Fibronectin Gradient Research Articles

A fibronectin gradient remodels mixed-phase mesoderm.

Physical processes ultimately shape tissue during development. Two emerging proposals are that cells migrate toward stiffer tissue (durotaxis) and that the extent of cell rearrangements reflects tissue phase, but it is unclear whether and how these concepts are related. Here, we identify fibronectin-dependent tissue stiffness as a control variable that underlies and unifies these phenomena in vivo. In murine limb bud mesoderm, cells are either caged, move directionally, or intercalate as a function of their location along a stiffness gradient. A modified Landau phase equation that incorporates tissue stiffness accurately predicts cell diffusivity upon loss or gain of fibronectin. Fibronectin is regulated by WNT5A-YAP feedback that controls cell movements, tissue shape, and skeletal pattern. The results identify a key determinant of phase transition and show how fibronectin-dependent directional cell movement emerges in a mixed-phase environment in vivo.

Spatial Gradients of E-Cadherin and Fibronectin in TGF-β1-Treated Epithelial Colonies Are Independent of Fibronectin Fibril Assembly.

Epithelial to Mesenchymal Transition (EMT) is a dynamic, morphogenetic process characterized by a phenotypic shift in epithelial cells towards a motile and often invasive mesenchymal phenotype. We have previously demonstrated that EMT is associated with an increase in assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils. We have also demonstrated that Transforming Growth Factor-β1 (TGF-β1) localizes to FN fibrils, and disruption of FN assembly or disruption of TGF-β1 localization to FN fibrils attenuates EMT. Previous studies have shown that TGF-β1 induces spatial gradients of EMT in mammary epithelial cells cultured on FN islands, with cells at free edges of the island preferentially undergoing EMT. In the current work, we sought to investigate: (a) whether FN fibril assembly is also spatially patterned in response to TGF-β1, and (b) what effects FN fibril inhibition has on spatial gradients of E-Cadherin and FN fibrillogenesis. We demonstrate that mammary epithelial cells cultured on square micropatterns have fewer E-Cadherin-containing adherens junctions and assemble more FN fibrils at the periphery of the micropattern in response to increasing TGF-β1 concentration, indicating that TGF-β1 induces a spatial gradient of both E-Cadherin and FN fibrils. Inhibition of FN fibril assembly globally diminished E-Cadherin-containing adherens junctions and FN fibrillogenesis, but did not eliminate the spatial gradient of either. This suggests that global inhibition of FN reduces the degree of both FN fibrillogenesis and E-Cadherin-containing adherens junctions, but does not eliminate the spatial gradient of either, suggesting that spatial gradients of EMT and FN fibrillogenesis are influenced by additional factors.

Manipulating electrostatic field to control the distribution of bioactive proteins or polymeric microparticles on planar surfaces for guiding cell migration

We report a general strategy to generate linear and circular gradients of active proteins or polymeric microparticles on planar surfaces by controlling the distribution of electrostatic field during electrohydrodynamic jet printing or electrospray process. Taking fibronectin as an example, we generated a circular gradient of fibronectin and investigated its effect on accelerating the migration of fibroblasts to suit for use in wound closure. In another demonstration, we created linear gradients of laminin in unidirectional and bidirectional patterns, respectively. We showed that such gradations significantly promoted the migration of human neuroblastoma cells with the increase of laminin content. When we changed fibronectin/laminin to electrosprayed poly(lactic-co-glycolic acid) (PLGA) microparticles, we found similar results in terms of guiding cell migration, except that the guidance cues varied from biological signal to topographic structure. Taken together, this method for generating linear/circular gradients of fibronectin/laminin and PLGA microparticles can be readily extended to different types of bioactive proteins and polymeric microparticles to suit wound closure, nerve repair, and related applications involving cell migration.

