Extracellular Matrix Cues Research Articles

Directing hMSCs fate through geometrical cues and mimetics peptides.

The native microenvironment of mesenchymal stem cells (hMSCs)-the extracellular matrix (ECM), is a complex and heterogenous environment structured at different scales. The present study aims at mimicking the hierarchical microorganization of proteins or growth factors within the ECM using the photolithography technique. Polyethylene terephthalate substrates were used as a model material to geometrically defined regions of RGD + BMP-2 or RDG + OGP mimetic peptides. These ECM-derived ligands are under research for regulation of mesenchymal stem cells osteogenic differentiation in a synergic manner. The hMSCs osteogenic differentiation was significantly affected by the spatial distribution of dually grafted peptides on surfaces, and hMSCs cells reacted differently according to the shape and size of peptide micropatterns. Our study demonstrates the presence of a strong interplay between peptide geometric cues and stem cell differentiation toward the osteoblastic lineage. These tethered surfaces provide valuable tools to investigate stem cell fate mechanisms regulated by multiple ECM cues, thereby contributing to the design of new biomaterials and improving hMSCs differentiation cues.

3D extracellular matrix microenvironment in bioengineered tissue models of primary pediatric and adult brain tumors

Dynamic alterations in the unique brain extracellular matrix (ECM) are involved in malignant brain tumors. Yet studies of brain ECM roles in tumor cell behavior have been difficult due to lack of access to the human brain. We present a tunable 3D bioengineered brain tissue platform by integrating microenvironmental cues of native brain-derived ECMs and live imaging to systematically evaluate patient-derived brain tumor responses. Using pediatric ependymoma and adult glioblastoma as examples, the 3D brain ECM-containing microenvironment with a balance of cell-cell and cell-matrix interactions supports distinctive phenotypes associated with tumor type-specific and ECM-dependent patterns in the tumor cells’ transcriptomic and release profiles. Label-free metabolic imaging of the composite model structure identifies metabolically distinct sub-populations within a tumor type and captures extracellular lipid-containing droplets with potential implications in drug response. The versatile bioengineered 3D tumor tissue system sets the stage for mechanistic studies deciphering microenvironmental role in brain tumor progression.

Cancer Cells Sense Fibers by Coiling on them in a Curvature-Dependent Manner.

SummaryMetastatic cancer cells sense the complex and heterogeneous fibrous extracellular matrix (ECM) by formation of protrusions, and our knowledge of how cells physically recognize these fibers remains in its infancy. Here, using suspended ECM-mimicking isodiameter fibers ranging from 135 to 1,000 nm, we show that metastatic breast cancer cells sense fiber diameters differentially by coiling (wrapping-around) on them in a curvature-dependent manner, whereas non-tumorigenic cells exhibit diminished coiling. We report that coiling occurs at the tip of growing protrusions and the coil width and coiling rate increase in a curvature-dependent manner, but time to maximum coil width occurs biphasically. Interestingly, bundles of 135-nm diameter fibers recover coiling width and rate on 1,000-nm-diameter fibers. Coiling also coincides with curvature-dependent persistent and ballistic transport of endogenous granules inside the protrusions. Altogether, our results lay the groundwork to link biophysical sensing with biological signaling to quantitate pro- and anti-invasive fibrous environments.Video

Optimizing the alignment of thermoresponsive poly(N-isopropyl acrylamide) electrospun nanofibers for tissue engineering applications: A factorial design of experiments approach.

