PTEN and HOXA9 levels were lowest, and high miR-18a expression predicted poor prognosis in patients with luminal breast cancers. Our findings identify a mechanically regulated microRNA circuit that can promote malignancy and suggest potential prognostic roles for HOXA9 and miR-18a levels in stratifying patients with luminal breast cancers.

FGF-2 and VEGF functionalization of starPEG―heparin hydrogels to modulate biomolecular and physical cues of angiogenesis

Tissue engineering therapies require biomaterials capable of encouraging an angiogenic response. To dissect the influence of different pro-angiogenic stimuli a set of starPEG-heparin hydrogels with varied physicochemical properties was used as a highly efficient reservoir and tunable delivery system for basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF). The engineered gel materials could be precisely tailored by decoupling the biomolecular functionalization from the variation of the viscoelastic matrix characteristics. Culture experiments with human umbilical vein endothelial cells (HUVECs) revealed the interplay of growth factor presentation, adhesive characteristics and elasticity of the gel matrices in triggering differential cellular behavior which allowed identifying effective pro-angiogenic conditions.

Sustained delivery of SDF-1α from heparin-based hydrogels to attract circulating pro-angiogenic cells.

Enrichment of progenitor cells in ischemic tissue has become a promising therapeutic strategy in the treatment of myocardial infarction. Towards this aim, we report a biology-inspired concept using sulfated glycosaminoglycans to sustainably generate chemokine gradients for the localized accumulation of early endothelial progenitor cells (eEPCs). StarPEG-heparin hydrogels, which have been previously demonstrated to support angiogenesis, were functionalized with SDF-1α, a potent chemoattractant known to act on EPCs. The gels were quantitatively shown to release the chemokine in amounts that are adjustable by the choice of loading concentrations and by matrix metalloprotease (MMP) mediated hydrogel cleavage. Transwell assays confirmed significantly enhanced migration of early EPCs towards concentration gradients of hydrogel-delivered SDF-1α in vitro. Subcutaneous implantation of SDF-1α-releasing gels in mice resulted in massive infiltration of early EPCs and subsequently improved vascularization. In conclusion, sustained delivery of SDF-1α from pro-angiogenic starPEG-heparin hydrogels can effectively attract early EPCs, offering a powerful means to trigger endogenous mechanisms of cardiac regeneration.

Defined Polymer–Peptide Conjugates to Form Cell‐Instructive starPEG–Heparin Matrices In Situ

Poly(ethylene glycol)-peptide- and glycosaminoglycan-peptide conjugates obtained by a regio-selective amino acid protection strategy are converted into cell-instructive hydrogel matrices capable of inducing morphogenesis in embedded human vascular endothelial cells and dorsal root ganglia.

Dual independent delivery of pro-angiogenic growth factors from starPEG-heparin hydrogels.

Effective vascularization is a prerequisite for the success of various different tissue engineering concepts. While simultaneous administration of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) has been previously demonstrated to boost angiogenesis, the combined long-term delivery of both growth factors from biomaterials is still a major challenge. In this work, two important heparin binding cytokines were delivered in parallel from a modular starPEG (multi-armed polyethylene glycol)--heparin hydrogel system to human umbilical vein endothelial cells (HUVECs) grown in culture and in a chicken embryo chorioallantoic membrane (CAM) model. As the utilized gels contain high quantities of heparin, loading and subsequent release of both growth factors (as determined by radiolabeling studies and Enzyme-Linked Immunosorbent Assay [ELISA]) occurred independently from each other. The combined delivery of FGF-2 and VEGF through starPEG-heparin hydrogels resulted in pro-angiogenic effects in vitro (study of cell survival/proliferation, morphology and migration) and in vivo (quantification of CAM vascularization) being clearly superior over those of the administration of single factors. Consequently, the independent delivery of growth factor combinations by biohybrid starPEG-heparin matrices allows for the precise multifactorial control of cellular processes critically determining regeneration.