Collective migration during a gap closure in a two-dimensional haptotactic model

The ability of cells to respond to substrate-bound protein gradients is crucial for many physiological processes, such as immune response, neurogenesis and cancer cell migration. However, the difficulty to produce well-controlled protein gradients has long been a limitation to our understanding of collective cell migration in response to haptotaxis. Here we use a photopatterning technique to create circular, square and linear fibronectin (FN) gradients on two-dimensional (2D) culture substrates. We observed that epithelial cells spread preferentially on zones of higher FN density, creating rounded or elongated gaps within epithelial tissues over circular or linear FN gradients, respectively. Using time-lapse experiments, we demonstrated that the gap closure mechanism in a 2D haptotaxis model requires a significant increase of the leader cell area. In addition, we found that gap closures are slower on decreasing FN densities than on homogenous FN-coated substrate and that fresh closed gaps are characterized by a lower cell density. Interestingly, our results showed that cell proliferation increases in the closed gap region after maturation to restore the cell density, but that cell–cell adhesive junctions remain weaker in scarred epithelial zones. Taken together, our findings provide a better understanding of the wound healing process over protein gradients, which are reminiscent of haptotaxis.

Covalent Binding of Bone Morphogenetic Protein-2 and Transforming Growth Factor-β3 to 3D Plotted Scaffolds for Osteochondral Tissue Regeneration.

Engineering the osteochondral tissue presents some challenges mainly relying in its function of transition from the subchondral bone to articular cartilage and the gradual variation in several biological, mechanical, and structural features. A possible solution for osteochondral regeneration might be the design and fabrication of scaffolds presenting a gradient able to mimic this transition. Covalent binding of biological factors proved to enhance cell adhesion and differentiation in two-dimensional culture substrates. Here, we used polymer brushes as selective linkers of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β3 (TGF-β3) on the surface of 3D scaffolds fabricated via additive manufacturing (AM) and subsequent controlled radical polymerization. These growth factors (GFs) are known to stimulate the differentiation of human mesenchymal stromal cells (hMSCs) toward the osteogenic and chondrogenic lineages, respectively. BMP-2 and TGF-β3 were covalently bound both homogeneously within a poly(ethylene glycol) (PEG)-based brush-functionalized scaffolds, and following a gradient composition by varying their concentration along the axial section of the 3D constructs. Following an approach previously developed by our group and proved to be successful to generate fibronectin gradients, opposite brush-supported gradients of BMP-2 and TGF-β3 were finally generated and subsequently tested to differentiate cells in a gradient fashion. The brush-supported GFs significantly influenced hMSCs osteochondral differentiation when the scaffolds were homogenously modified, yet no effect was observed in the gradient scaffolds. Therefore, this technique seems promising to maintain the biological activity of growth factors covalently linked to 3D scaffolds, but needs to be further optimized in case biological gradients are desired.

Ovarian and Breast Cancer Migration Dynamics on Laminin and Fibronectin Bidirectional Gradient Fibers Fabricated via Multiphoton Excited Photochemistry.

Migration mis-regulation is a hallmark of cancer, and remains an important problem in cancer biology. We postulate the needs for better in vitro models to understand the details of cell-matrix interactions. Here, we utilized multiphoton excited (MPE) photochemistry to fabricate models to systematically study migration dynamics operative in breast and ovarian cancer. Gradients are a convenient means to modulate concentration and also have been implicated in metastases. We specifically pattern sub-micron structured gradients from laminin and fibronectin whose up-regulation is associated with increased metastasis and poor prognosis. We developed a new continuous linear bi-directional gradient design, permitting exploration of the underlying cell-matrix interactions of migration, including speed, directness, and f-actin cytoskeleton alignment as a function of concentration. These new models provide both contact guidance and ECM binding cues, and provide a more relevant environment than possible with existing technologies such as flow chambers or 2D printed surfaces. We found an overall increase in these processes with increasing concentration on both laminin and fibronectin gradients for a series of ovarian and breast cancer lines. Moreover, directness was higher for more metastatic cells, indicating that epithelial or mesenchymal state of the cell type governs the dynamics. However, the specifics of the speed and directedness depend on both the cell type and protein, thus we found that we must consider these processes collectively to obtain a self-consistent picture of the migration. For this purpose, we performed a linear discriminate analysis (LDA) and successfully classified the different cell types on the two protein gradients without molecular biology analysis. The bi-gradient structures are versatile tools to performing detailed studies of cell migration, specifically haptotxis. We further suggest the can be used in assessing efficacy of drug treatments targeted at specific matrix components.

Ankyrin-B is a PI3P effector that promotes polarized α5β1-integrin recycling via recruiting RabGAP1L to early endosomes.