Thermoresponsive polymers, such as poly(N-isopropyl acrylamide) (PNIPAM), have been identified and used as cell culture substrates, taking advantage of the polymer’s lower critical solution temperature (LCST) to mechanically harvest cells. This technology bypasses the use of biochemical enzymes that cleave important cell-cell and cell-matrix interactions. In this study, the process of electrospinning is used to fabricate and characterize aligned PNIPAM nanofiber scaffolds that are biocompatible and thermoresponsive. Nanofiber scaffolds produced by electrospinning possess a 3D architecture that mimics native extracellular matrix, providing physical and chemical cues to drive cell function and phenotype. We present a factorial design of experiments (DOE) approach to systematically determine the effects of different electrospinning process parameters on PNIPAM nanofiber diameter and alignment. Results show that high molecular weight PNIPAM can be successfully electrospun into both random and uniaxially aligned nanofiber mats with similar fiber diameters by simply altering the speed of the rotating mandrel collector from 10,000 to 33,000 RPM. PNIPAM nanofibers were crosslinked with OpePOSS, which was verified using FTIR. The mechanical properties of the scaffolds were characterized using dynamic mechanical analysis, revealing an order of magnitude difference in storage modulus (MPa) between cured and uncured samples. In summary, cross-linked PNIPAM nanofiber scaffolds were determined to be stable in aqueous culture, biocompatible, and thermoresponsive, enabling their use in diverse cell culture applications.

Surface Micro- and Nanoengineering: Applications of Layer-by-Layer Technology as a Versatile Tool to Control Cellular Behavior.

Extracellular matrix (ECM) cues have been widely investigated for their impact on cellular behavior. Among mechanics, physics, chemistry, and topography, different ECM properties have been discovered as important parameters to modulate cell functions, activating mechanotransduction pathways that can influence gene expression, proliferation or even differentiation. Particularly, ECM topography has been gaining more and more interest based on the evidence that these physical cues can tailor cell behavior. Here, an overview of bottom-up and top-down approaches reported to produce materials capable of mimicking the ECM topography and being applied for biomedical purposes is provided. Moreover, the increasing motivation of using the layer-by-layer (LbL) technique to reproduce these topographical cues is highlighted. LbL assembly is a versatile methodology used to coat materials with a nanoscale fidelity to the geometry of the template or to produce multilayer thin films composed of polymers, proteins, colloids, or even cells. Different geometries, sizes, or shapes on surface topography can imply different behaviors: effects on the cell adhesion, proliferation, morphology, alignment, migration, gene expression, and even differentiation are considered. Finally, the importance of LbL assembly to produce defined topographical cues on materials is discussed, highlighting the potential of micro- and nanoengineered materials to modulate cell function and fate.

Oxidized alginate hydrogels with the GHK peptide enhance cord blood mesenchymal stem cell osteogenesis: A paradigm for metabolomics-based evaluation of biomaterial design

Oxidized alginate hydrogels are appealing alternatives to natural alginate due to their favourable biodegradability profiles and capacity to self-crosslink with amine containing molecules facilitating functionalization with extracellular matrix cues, which enable modulation of stem cell fate, achieve highly viable 3-D cultures, and promote cell growth. Stem cell metabolism is at the core of cellular fate (proliferation, differentiation, death) and metabolomics provides global metabolic signatures representative of cellular status, being able to accurately identify the quality of stem cell differentiation. Herein, umbilical cord blood mesenchymal stem cells (UCB MSCs) were encapsulated in novel oxidized alginate hydrogels functionalized with the glycine-histidine-lysine (GHK) peptide and differentiated towards the osteoblastic lineage. The ADA-GHK hydrogels significantly improved osteogenic differentiation compared to gelatin-containing control hydrogels, as demonstrated by gene expression, alkaline phosphatase activity and bone extracellular matrix deposition. Metabolomics revealed the high degree of metabolic heterogeneity in the gelatin-containing control hydrogels, captured the enhanced osteogenic differentiation in the ADA-GHK hydrogels, confirmed the similar metabolism between differentiated cells and primary osteoblasts, and elucidated the metabolic mechanism responsible for the function of GHK. Our results suggest a novel paradigm for metabolomics-guided biomaterial design and robust stem cell bioprocessing. Statement of SignificanceProducing high quality engineered bone grafts is important for the treatment of critical sized bone defects. Robust and sensitive techniques are required for quality assessment of tissue-engineered constructs, which result to the selection of optimal biomaterials for bone graft development. Herein, we present a new use of metabolomics signatures in guiding the development of novel oxidised alginate-based hydrogels with umbilical cord blood mesenchymal stem cells and the glycine-histidine-lysine peptide, demonstrating that GHK induces stem cell osteogenic differentiation. Metabolomics signatures captured the enhanced osteogenesis in GHK hydrogels, confirmed the metabolic similarity between differentiated cells and primary osteoblasts, and elucidated the metabolic mechanism responsible for the function of GHK. In conclusion, our results suggest a new paradigm of metabolomics-driven design of biomaterials.

