Lauren E. Barney

Thermal-Responsive Behavior of a Cell Compatible Chitosan/Pectin Hydrogel

Biopolymer hydrogels are important materials for wound healing and cell culture applications. While current synthetic polymer hydrogels have excellent biocompatibility and are nontoxic, they typically function as a passive matrix that does not supply any additional bioactivity. Chitosan (CS) and pectin (Pec) are natural polymers with active properties that are desirable for wound healing. Unfortunately, the synthesis of CS/Pec materials have previously been limited by harsh acidic synthesis conditions, which further restricted their use in biomedical applications. In this study, a zero-acid hydrogel has been synthesized from a mixture of chitosan and pectin at biologically compatible conditions. For the first time, we demonstrated that salt could be used to suppress long-range electrostatic interactions to generate a thermoreversible biopolymer hydrogel that has temperature-sensitive gelation. Both the hydrogel and the solution phases are highly elastic, with a power law index of close to -1. When dried hydrogels were placed into phosphate buffered saline solution, they rapidly rehydrated and swelled to incorporate 2.7× their weight. As a proof of concept, we removed the salt from our CS/Pec hydrogels, thus, creating thick and easy to cast polyelectrolyte complex hydrogels, which proved to be compatible with human marrow-derived stem cells. We suggest that our development of an acid-free CS/Pec hydrogel system that has excellent exudate uptake, holds potential for wound healing bandages.

Comparative Study of Multicellular Tumor Spheroid Formation Methods and Implications for Drug Screening

Improved in vitro models are needed to better understand cancer progression and bridge the gap between in vitro proof-of-concept studies, in vivo validation, and clinical application. Multicellular tumor spheroids (MCTS) are a popular method for three-dimensional (3D) cell culture, because they capture some aspects of the dimensionality, cell-cell contact, and cell-matrix interactions seen in vivo. Many approaches exist to create MCTS from cell lines, and they have been used to study tumor cell invasion, growth, and how cells respond to drugs in physiologically relevant 3D microenvironments. However, there are several discrepancies in the observations made of cell behaviors when comparing between MCTS formation methods. To resolve these inconsistencies, we created and compared the behavior of breast, prostate, and ovarian cancer cells across three MCTS formation methods: in polyNIPAAM gels, in microwells, or in suspension culture. These methods formed MCTS via proliferation from single cells or passive aggregation, and therefore showed differential reliance on genes important for cell-cell or cell-matrix interactions. We also found that the MCTS formation method dictated drug sensitivity, where MCTS formed over longer periods of time via clonal growth were more resistant to treatment. Toward clinical application, we compared an ovarian cancer cell line MCTS formed in polyNIPAAM with cells from patient-derived malignant ascites. The method that relied on clonal growth (PolyNIPAAM gel) was more time and cost intensive, but yielded MCTS that were uniformly spherical, and exhibited the most reproducible drug responses. Conversely, MCTS methods that relied on aggregation were faster, but yielded MCTS with grapelike, lobular structures. These three MCTS formation methods differed in culture time requirements and complexity, and had distinct drug response profiles, suggesting the choice of MCTS formation method should be carefully chosen based on the application required.

Acetyl-CoA promotes glioblastoma cell adhesion and migration through Ca2+–NFAT signaling