Membrane raft association is a determinant of plasma membrane localization

Significance The organization of metazoan membranes into functional domains is a key feature of their physiology. A subset of these domains, known as “membrane rafts,” has been implicated in a large variety of cellular processes; however, the molecular mechanisms by which raft domains regulate cell function remain elusive. Here, we demonstrate that membrane raft association is a necessary and sufficient sorting signal for cell-surface localization of a specific single-pass membrane protein, linker for activation of T-cells, and that this raft association is governed by the properties of the protein transmembrane domain. These results begin to define the physical bases for protein raft affinity and establish lateral membrane domains as mediators of protein sorting in mammalian cells. The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting.

A simple indentation device for measuring micrometer-scale tissue stiffness

Mechanical properties of cells and extracellular matrices are critical determinants of function in contexts including oncogenic transformation, neuronal synapse formation, hepatic fibrosis and stem cell differentiation. The size and heterogeneity of biological specimens and the importance of measuring their mechanical properties under conditions that resemble their environments in vivo present a challenge for quantitative measurement. Centimeter-scale tissue samples can be measured by commercial instruments, whereas properties at the subcellular (nm) scale are accessible by atomic force microscopy, optical trapping, or magnetic bead microrheometry; however many tissues are heterogeneous on a length scale between micrometers and millimeters which is not accessible to most current instrumentation. The device described here combines two commercially available technologies, a micronewton resolution force probe and a micromanipulator for probing soft biological samples at sub-millimeter spatial resolution. Several applications of the device are described. These include the first measurement of the stiffness of an intact, isolated mouse glomerulus, quantification of the inner wall stiffness of healthy and diseased mouse aortas, and evaluation of the lateral heterogeneity in the stiffness of mouse mammary glands and rat livers with correlation of this heterogeneity with malignant or fibrotic pathology as evaluated by histology.

Polyunsaturated Lipids Regulate Membrane Domain Stability by Tuning Membrane Order

The plasma membrane (PM) serves as the functional interface between a cell and its environment, hosting extracellular signal transduction and nutrient transport among a variety of other processes. To support this extensive functionality, PMs are organized into lateral domains, including ordered, lipid-driven assemblies termed lipid rafts. Although the general requirements for ordered domain formation are well established, how these domains are regulated by cell-endogenous mechanisms or exogenous perturbations has not been widely addressed. In this context, an intriguing possibility is that dietary fats can incorporate into membrane lipids to regulate the properties and physiology of raft domains. Here, we investigate the effects of polyunsaturated fats on the organization of membrane domains across a spectrum of membrane models, including computer simulations, synthetic lipid membranes, and intact PMs isolated from mammalian cells. We observe that the ω-3 polyunsaturated fatty acid docosahexaenoic acid is robustly incorporated into membrane lipids, and this incorporation leads to significant remodeling of the PM lipidome. Across model systems, docosahexaenoic acid-containing lipids enhance the stability of ordered raft domains by increasing the order difference between them and coexisting nonraft domains. The relationship between interdomain order disparity and the stability of phase separation holds for a spectrum of different perturbations, including manipulation of cholesterol levels and high concentrations of exogenous amphiphiles, suggesting it as a general feature of the organization of biological membranes. These results demonstrate that polyunsaturated fats affect the composition and organization of biological membranes, suggesting a potential mechanism for the extensive effects of dietary fat on health and disease.

Two-tier hydrogel degradation to boost endothelial cell morphogenesis.

Cell-responsive degradation of biofunctional scaffold materials is required in many tissue engineering strategies and commonly achieved by the incorporation of protease-sensitive oligopeptide units. In extension of this approach, we combined protease-sensitive and -insensitive cleavage sites for the far-reaching control over degradation rates of starPEG-heparin hydrogel networks with orthogonally modulated elasticity, RGD presentation and VEGF delivery. Enzymatic cleavage was massively accelerated when the accessibility of the gels for proteases was increased through non-enzymatic cleavage of ester bonds. The impact of gel susceptibility to degradation was explored for the 3-dimensional ingrowth of human endothelial cells. Gels with accelerated degradation and VEGF release resulted in strongly enhanced endothelial cell invasion in vitro as well as blood vessel density in the chicken chorioallantoic membrane assay in vivo. Thus, combination of protease-sensitive and -insensitive cleavage sites can amplify the degradation of bioresponsive gel materials in ways that boost endothelial cell morphogenesis.