Endosomal membrane trafficking requires coordination between phosphoinositide lipids, Rab GTPases, and microtubule-based motors to dynamically determine endosome identity and promote long-range organelle transport. Here we report that ankyrin-B (AnkB), through integrating all three systems, functions as a critical node in the protein circuitry underlying polarized recycling of α5β1-integrin in mouse embryonic fibroblasts, which enables persistent fibroblast migration along fibronectin gradients. AnkB associates with phosphatidylinositol 3-phosphate (PI3P)-positive organelles in fibroblasts and binds dynactin to promote their long-range motility. We demonstrate that AnkB binds to Rab GTPase Activating Protein 1-Like (RabGAP1L) and recruits it to PI3P-positive organelles, where RabGAP1L inactivates Rab22A, and promotes polarized trafficking to the leading edge of migrating fibroblasts. We further determine that α5β1-integrin depends on an AnkB/RabGAP1L complex for polarized recycling. Our results reveal AnkB as an unexpected key element in coordinating polarized transport of α5β1-integrin and likely of other specialized endocytic cargos.

MenaINV mediates synergistic cross-talk between signaling pathways driving chemotaxis and haptotaxis.

Directed cell migration, a key process in metastasis, arises from the combined influence of multiple processes, including chemotaxis-the directional movement of cells to soluble cues-and haptotaxis-the migration of cells on gradients of substrate-bound factors. However, it is unclear how chemotactic and haptotactic pathways integrate with each other to drive overall cell behavior. MenaINV has been implicated in metastasis by driving chemotaxis via dysregulation of phosphatase PTP1B and more recently in haptotaxis via interaction with integrin α5β1. Here we find that MenaINV-driven haptotaxis on fibronectin (FN) gradients requires intact signaling between α5β1 integrin and the epidermal growth factor receptor (EGFR), which is influenced by PTP1B. Furthermore, we show that MenaINV-driven haptotaxis and ECM reorganization both require the Rab-coupling protein RCP, which mediates α5β1 and EGFR recycling. Finally, MenaINV promotes synergistic migratory response to combined EGF and FN in vitro and in vivo, leading to hyperinvasive phenotypes. Together our data demonstrate that MenaINV is a shared component of multiple prometastatic pathways that amplifies their combined effects, promoting synergistic cross-talk between RTKs and integrins.

Abstract 5057: Mena at the nexus of chemotaxis and haptotaxis during tumor progression

Abstract Directed-cell migration is key step in the metastatic cascade, and multiple cues can promote local invasion. The most well studied mode of cell motility for metastasizing tumor cells is chemotaxis, the directional movement of cells attracted to a source of soluble cues. In contrast, haptotaxis, migration of cells on gradients of substrate-bound factors, remains poorly understood. Mena, an actin regulatory protein, plays an important role in cell motility and is upregulated in various cancers, driving migration in response to chemotactic and haptotactic cues. We have recently shown expression of the pro-metastatic isoform of Mena, MenaINV, increases sensitivity to growth factors (EGF, HGF and IGF) via dysregulation of the phosphatase PTP1B, as well as high concentrations of fibronectin (FN) via its interaction with the integrin α5β1. We hypothesized that MenaINV may also promote crosstalk between these two pathways to drive metastasis. First, we show that MenaINV-driven haptotaxis on FN gradients and FN-driven invasion requires crosstalk between α5β1 and EGFR. Indeed, we show evidence that MenaINV expression is associated high phosphorylation of EGFR in high FN environments in vitro and in human breast cancer databases. Second, we find that MenaINV promotes synergy between EGF and FN in vitro and in vivo, leading to hyper-invasive phenotypes that drive more invasion than which each cue alone. Finally, we show that MenaINV-driven haptotaxis, ECM reorganization and 3D invasion requires RCP-dependent recycling of α5β1 and EGFR. Together, our data demonstrate that MenaINV is a shared component of the machinery that mediates both haptotactic responses to FN and chemotactic responses to EGF, two pathways that promote tumor progression. Citation Format: Madeleine J. Oudin, Miles A. Miller, Tatsiana Kosciuk, Joelle Klazen, Alisha Lussiez, Douglas Lauffenburger, James E. Bear, Frank B. Gertler. Mena at the nexus of chemotaxis and haptotaxis during tumor progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5057.