Synergy of Paracrine Signaling During Early-Stage Mouse Ovarian Follicle Development In Vitro.

Paracrine signals, such as soluble cytokines and extracellular matrix cues, are essential for the survival and development of multicellular ovarian follicles. While it is well established that hydrogel-based culture systems successfully support the growth of late-stage follicles for fertility preservation, growing small, early-stage ovarian follicles still proves to be challenging. We hypothesized that paracrine factors secreted from neighboring follicles may be crucial for improving the survival of early-stage follicles in vitro. To test our hypothesis, we investigated the bi-directional crosstalk of the paracrine signals, such as cell-secreted cytokines, sex hormones and transcription factors (TFs), in follicles encapsulated and cultured for 12days in alginate in groups of five (5×) and ten (10×). The differential profiles of TF activity and secretome during folliculogenesis were analyzed using TRanscriptional Activity CEllular aRray (TRACER) and data-driven multivariate modeling approach. The mechano- and oxygen-responsive TFs, NF-κB and HIF1, exhibited a unique upregulation signature in 10× follicles. Consistently, levels of proangiogenic factors, such as VEGF-A and angiopoietin-2, were significantly higher in 10× follicles than those in 5× follicles, reaching 269.77 and 242.82pg/mL on the last day of culture. The analysis of TRACER and secreted cytokines also revealed critical early interactions between cytokines and TFs, correlating with the observed phenotypical and functional differences between conditions. We identified unique signatures of synergism during successful early-stage ovarian follicle development. These findings bring us closer to understanding of mechanisms underlying the downstream effects of interactions between the extracellular microenvironment and early-stage folliculogenesis in vitro.

3081 - Geometrical Control of Stem Cell Fate by Self-Organization of Cytoskeleton

The commitment of mesenchymal stem cells (MSCs) differentiation lineage can be controlled by physical signals, such as geometric cues of extracellular matrix (ECM). It is well known that the physical stimuli drive focal adhesions (FAs) maturation, actin polymerization and stress fiber formation, yet how cell shape controls MSC differentiation through the FA-mediated signals or FA-cytoskeleton organization remains largely unknown. To determine how geometric cues direct MSC lineage commitment, a technique of microcontact printing is performed to shape MSCs. We find that the geometric cues control osteogenic-adipogenic switch in MSC lineage commitment through regulating the organization of FAs, actin filaments, intermediate filaments, microtubules and nucleus, which further influences the distribution of cellular visco-elasticity and traction forces. Our data demonstrate a role for FA signaling in dictating MSC differentiation regulated by geometric cues.

Tissue engineered bone mimetics to study bone disorders ex vivo: Role of bioinspired materials

Recent advances in materials development and tissue engineering has resulted in a substantial number of bioinspired materials that recapitulate cardinal features of bone extracellular matrix (ECM) such as dynamic inorganic and organic environment(s), hierarchical organization, and topographical features. Bone mimicking materials, as defined by its self-explanatory term, are developed based on the current understandings of the natural bone ECM during development, remodeling, and fracture repair. Compared to conventional plastic cultures, biomaterials that resemble some aspects of the native environment could elicit a more natural molecular and cellular response relevant to the bone tissue. Although current bioinspired materials are mainly developed to assist tissue repair or engineer bone tissues, such materials could nevertheless be applied to model various skeletal diseases in vitro. This review summarizes the use of bioinspired materials for bone tissue engineering, and their potential to model diseases of bone development and remodeling ex vivo. We largely focus on biomaterials, designed to re-create different aspects of the chemical and physical cues of native bone ECM. Employing these bone-inspired materials and tissue engineered bone surrogates to study bone diseases has tremendous potential and will provide a closer portrayal of disease progression and maintenance, both at the cellular and tissue level. We also briefly touch upon the application of patient-derived stem cells and introduce emerging technologies such as organ-on-chip in disease modeling. Faithful recapitulation of disease pathologies will not only offer novel insights into diseases, but also lead to enabling technologies for drug discovery and new approaches for cell-based therapies.