The metabolite acetyl-coenzyme A (acetyl-CoA) is the required acetyl donor for lysine acetylation and thereby links metabolism, signaling, and epigenetics. Nutrient availability alters acetyl-CoA levels in cancer cells, correlating with changes in global histone acetylation and gene expression. However, the specific molecular mechanisms through which acetyl-CoA production impacts gene expression and its functional roles in promoting malignant phenotypes are poorly understood. Here, using histone H3 Lys27 acetylation (H3K27ac) ChIP-seq (chromatin immunoprecipitation [ChIP] coupled with next-generation sequencing) with normalization to an exogenous reference genome (ChIP-Rx), we found that changes in acetyl-CoA abundance trigger site-specific regulation of H3K27ac, correlating with gene expression as opposed to uniformly modulating this mark at all genes. Genes involved in integrin signaling and cell adhesion were identified as acetyl-CoA-responsive in glioblastoma cells, and we demonstrate that ATP citrate lyase (ACLY)-dependent acetyl-CoA production promotes cell migration and adhesion to the extracellular matrix. Mechanistically, the transcription factor NFAT1 (nuclear factor of activated T cells 1) was found to mediate acetyl-CoA-dependent gene regulation and cell adhesion. This occurs through modulation of Ca2+ signals, triggering NFAT1 nuclear translocation when acetyl-CoA is abundant. The findings of this study thus establish that acetyl-CoA impacts H3K27ac at specific loci, correlating with gene expression, and that expression of cell adhesion genes are driven by acetyl-CoA in part through activation of Ca2+-NFAT signaling.

Mechanosensing of Integrin α6 and EGFR Converges at Calpain 2

Cells sense and respond to mechanical cues from the extracellular matrix (ECM) via integrins. ECM stiffening is known to increase integrin clustering and sensitivity to epidermal growth factor (EGF), but we lack information on when or if these mechanosensitive growth factor receptors and integrins converge intracellularly. Towards closing this knowledge gap, we combined a biomaterial platform with transcriptomics, molecular biology, and functional assays to link integrin-mediated mechanosensing and epidermal growth factor receptor (EGFR) signaling. We found that high integrin α6 expression controlled breast cancer cell adhesion and motility on soft, laminin-coated substrates, and this mimicked the response of cells to EGF stimulation. The mechanisms that drove both mechanosensitive cell adhesion and motility converged on calpain 2, an intracellular protease important for talin cleavage and focal adhesion turnover. EGF stimulation enhanced adhesion and motility on soft substrates, but required integrin α6 and calpain 2 signaling. In sum, we identified a new role for integrin α6 mechanosensing in breast cancer, wherein cell adhesion to laminin on soft substrates mimicked EGF stimulation. We identified calpain 2, downstream of both integrin α6 engagement and EGFR phosphorylation, as a common intracellular signaling node, and implicate integrin α6 and calpain 2 as potential targets to inhibit the migration of cancer cells in stiff tumor environments.Cells sense and respond to mechanical cues from the extracellular matrix (ECM) via integrins. ECM stiffness is known to enhance integrin clustering and response to epidermal growth factor (EGF), but we lack information on when or if these mechanosensitive receptors converge intracellularly. Towards closing this knowledge gap, we combined a biomaterial platform with transcriptomics, molecular biology, and functional assays to link integrin-mediated mechanosensing and epidermal growth factor receptor (EGFR) signaling. We found that high integrin α6 expression controlled cell adhesion and motility on soft, laminin-coated substrates, and this mimicked the response of cells to EGF stimulation. Signaling pathways downstream of mechanosensitive cell adhesion and motility converged on calpain 2, an intracellular protease important for talin cleavage and focal adhesion turnover. Inhibiting calpain 2 shifted the biphasic dependence of cell migration on substrate stiffness. EGF stimulation enhanced both adhesion and motility on soft substrates, but required the presence of both integrin α6 and calpain 2. In sum, we identified a new role for integrin α6 mechanosensing, where high integrin α6 binding to laminin mimicked the effect of EGF stimulation. Downstream of both integrin α6 and EGFR, calpain 2 is known to control focal adhesion dynamics and motility, implicating integrin α6 and calpain 2 as potential targets to inhibit the migration of cancer cells in stiff tumor environments.

