Claudia Fischbach

Cancer cell angiogenic capability is regulated by 3D culture and integrin engagement

Three-dimensional culture alters cancer cell signaling; however, the underlying mechanisms and importance of these changes on tumor vascularization remain unclear. A hydrogel system was used to examine the role of the transition from 2D to 3D culture, with and without integrin engagement, on cancer cell angiogenic capability. Three-dimensional culture recreated tumor microenvironmental cues and led to enhanced interleukin 8 (IL-8) secretion that depended on integrin engagement with adhesion peptides coupled to the polymer. In contrast, vascular endothelial growth factor (VEGF) secretion was unaffected by 3D culture with or without substrate adhesion. IL-8 diffused greater distances and was present in higher concentrations in the systemic circulation, relative to VEGF. Implantation of a polymeric IL-8 delivery system into GFP bone marrow-transplanted mice revealed that localized IL-8 up-regulation was critical to both the local and systemic control of tumor vascularization in vivo. In summary, 3D integrin engagement within tumor microenvironments regulates cancer cell angiogenic signaling, and controlled local and systemic blockade of both IL-8 and VEGF signaling may improve antiangiogenic therapies.

Dense type I collagen matrices that support cellular remodeling and microfabrication for studies of tumor angiogenesis and vasculogenesis in vitro

Type I collagen is a favorable substrate for cell adhesion and growth and is remodelable by many tissue cells; these characteristics make it an attractive material for the study of dynamic cellular processes. Low mass fraction (1.0-3.0 mg/ml), hydrated collagen matrices used for three-dimensional cell culture permit cellular movement and remodeling, but their microstructure and mechanics fail to mimic characteristics of many extracellular matrices in vivo and limit the definition of fine-scale geometrical features (<1 mm) within scaffolds. In this study, we worked with hydrated type I collagen at mass fractions between 3.0 and 20 mg/ml to define the range of densities over which the matrices support both microfabrication and cellular remodeling. We present pore and fiber dimensions based on confocal microscopy and longitudinal modulus and hydraulic permeability based on confined compression. We demonstrate faithful reproduction of simple pores of 50 μm-diameter over the entire range and formation of functional microfluidic networks for mass fractions of at least 10.0 mg/ml. We present quantitative characterization of the rate and extent of cellular remodelability using human umbilical vein endothelial cells. Finally, we present a co-culture with tumor cells and discuss the implications of integrating microfluidic control within scaffolds as a tool to study spatial and temporal signaling during tumor angiogenesis and vascularization of tissue engineered constructs.

Integrated approach to designing growth factor delivery systems

Growth factors have been widely used in strategies to regenerate and repair diseased tissues, but current therapies that go directly from bench to bedside have had limited clinical success. We hypothesize that engineering successful therapies with recombinant proteins will often require specific quantitative information of the spatiotemporal role of the factors and the development of sophisticated delivery approaches that provide appropriate tissue exposures. This hypothesis was tested in the context of therapeutic angiogenesis. An in vitro model of angiogenesis was adapted to quantify the role of the concentration/gradient of vascular endothelial growth factor [VEGF (165)] on microvascular endothelial cells, and a delivery system was then designed, based on a mathematical model, to provide the desired profile in ischemic mice hindlimbs. This system significantly enhanced blood vessel formation, and perfusion and recovery from severe ischemia. This general approach may be broadly applicable to growth factor therapies.— Chen R. R., Silva, E. A., Yuen, W. W., Brock, A. A., Fischbach, C., Lin, A. S., Guldberg, R. E., Mooney D. J. Integrated approach to designing growth factor delivery systems. FASEB J. 21, 3896–3903 (2007)