Complementary, Semiautomated Methods for Creating Multidimensional PEG-Based Biomaterials.

Tunable biomaterials that mimic selected features of the extracellular matrix (ECM) such as its stiffness, protein composition, and dimensionality are increasingly popular for studying how cells sense and respond to ECM cues. In the field, there exists a significant trade-off for how complex and how well these biomaterials represent the in vivo microenvironment versus how easy they are to make and how adaptable they are to automated fabrication techniques. To address this need to integrate more complex biomaterials design with high-throughput screening approaches, we present several methods to fabricate synthetic biomaterials in 96-well plates and demonstrate that they can be adapted to semiautomated liquid handling robotics. These platforms include (1) glass bottom plates with covalently attached ECM proteins and (2) hydrogels with tunable stiffness and protein composition with either cells seeded on the surface or (3) laden within the three-dimensional hydrogel matrix. This study includes proof-of-concept results demonstrating control over breast cancer cell line phenotypes via these ECM cues in a semiautomated fashion. We foresee the use of these methods as a mechanism to bridge the gap between high-throughput cell-matrix screening and engineered ECM-mimicking biomaterials.

C-Src activity is differentially required by cancer cell motility modes

Cancer cell migration requires that cells respond and adapt to their surroundings. In the absence of extracellular matrix cues, cancer cells will undergo a mesenchymal to ameboid transition, whereas a highly confining space will trigger a switch to “leader bleb-based” migration. To identify oncogenic signaling pathways mediating these transitions, we undertook a targeted screen using clinically useful inhibitors. Elevated Src activity was found to change actin and focal adhesion dynamics, whereas inhibiting Src triggered focal adhesion disassembly and blebbing. On non-adherent substrates and in collagen matrices, amoeboid-like, blebbing cells having high Src activity formed protrusions of the plasma membrane. To evaluate the role of Src in confined cells, we use a novel approach that places cells under a slab of polydimethylsiloxane (PDMS), which is held at a defined height. Using this method, we find that leader bleb-based migration is resistant to Src inhibition. High Src activity was found to markedly change the architecture of cortical actomyosin, reduce cell mechanical properties, and the percentage of cells that undergo leader bleb-based migration. Thus, Src is a signal transducer that can potently influence transitions between migration modes with implications for the rational development of metastasis inhibitors.

Double-exclusive liquid repellency (double-ELR): an enabling technology for rare phenotype analysis.

Double-exclusive liquid repellency (double-ELR) is an extreme wettability phenomenon in which adjacent regions selectively and completely repel immiscible liquids with different surface chemistries on a non-textured substrate (i.e., a substrate in absence of micro/nano-structures). Under double-ELR conditions, each liquid exhibits no physical contact (contact angle of 180°) with its non-preferred surface chemistry, thus enabling complete partitioning of adjacent fluidic volumes (e.g., between water and oil). This enables a new type of cell culture-based assay, where cell loss from common failure modes (e.g., biofouling from inadvertent cell adhesion, detrimental moisture loss/gain, and liquid handling dead volumes) is significantly mitigated. Importantly, the principles of double-ELR were leveraged to achieve underoil sweep patterning, a no-loss, robust and high-throughput distribution of sub-microliter volumes of aqueous media (and cells). In addition to high-efficiency distribution via sweep patterning, double-ELR can be used to construct "modular" (i.e., easily implemented and/or linked together with spatial and temporal control) higher-order architectures for in vitro imitation of physiologically relevant microenvironments that are of particular interest within the cell assay community, including multi-phenotype cultures with excellent spatial and temporal control, three-dimensional layered multi-phenotype cultures, cultures with selective mechanical cues of extracellular matrix (i.e., collagen fiber alignment), and spheroid cultures. Together, these features of double-ELR uniquely facilitate culture and high content analysis of limited cellular samples (e.g., a few hundred to a few thousand cells).