Integrin α6 and EGFR signaling converge at mechanosensitive calpain 2

Cells sense and respond to mechanical cues from the extracellular matrix (ECM) via integrins. ECM stiffness is known to enhance integrin clustering and response to epidermal growth factor (EGF), but we lack information on when or if these mechanosensitive growth factor receptors and integrins converge intracellularly. Towards closing this knowledge gap, we combined a biomaterial platform with transcriptomics, molecular biology, and functional assays to link integrin-mediated mechanosensing and epidermal growth factor receptor (EGFR) signaling. We found that high integrin α6 expression controlled breast cancer cell adhesion and motility on soft, laminin-coated substrates, and this mimicked the response of cells to EGF stimulation. The mechanisms that drove both mechanosensitive cell adhesion and motility converged on calpain 2, an intracellular protease important for talin cleavage and focal adhesion turnover. EGF stimulation enhanced adhesion and motility on soft substrates, but required integrin α6 and calpain 2 signaling. In sum, we identified a new role for integrin α6 mechanosensing in breast cancer, wherein cell adhesion to laminin on soft substrates mimicked EGF stimulation. We identified calpain 2, downstream of both integrin α6 engagement and EGFR phosphorylation, as a common intracellular signaling node, and implicate integrin α6 and calpain 2 as potential targets to inhibit the migration of cancer cells in stiff tumor environments.

Anomalously Diffusing and Persistently Migrating Cells in 2D and 3D Culture Environments

Appropriately chosen descriptive models of cell migration in biomaterials will allow researchers to characterize and ultimately predict the movement of cells in engineered systems for a variety of applications in tissue engineering. The persistent random walk (PRW) model accurately describes cell migration on two-dimensional (2D) substrates. However, this model inherently cannot describe subdiffusive cell movement, i.e., migration paths in which the root mean square displacement increases more slowly than the square root of the time interval. Subdiffusivity is a common characteristic of cells moving in confined environments, such as three-dimensional (3D) porous scaffolds, hydrogel networks, and in vivo tissues. We demonstrate that a generalized anomalous diffusion (AD) model, which uses a simple power law to relate the mean square displacement to time, more accurately captures individual cell migration paths across a range of engineered 2D and 3D environments than does the more commonly used PRW model. We used the AD model parameters to distinguish cell movement profiles on substrates with different chemokinetic factors, geometries (2D vs 3D), substrate adhesivities, and compliances. Although the two models performed with equal precision for superdiffusive cells, we suggest a simple AD model, in lieu of PRW, to describe cell trajectories in populations with a significant subdiffusive fraction, such as cells in confined, 3D environments.

Anomalous Diffusion as a Descriptive Model of Cell Migration

Appropriately chosen descriptive models of cell migration in biomaterials will allow researchers to characterize and ultimately predict the movement of cells in engineered systems for a variety of applications in tissue engineering. The persistent random walk (PRW) model accurately describes cell migration on two-dimensional (2D) substrates. However, this model inherently cannot describe subdiffusive cell movement, i.e. migration paths in which the root mean square displacement increases more slowly than the square root of the time interval. Subdiffusivity is a common characteristic of cells moving in confined environments, such as three-dimensional (3D) porous scaffolds, hydrogel networks, and in vivo tissues. We demonstrate that a generalized anomalous diffusion (AD) model, which uses a simple power law to relate the mean square displacement (MSD) to time, more accurately captures individual cell migration paths across a range of engineered 2D and 3D environments than does the more commonly used PRW model. We used the AD model parameters to distinguish cell movement profiles on substrates with different chemokinetic factors, geometries (2D vs 3D), substrate adhesivities, and compliances. Although the two models performed with equal precision for superdiffusive cells, we suggest a simple AD model, in lieu of PRW, to describe cell trajectories in populations with a significant subdiffusive fraction, such as cells in confined, 3D environments.