Formation of microvascular networks in vitro

This protocol describes how to form a 3D cell culture with explicit, endothelialized microvessels. The approach leads to fully enclosed, perfusable vessels in a bioremodelable hydrogel (type I collagen). The protocol uses microfabrication to enable user-defined geometries of the vascular network and microfluidic perfusion to control mass transfer and hemodynamic forces. These microvascular networks (μVNs) allow for multiweek cultures of endothelial cells or cocultures with parenchymal or tissue cells in the extra-lumen space. The platform enables real-time fluorescence imaging of living engineered tissues, in situ confocal fluorescence of fixed cultures and transmission electron microscopy (TEM) imaging of histological sections. This protocol enables studies of basic vascular and blood biology, provides a model for diseases such as tumor angiogenesis or thrombosis and serves as a starting point for constructing prevascularized tissues for regenerative medicine. After one-time microfabrication steps, the system can be assembled in less than 1 d and experiments can run for weeks.

Implanted adipose progenitor cells as physicochemical regulators of breast cancer

Multipotent adipose-derived stem cells (ASCs) are increasingly used for regenerative purposes such as soft tissue reconstruction following mastectomy; however, the ability of tumors to commandeer ASC functions to advance tumor progression is not well understood. Through the integration of physical sciences and oncology approaches we investigated the capability of tumor-derived chemical and mechanical cues to enhance ASC-mediated contributions to tumor stroma formation. Our results indicate that soluble factors from breast cancer cells inhibit adipogenic differentiation while increasing proliferation, proangiogenic factor secretion, and myofibroblastic differentiation of ASCs. This altered ASC phenotype led to varied extracellular matrix (ECM) deposition and contraction thereby enhancing tissue stiffness, a characteristic feature of breast tumors. Increased stiffness, in turn, facilitated changes in ASC behavior similar to those observed with tumor-derived chemical cues. Orthotopic mouse studies further confirmed the pathological relevance of ASCs in tumor progression and stiffness in vivo. In summary, altered ASC behavior can promote tumorigenesis and, thus, their implementation for regenerative therapy should be carefully considered in patients previously treated for cancer.

Basic fibroblast growth factor enhances PPARγ ligand-induced adipogenesis of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are capable of differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and differentiation factors. It is widely acknowledged that basic fibroblast growth factor (bFGF) modulates chondrogenic and osteogenic differentiation of MSCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate on the cellular and molecular level that supplementation of bFGF in different phases of cell culture leads to a strong enhancement of adipogenesis of MSCs, as induced by an adipogenic hormonal cocktail. In cultures receiving bFGF, mRNA expression of peroxisome proliferator‐activated receptor γ2 (PPARγ2), a key transcription factor in adipogenesis, was upregulated even prior to adipogenic induction. In order to investigate the effects of bFGF on PPARγ ligand‐induced adipogenic differentiation, the thiazolidinedione troglitazone was administered as a single adipogenic inducer. Basic FGF was demonstrated to also strongly increase adipogenesis induced by troglitazone, that is, bFGF clearly increased the responsiveness of MSCs to a PPARγ ligand.

Poly(D,L-lactic acid)-Poly(ethylene glycol)-Monomethyl Ether Diblock Copolymers Control Adhesion and Osteoblastic Differentiation of Marrow Stromal Cells

Biodegradable polymers, such as poly(lactic acid) (PLA) and poly(lactic-coglycolic acid) (PLGA), are attractive materials for tissue engineering because of their degradative and mechanical properties, which permit scaffolds to be tailored to the individual requirements of different tissues. Although these materials support tissue development, their chemical properties offer no control of cell adhesion or function because their surfaces become immediately masked by adsorbing serum proteins when the materials come into contact with body fluids. Furthermore, adhesion proteins undergo conformational changes and a decrease in bioactivity when adsorbed to hydrophobic materials, such as PLA. To overcome these limitations, we modified the properties of PLA by synthesizing a diblock copolymer with poly(ethylene glycol) (PEG), which is known to reduce the amount of adsorbed proteins and to modify their conformation. By altering the PEG content of these diblock copolymers we were able to control the adsorption of adhesion proteins and, because cell adhesion takes place only in the presence of serum proteins, to control cell adhesion and cell shape. Marrow stromal cell differentiation to the osteoblastic phenotype was strongly improved on PEG-PLA compared with PLA, PLGA and tissue culture polystyrene and led to a 2-fold increase in alkaline phosphatase activity and mineralization.