Collagen vitrigels with low-fibril density enhance human embryonic stem cell-derived retinal pigment epithelial cell maturation.

Structural and biochemical cues of extracellular matrix can substantially influence the differentiation and maturation of cultured retinal pigment epithelial (RPE) cells. In this study, thin collagen vitrigels were engineered to create collagen nanofibrillar structures of different fibril densities in an effort to evaluate the maturation of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells. The ultrastructure of the different collagen vitrigels was characterized by transmission electron microscopy, and the mechanical properties were evaluated by tensile testing. The pigmentation and polarization of cells, in addition to key RPE marker gene and protein expression levels, were analyzed to determine the differentiation of hESCs on the gels. The hESC-RPE differentiation was most significant in collagen vitrigels with low fibril density with mature collagen fibrils with diameter of around 60nm and Young's modulus of 2.41±0.13MPa. This study provides insight into the influence of collagen nanofibrillar structures on hESC-RPE maturation and presents a potential bioengineered substratum for hESC-RPE for future preclinical and clinical applications.

Influence of hippocampal niche signals on neural stem cell functions during aging.

The genesis of new neurons from neural stem cells in the adult brain offers the hope that this mechanism of plasticity can be harnessed for the treatment of brain injuries and diseases. However, neurogenesis becomes impaired during the normal course of aging; this is also the primary risk factor for most neurodegenerative diseases. The local microenvironment that regulates the function of resident neural stem cells (the "neurogenic niche") is a particularly complex network of various signaling mechanisms, rendering it especially challenging for the dissection of the control of these cells but offering the potential for the advancement of our understanding of the regulation/misregulation of neurogenesis. In this review, we examine the factors that control neurogenesis in an age-dependent manner, and we define these signals by the extrinsic mechanism through which they are presented to the neural stem cells. Secreted signals, cell-contact-dependent signals, and extracellular matrix cues all contribute to the regulation of the aging neurogenic niche and offer points of therapeutic intervention.

Preparation and characterization of acellular adipose tissue matrix using a combination of physical and chemical treatments.

Decellularized adipose extracellular matrix (ECM) has been used in the clinic to support the regeneration of adipose tissues. The methods used to produce adipose tissue ECM scaffolds exhibit distinct effects upon the structural and functional components of the resultant scaffold material. The current study presents an acellular ECM scaffold from human adipose tissues derived using successive physical and chemical treatments, including repeated freeze-thaw cycles followed by centrifugation, polar solvent extraction and enzymatic digestion. Cellular components, including nucleic acids were effectively removed without significant disruption of the morphology or structure of the ECM. The compositions of major ECM components were evaluated, including acid/pepsin soluble collagen, sulfated glycosaminoglycan and laminin. The decellularized ECM exhibited satisfactory mechanical properties. Cell seeding experiments involving human adipose-derived stem cells indicated that the decellularized ECM provided an inductive microenvironment for adipogenesis without the need for exogenous differentiation factors. Higher levels of glycerol-3-phosphate dehydrogenase activity were observed among induced cells in the ECM scaffolds when compared with induced cells in collagen type I scaffolds. In conclusion, the results suggested that the decellularized ECM, containing biological and chemical cues of native human ECM, may be an ideal scaffold material for autologous and allograft tissue engineering.

Astrocyte spreading and migration on aggrecan-laminin dot gradients.