Tumor Cell-Driven Extracellular Matrix Remodeling Drives Haptotaxis during Metastatic Progression.

Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here, we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and MENA, an actin regulator, and involves increases in focal complex signaling and tumor cell-mediated extracellular matrix (ECM) remodeling. Compared with MENA, higher levels of the prometastatic MENA(INV) isoform associate with α5, which enables 3-D haptotaxis of tumor cells toward the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MENA(INV) and FN levels were correlated in two breast cancer cohorts, and high levels of MENA(INV) were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor cell-intrinsic mechanism that promotes metastasis through ECM remodeling and ECM-guided directional migration. Here, we provide new insight into how tumor cell:ECM interactions generate signals and structures that promote directed tumor cell migration, a critical component of metastasis. Our results identify a tumor cell-intrinsic mechanism driven by the actin regulatory protein MENA that promotes ECM remodeling and haptotaxis along FN gradients. Cancer Discov; 6(5); 516-31. ©2016 AACR.See related commentary by Santiago-Medina and Yang, p. 474This article is highlighted in the In This Issue feature, p. 461.

In This Issue

In This Issue

MENA Promotes Tumor-Intrinsic Metastasis through ECM Remodeling and Haptotaxis.

Oudin and colleagues report a novel and specific function of MENA in mediating directional migration of breast cancer cells toward a fibronectin gradient of increasing concentration. This MENA-mediated haptotactic response depends on the binding of MENA to the α5β1 integrin receptor, adhesion protein signaling, and fibronectin fibrillogenesis. Cancer Discov; 6(5); 474-6. ©2016 AACRSee related article by Oudin et al., p. 516.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 437: Mena at the nexus of chemotaxis and haptotaxis during tumor progression

Abstract The most well studied mode of cell motility for metastasizing tumor cells is chemotaxis, the directional movement of cells attracted to a source of soluble cues. In contrast, haptotaxis, migration of cells on gradients of substrate-bound factors, remains poorly understood. Due to the high expression of extracellular matrix (ECM) proteins such as collagen and fibronectin (FN) in metastatic tumors, it is possible that haptotaxis along ECM gradients could also be driving invasion. Mena, an actin regulatory protein, plays an important role in cell motility and is upregulated in various cancers, where Mena, and in particular the MenaINV isoform, potentiates chemotactic and invasive responses to EGF. All Mena isoforms bind directly to the α5 subunit of the α5β1 integrin, a FN receptor. We hypothesized that the pro-metastatic effect of MenaINV arises from increases in tumor cell responses to specific chemotactic and haptotactic cues, including EGF and FN. To study haptotaxis, MDA-MB231 breast adenocarcinoma cells were plated on gradients of fluorescently-labeled FN in 2D or in 3D collagen gels, generated within a PDMS microfluidic device. Wild type cells expressing low levels of endogenous Mena failed to respond to a FN gradient, but migrated equally well in all directions irrespective of the FN gradient. Surprisingly, ectopic expression of Mena in these cells conferred the ability to respond to FN gradients, with cells exhibiting haptotactic response towards higher concentrations of FN in 2D and 3D. Interestingly, cells expressing MenaINV were able to haptotax towards FN concentrations 4 times higher then cells expressing Mena, while reorganizing and recruiting both collagen and FN surrounding them. MenaINV-dependent haptotaxis was ablated by deletion of the α5 binding site within Mena or treatment with an α5 function blocking antibody, but was unaffected by inhibition of another FN receptor, αVβ3. Using intravital imaging of mouse mammary tumors, we visualized haptotaxis of MenaINV-expressing cells towards gradients of fluorescently labelled FN released locally from implanted microsccale devices. Tumor cells expressing MenaINV lacking the α5 binding site did not haptotax, displayed decreased motility and metastasis to the lung. This was also accompanied by a striking change in the organization of the ECM surrounding the tumor. While investigating the mechanism of haptotaxis, we found that MenaINV-driven invasive responses to FN were also dependent on EGFR signalling and mediated by the phosphatase PTP1B. Finally, using a combination of in vitro and in vivo invasion assays, we show that MenaINV expression promoted synergistic invasive responses to combinations of EGF and FN and also caused increased phosphorylation of Paxilin and FAK at focal adhesions. Together, our data demonstrate that MenaINV is a shared component of the machinery that mediates both haptotactic responses to FN and chemotactic responses to EGF, two pathways that promote tumor progression. Citation Format: Madeleine J. Oudin, Oliver Jonas, Tatiana Kosciuk, Liliane C. Broye, Jeff Wyckoff, Miles A. Miller, Alisha Lussiez, Sreeja Asokan, Robert Langer, Douglas Lauffenburger, James E. Bear, Frank B. Gertler. Mena at the nexus of chemotaxis and haptotaxis during tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 437. doi:10.1158/1538-7445.AM2015-437