Abstract PR14: Extracellular matrix control of metastasis and dormancy

Abstracts: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Introduction: Pagets classical seed and soil hypothesis states that specific interactions between the metastatic cell (the seed) and the recipient tissue microenvironment (the soil) mediate the non-random patterns of spread observed in breast cancer (tropism). We hypothesize that the ability of metastatic cells to survive dormancy, exit quiescence, and colonize a specific tissue depends upon the ability of the soil to first sustain survival, and subsequently trigger outgrowth. To this end, we created a biomaterial platform that varies extracellular matrix (ECM) density and composition, and used integrin-mediated phenotyping to study how highly metastatic cells differentiate between secondary sites via integrin binding. With this relationship in hand, we have found that both the ECM and growth factors also mediate dormancy in cells that display metastatic latency in vivo. Methods: ECM microenvironments were created by linking combinations of ECM proteins to glass coverslips using silane chemistry. Human breast cancer cell lines were seeded onto the surfaces, and we quantified initial cell adhesion and long-term migration via microscopy. Using cell lines that only metastasize to the bone, brain, or lung (provided by J. Massague), we created fingerprints of behavior characteristic of each tropism, and compared these with the phenotypes of other cell lines with known metastasis. Quiescence was induced on ECMs via culture in serum free medium for up to 14 days, and subsequent serum-stimulation for allowed for recovery and growth. Proliferation was assayed via immunofluorescent staining for Ki67, and cell number was determined via DAPI staining. Results: We identified unique phenotypes of bone, brain, and lung tropic subpopulations, and confirmed these in seven of nine other known, more heterogeneous, metastatic cell lines, establishing a connection between in vitro phenotype and in vivo fate. Function-affecting antibodies to integrins each shift a cell lines predicted tropism in vitro, and these in vitro phenotyping results mimic the clinical metastasis patterns dictated by both α2 and α6 integrin gene expression. Interestingly, however, our functional predictions regarding β1 integrin actually provide insights that are largely independent of clinical gene expression, potentially identifying new metastatic therapeutic targets, and emphasizing the need for this type of functional analysis. However, when we applied this approach to cell lines that display latency in vivo (e.g., ZR-75-1), our predictions were not correct. These cells were generally insensitive to ECM and growth factors in adhesion and motility, but instead, we found that these factors regulate entrance into dormancy. We uniformly observe that a laminin-coupled ECM is unable to permit survival of dormant cells, whereas binding to collagen I maintains cell survival for at least 14 days. Epidermal growth factor supplementation improves the entrance into dormancy on collagen alone. We have found that this collagen-specific, EGF-enhanced survival ability is mediated by β1 integrin binding and activation of ERK1/2, suggesting that β1 integrin function is critical both for aggressive metastasis and for permitting dormancy. Conclusions: We are the first to report that bone, brain, and lung tropism can be distinguished via simple integrin-mediated phenotyping, and that this approach can be used to predict in vivo tropism. We suggest that functional β1 integrin binding mediates both aggressive and latent metastasis. In sum, we propose that this functional screen of cell-matrix interactions can predict in vivo outcomes, and thus, is a valuable tool to provide insight toward integrins as druggable targets for metastatic disease. Citation Format: Lauren E. Barney, Ari Gilman, Arthur Mercurio, Shelly R. Peyton. Extracellular matrix control of metastasis and dormancy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR14.

Integrin binding uniquely regulates tropic breast cancer cell phenotypes in vitro

Breast cancer preferentially spreads to the bone, brain, liver, and lung, and this tissue-specific spread (tropism) is not well understood. The diversity of tissue sites often recipient of metastatic colonization suggests a role for the extracellular matrix (ECM) in tropism. We created a biomaterial platform that varies ECM type and composition, inspired by the bone, brain, and lung tissues, in order to study integrin-mediated adhesion and motility phenotypes. We report that bone, brain, and lung tropic breast cancer cells respond to ECM variations uniquely, implicating integrin binding in tropism. We confirmed these phenotypes in other metastatic cell lines, establishing a connection between in vitro phenotype and in vivo fate. We report that integrin-targeting alters the tropic phenotype, and provide insight toward integrins as druggable targets for metastatic disease. In sum, we emphasize the importance of the ECM and engineered microenvironments in order to understand breast cancer spread.