The surface concentration gradient of two extracellular matrix (ECM) macromolecules was developed to study the migratory and morphological responses of astrocytes to molecular cues typically found in the central nervous system injury environment. The gradient, prepared using microcontact printing, was composed of randomly positioned micrometer-sized dots of aggrecan (AGG) printed on a substrate uniformly coated with laminin (LN). AGG dots were printed in an increasing number along the 1000 μm long and 50 μm wide gradient area which had on each end either a full surface coverage of AGG or LN. Each dot gradient was surrounded by a 100 μm-wide uniform field of AGG printed over laminin. Seeded astrocytes were found to predominantly attach to LN regions on the gradient. Cellular extensions of these cells were longer than the similar processes for cells seeded on uniform substrates of AGG or LN serving as controls. Astrocyte extensions were the largest and spanned a distance of 150 μm when the cells were attached to the mixed AGG+LN patches on the gradient. As evidenced by their increased area and perimeter, the cells extended processes in a stellate fashion upon initial attachment and maintained extensions when seeded in AGG+LN regions but not on uniform laminin controls. The cells migrated short distances, ∼20-35 μm, over 24 h and in doing so preferentially shifted from AGG areas to higher LN surface coverage regions. The results indicated that presenting mixed ECM cues caused astrocytes to sample larger areas of the substrate and made the cells to preferentially relocate to a more permissive ECM region.

Data-Modeling Identifies Conflicting Signaling Axes Governing Myoblast Proliferation and Differentiation Responses to Diverse Ligand Stimuli.

Skeletal muscle tissue development and regeneration relies on the proliferation, maturation and fusion of muscle progenitor cells (myoblasts), which arise transiently from muscle stem cells (satellite cells). Following muscle damage, myoblasts proliferate and differentiate in response to temporally-varying inflammatory cytokines, growth factors, and extracellular matrix cues, which stimulate a shared network of intracellular signaling pathways. Here we present an integrated data-modeling approach to elucidate synergies and antagonisms among proliferation and differentiation signaling axes in myoblasts stimulated by regeneration-associated ligands. We treated mouse primary myoblasts in culture with combinations of eight regeneration-associated growth factors and cytokines in mixtures that induced additive, synergistic, and antagonistic effects on myoblast proliferation and differentiation responses. For these combinatorial stimuli, we measured the activation dynamics of seven signal transduction pathways using multiplexed phosphoprotein assays and scored proliferation and differentiation responses based on expression of myogenic commitment factors to assemble a cue-signaling-response data compendium. We interrogated the relationship between these signals and responses by partial least-squares (PLS) regression modeling. Partial least-squares data-modeling accurately predicted response outcomes in cross-validation on the training compendium (cumulative R 2=0.96). The PLS model highlighted signaling axes that distinctly govern myoblast proliferation (MEK-ERK, Stat3) and differentiation (JNK) in response to these combinatorial cues, and we confirmed these signal-response associations with small molecule perturbations. Unexpectedly, we observed that a negative feedback circuit involving the phosphatase DUSP6/MKP-3 auto-regulates MEK-ERK signaling in myoblasts. This data-modeling approach identified conflicting signaling axes that underlie muscle progenitor cell proliferation and differentiation.

Interplay of Geometric Cues and RGD/BMP-2 Crosstalk in Directing Stem Cell Fate.

Within the native microenvironment, extracellular matrix (ECM) components are thought to display a complex and heterogeneous distribution, spanning several length scales. Herein, the objective is to mimic, in vitro, the hierarchical organization of proteins and growth factors as well as their crosstalk. Photolithography technique was used to adjacently pattern geometrically defined regions of RGD and BMP-2 mimetic peptides onto glass substrates. These ECM-derived ligands are known to jointly regulate mesenchymal stem cells (MSCs) osteogenic differentiation. By manipulating the spatial distribution of dually grafted peptides, the extent of human MSCs osteogenic differentiation was significantly affected, depending on the shape of peptide micropatterns. Our data highlight the existence of a strong interplay between geometric cues and biochemical signals. Such in vitro systems provide a valuable tool to investigate mechanisms by which multiple ECM cues overlap to regulate stem cell fate, thereby contributing to the design of bioinspired biomaterials for bone tissue engineering applications.