GMFβ controls branched actin content and lamellipodial retraction in fibroblasts.

The lamellipodium is an important structure for cell migration containing branched actin nucleated via the Arp2/3 complex. The formation of branched actin is relatively well studied, but less is known about its disassembly and how this influences migration. GMF is implicated in both Arp2/3 debranching and inhibition of Arp2/3 activation. Modulation of GMFβ, a ubiquitous GMF isoform, by depletion or overexpression resulted in changes in lamellipodial dynamics, branched actin content, and migration. Acute pharmacological inhibition of Arp2/3 by CK-666, coupled to quantitative live-cell imaging of the complex, showed that depletion of GMFβ decreased the rate of branched actin disassembly. These data, along with mutagenesis studies, suggest that debranching (not inhibition of Arp2/3 activation) is a primary activity of GMFβ in vivo. Furthermore, depletion or overexpression of GMFβ disrupted the ability of cells to directionally migrate to a gradient of fibronectin (haptotaxis). These data suggest that debranching by GMFβ plays an important role in branched actin regulation, lamellipodial dynamics, and directional migration.

Abstract 1988: MenaINV interaction with α5β1 promotes tumor cell invasion in response to gradients of growth factors and fibronectin

Abstract To metastasize and disseminate, carcinoma cells must invade the surrounding extracellular matrix (ECM) and migrate to reach blood or lymphatic vessels. Collagen (CN) and fibronectin (FN), two components of the ECM surrounding tumors, are highly expressed in metastatic tumors and their patterns of expression can correlate with metastatic outcome in patient samples. Mena, an actin regulatory protein, is upregulated in breast cancer and is alternatively spliced to produce protein isoforms with distinct functions during tumor progression. The invasion-specific MenaINV isoform increases metastasis by potentiating tumor cell motility and invasion responses to various growth factors. All Mena isoforms bind directly to the α5 subunit of the α5β1 integrin, an FN receptor. We hypothesized that the interaction between MenaINV and α5β1 plays an important role in integrating signals from the ECM as well as growth factors to drive invasion. MDA-MB-231 breast cancer cells expressing MenaINV showed increased adhesion to FN alone as well as mixture of FN and CN, compared to control cells. MenaINV cells also had an increased number of α5-positive adhesions as well as increased signalling at focal adhesions as measured by phospho-Paxilin, compared to control. In 3D invasion assays, addition of FN to a CN gel drove invasion of MenaINV-expressing cells even in the absence of any growth factor ligand, an effect driven by its interaction with α5 and dependent on signalling via EGFR and Met. Furthermore, addition of FN caused an even greater potentiation of EGF-induced invasion by MenaINV, an effect that was dependent upon its interaction with α5. Next, we examined how MenaINV affects tumor cell responses to a gradient of FN within a 3D collagen gel. Control cells failed to respond to an FN gradient, while cells expressing MenaINV migrated preferentially towards FN and significantly reorganized both the collagen and FN surrounding them. Deletion of the α5 binding site or treatment with an α5 function blocking antibody ablated the effects MenaINV on movement towards a FN gradient as well ECM reorganization. Overall, these results suggest that some aspects of the pro-metastatic effects of MenaINV involve its ability to bind α5 and regulate bidirectional signaling with FN and growth factors in the tumor microenvironment. Citation Format: Madeleine J. Oudin, Liliane C. Broye, Alisha Lussiez, Sreeja B. Asokan, Miles A. Miller, Douglas A. Lauffenburger, James E. Bear, Frank B. Gertler. MenaINV interaction with α5β1 promotes tumor cell invasion in response to gradients of growth factors and fibronectin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1988. doi:10.1158/1538-7445.AM2014-1988