Abstract 186: Heart Disease Associated Lamin A/C Mutations and Resultant Defects in Cell and Nuclei Structure

Changes in cell organelle structure or shape, which can be associated with a variety of diseases, can be caused by inherited genetic mutations. For example, the progeria disease is associated with a mutation to the LMNA gene, and it leads to a devastating early aging and death. However, other LMNA gene mutations do not cause early aging, but instead have a more subtle effect with patients presenting only with heart disease symptoms. One way to study the mechanisms that lead to these differences is in vitro with patient specific cell-lines. Here we will present a study of 10 cell-lines negative for mutations or with four different types of LMNA mutations: 1) Hutchinson-Gilford progeria syndrome LMNA gene mutation, 2) LMNA splice-site mutation (c.357-2A>G , p.N120Lfs*5), 3) LMNA nonsense mutation (c.736 C>T, p.Q246X), and 4) LMNA missense mutation (c.1003C>T, p.R335W) in exon 6. Fibroblasts from each of these cells lines were cultured on either isotropic fibronectin or fibronectin patterned into anisotropic lines. The cells were then fixed and stained to visualize the nuclei, actin fibrils, and the underline remodeled fibronectin. To analyze this data, we applied existing algorithms to quantify the re-organization of actin in response to extracellular matrix cues by calculating both the actin and fibronectin orientational order parameter. Additionally, we have created a new algorithm to automatically identify nuclei defects, which is a known property of the progeria LMNA mutation. Using the new algorithm, it is possible to separately characterize the shape of normal and defective nuclei for each cell-line. Through this algorithm, we show the varied effects of the different mutations on the number of fibroblasts that present with nuclear defects and their properties. The current data is consistent with the known patient symptoms providing further avenues of study into the mechanisms triggered by these mutations. Furthermore, we probe whether the unique mechanical conditions present in the myocardium are responsible for the heart disease symptoms of some of the tested mutations.

Abstract 5779: Building a novel 3D hyaluronan-based tri-culture model of the bone metastatic microenvironment for cancer studies

Abstract The high attrition rate of potential anti-cancer drugs entering clinical trials predicates a need for more predictive pre-clinical model systems. Three-dimensional (3D) culture systems provide cells a more physiological microenvironment than traditional cell culture methods—including relevant extracellular matrix (ECM) cues and cell-cell interactions—while allowing greater experimental control and higher-throughput studies than animal models. Prevalent cancers, including breast and prostate cancer, metastasize preferentially to bone, where they become incurable. The bone marrow microenvironment is a complex network of interacting bone homeostatic and hematopoietic niches that contribute to osteotropic cancer homing, proliferation, and treatment resistance. We created a 3D in vitro tri-culture model of the bone metastatic microenvironment to serve as a new platform to study the “trialogue” between bone metastatic cancer, bone marrow stroma, and bone marrow endothelial cells. This tool can be used to inform drug discovery and to combat treatment resistance. Within the present study, the utility of peptide-functionalized hyaluronan hydrogels for supporting the self-organization of 3D bone marrow microvascular networks was explored. Gel crosslinking density was varied to examine the effect of matrix porosity on cell migration and organization over time. Tubule length and diameter were quantified and the endothelial structures were assessed for indicators of mature vasculature, including lumen formation, basement membrane production, and the recruitment of supporting stromal cells. Breast/prostate cancer cell growth, survival, and physical association with bone marrow stromal and bone marrow microvascular endothelial cell networks was examined in tri-culture models. Ongoing studies seek to identify and disrupt key cell signaling pathways that mediate cell-cell interaction and promote cancer cell proliferation and survival, providing new tools to combat bone metastatic disease. Citation Format: Lindsey K. Sablatura, Daniel A. Harrington, Mary C. Farach-Carson. Building a novel 3D hyaluronan-based tri-culture model of the bone metastatic microenvironment for cancer studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5779. doi:10.1158/1538-7445.AM2017-5779