Human Embryonic Stem Cell-Derived Cardiomyocytes Migrate in Response to Gradients of Fibronectin and Wnt5a

An improved understanding of the factors that regulate the migration of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) would provide new insights into human heart development and suggest novel strategies to improve their electromechanical integration after intracardiac transplantation. Since nothing has been reported as to the factors controlling hESC-CM migration, we hypothesized that hESC-CMs would migrate in response to the extracellular matrix and soluble signaling molecules previously implicated in heart morphogenesis. To test this, we screened candidate factors by transwell assay for effects on hESC-CM motility, followed by validation via live-cell imaging and/or gap-closure assays. Fibronectin (FN) elicited a haptotactic response from hESC-CMs, with cells seeded on a steep FN gradient showing nearly a fivefold greater migratory activity than cells on uniform FN. Studies with neutralizing antibodies indicated that adhesion and migration on FN are mediated by integrins α-5 and α-V. Next, we screened 10 soluble candidate factors by transwell assay and found that the noncanonical Wnt, Wnt5a, elicited an approximately twofold increase in migration over controls. This effect was confirmed using the gap-closure assay, in which Wnt5a-treated hESC-CMs showed approximately twofold greater closure than untreated cells. Studies with microfluidic-generated Wnt5a gradients showed that this factor was chemoattractive as well as chemokinetic, and Wnt5a-mediated responses were inhibited by the Frizzled-1/2 receptor antagonist, UM206. In summary, hESC-CMs show robust promigratory responses to FN and Wnt5a, findings that have implications on both cardiac development and cell-based therapies.

Abstract 4339: RGD-conjugated nanoparticles for targeted inhibition of adhesion and migration of integrin αvβ3-overexpressing breast cancer cells.

Abstract Background: Tumor cell adhesion and migration are critical in the establishment of metastasis. This study aims to develop a RGD-conjugated nanoparticle system that is able to target αvβ3 integrin receptor-overexpressing breast tumor cells while evading the liver uptake, thus enhancing treatment of cancer metastasis through inhibition of adhesion and migration of cancer cells. Methods: The nanoparticle formulation was first optimized with a suitable concentration of RGD targeting moiety on the nanoparticle surface to maximize nanoparticle accumulation in tumor and minimize liver uptake. RGD-conjugated solid lipid nanoparticles (RGD-SLNs) were synthesized with a fatty acid lipid core and a poly(ethylene glycol) corona and decorated with varying concentrations of RGD peptides (0%, 0.5%, 1%, 5% and 10% mol/mol ratio). The quantity and kinetics of the nanoparticle binding to αvβ3 integrin receptor and in vitro cellular uptake on αvβ3 positive MDA-MB-231 cells were investigated by fluorescence microscopy and compared with the results with αvβ3 negative MCF-7 cells. The influence of RGD-SLNs on cell adhesion and migration via binding to αvβ3 integrin receptor was examined using standard adhesion and transwell migration assays. Whole animal biodistribution, tumor uptake and intratumoral distribution of quantum dot-loaded nanoparticles were investigated to identify optimized RGD-concentrations. Results: RGD-SLNs showed specific binding for αvβ3 integrin receptors on MDA-MB-231 cells as compared to the negative control (MCF-7 cells). The RGD-SLN formed clusters at the cellular membrane and then entered the cells at a lower pace than SLN. The RGD-SLNs also exhibited higher cellular uptake compared to SLN in MDA-MB-231 cells while there was no detectable difference in uptake in αvβ3 negative MCF-7 cells. RGD concentration of 1% on the SLN surface was found to have most tumor retention and low liver uptake among all formulations. Receptor mediated uptake of RGD-SLNs reduced cell adhesion and migration towards fibronectin gradient, attributable to the occupation of the receptors by the RGD-SLN and slow recycling of the receptors to the cell surface. Conclusions: We have optimized the RGD-SLN formulation to maximize tumor accumulation and minimize liver uptake. The RGD-SLN formulation has the potential to prevent metastasis through interference with cell adhesion and migration. Citation Format: Dan Shan, Ping Cai, Preethy Prasad, Andrew Michael Rauth, Xiao Yu Wu. RGD-conjugated nanoparticles for targeted inhibition of adhesion and migration of integrin αvβ3-overexpressing breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4339. doi:10.1158/1538-7445.AM2013-4339

Abstract C41: MenaINV dysregulation of receptor tyrosine kinase signaling

Abstract Mena, an actin regulatory protein, is upregulated in human breast cancer and its expression is associated with increased metastatic progression. An invasion-specific isoform, MenaINV, is upregulated selectively in invasive tumor cells and increases invasion and metastasis by potentiating tumor cell responses to EGF. MenaINV causes dysregulation of the tyrosine phosphatase PTP1B, leading to increased phosphorylation of EGFR and other downstream targets. We investigated whether MenaINV could similarly promote invasion through dysregulation of signaling downstream of other receptor tyrosine kinases (RTKs). The behavior of MDA-MB-231 triple negative breast cancer cells stably expressing MenaINV or the other cannonical, broadly expressed Mena lacking the “INV” exon was measured after growth factor treatment in two migration assays: a 2D protrusion assay on matrigel and collagen coated coverslips and a 3D invasion assay into collagen gels. We found that expression of MenaINV sensitized responses to other EGFR ligands including HB-EGF and amphiregulin. In addition, MenaINV expression enhanced responses mediated by two other RTKs associated with tumor progression, Met and IGFR, both known PTP1B substrates. Cells expressing MenaINV respond to 20-fold lower concentrations HGF and IGF than control cells. However, there was no effect of MenaINV on responses to the ERBB3 ligand neuregulin and the chemokine SDF-1. Recently we reported that Mena can regulate cell motility in fibroblasts via its interaction with α5β1 integrin, which is known to co-traffic with EGFR and other RTKs to regulate invasion in tumor cells expressing mutant p53 or upon inhibition of αvβ3. We asked whether the direct interaction of Mena with α5β1 also contributes to invasion. Disrupting the interaction between the Mena isoforms and α5β1 in MDA-MB-231 cells affected how cells responded to EGF in a 3D collagen gel. In addition, fibroblasts that were null for Mena were unable to respond to a fibronectin gradient in a haptotaxis assay. Overall, these results suggest that the effects of MenaINV on invasion and metastasis could be mediated via different RTKs, as well as through direct interaction with integrins and the extracellular matrix. Experiments are currently underway to investigate how MenaINV regulates signalling downstream of these RTKs and integrins, to understand the molecular mechanism underlying the pro-metastatic effect of MenaINV. Citation Format: Madeleine J. Oudin, Shannon K. Hughes-Alford, Miles Miller, Sreeja B. Asokan, James E. Bear, Doug A. Lauffenburger, Frank B. Gertler. MenaINV dysregulation of receptor tyrosine kinase signaling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C41.

Cell Adhesion on Micro-Structured Fibronectin Gradients Fabricated by Multiphoton Excited Photochemistry.

Concentration gradients of ECM proteins play active roles in many areas of cell biology including wound healing and metastasis. They may also form the basis of tissue engineering scaffolds, as these can direct cell adhesion and migration and promote new matrix synthesis. To better understand cell-matrix interactions on attractive gradients, we have used multiphoton excited (MPE) photochemistry to fabricate covalently linked micro-structured gradients from fibronectin (FN). The gradient design is comprised of a parallel series of individual linear gradients with overall dimensions of approximately 800 × 800 μm, where a linear dynamic range of nearly 10-fold in concentration was achieved. The adhesion dynamics of 3T3 fibroblasts were investigated, where the cell morphology and actin cytoskeleton became increasingly elongated and aligned with the direction of the gradient at increasing protein concentration. Moreover, the cell morphologies are distinct when adhered to regions of differing FN concentration but with similar topography. These results show that the fabrication approach allows investigating the roles of contact guidance and ECM cues on the cell-matrix interactions. We suggest this design overcomes some of the limitations with other fabrication methods, especially in terms of 3D patterning capabilities, and will serve as a new tool to study cell-matrix interactions.