Engineered culture models for studies of tumor-microenvironment interactions.

Heterogeneous microenvironmental conditions play critical roles in cancer pathogenesis and therapy resistance and arise from changes in tissue dimensionality, cell-extracellular matrix (ECM) interactions, soluble factor signaling, oxygen as well as metabolic gradients, and exogeneous biomechanical cues. Traditional cell culture approaches are restricted in their ability to mimic this complexity with physiological relevance, offering only partial explanation as to why novel therapeutic compounds are frequently efficacious in vitro but disappoint in preclinical and clinical studies. In an effort to overcome these limitations, physical sciences-based strategies have been employed to model specific aspects of the cancer microenvironment. Although these strategies offer promise to reveal the contributions of microenvironmental parameters on tumor initiation, progression, and therapy resistance, they, too, frequently suffer from limitations. This review highlights physicochemical and biological key features of the tumor microenvironment, critically discusses advantages and limitations of current engineering strategies, and provides a perspective on future opportunities for engineered tumor models.

Hydroxyapatite nanoparticle-containing scaffolds for the study of breast cancer bone metastasis

Breast cancer frequently metastasizes to bone, where it leads to secondary tumor growth, osteolytic bone degradation, and poor clinical prognosis. Hydroxyapatite Ca(10)(PO(4))(6)(OH)(2) (HA), a mineral closely related to the inorganic component of bone, may be implicated in these processes. However, it is currently unclear how the nanoscale materials properties of bone mineral, such as particle size and crystallinity, which change as a result of osteolytic bone remodeling, affect metastatic breast cancer. We have developed a two-step hydrothermal synthesis method to obtain HA nanoparticles with narrow size distributions and varying crystallinity. These nanoparticles were incorporated into gas-foamed/particulate leached poly(lactide-co-glycolide) scaffolds, which were seeded with metastatic breast cancer cells to create mineral-containing scaffolds for the study of breast cancer bone metastasis. Our results suggest that smaller, poorly-crystalline HA nanoparticles promote greater adsorption of adhesive serum proteins and enhance breast tumor cell adhesion and growth relative to larger, more crystalline nanoparticles. Conversely, the larger, more crystalline HA nanoparticles stimulate enhanced expression of the osteolytic factor interleukin-8 (IL-8). Our data suggest an important role for nanoscale HA properties in the vicious cycle of bone metastasis and indicate that mineral-containing tumor models may be excellent tools to study cancer biology and to define design parameters for non-tumorigenic mineral-containing or mineralized matrices for bone regeneration.

Glioblastoma Stem Cells Are Regulated by Interleukin-8 Signaling in a Tumoral Perivascular Niche

Glioblastoma multiforme contains a subpopulation of cancer stem-like cells (CSC) believed to underlie tumorigenesis and therapeutic resistance. Recent studies have localized CSCs in this disease adjacent to endothelial cells (EC) in what has been termed a perivascular niche, spurring investigation into the role of EC-CSC interactions in glioblastoma multiforme pathobiology. However, these studies have been limited by a lack of in vitro models of three-dimensional disease that can recapitulate the relevant conditions of the niche. In this study, we engineered a scaffold-based culture system enabling brain endothelial cells to form vascular networks. Using this system, we showed that vascular assembly induces CSC maintenance and growth in vitro and accelerates tumor growth in vivo through paracrine interleukin (IL)-8 signaling. Relative to conventional monolayers, endothelial cells cultured in this three-dimensional system not only secreted enhanced levels of IL-8 but also induced CSCs to upregulate the IL-8 cognate receptors CXCR1 and CXCR2, which collectively enhanced CSC migration, growth, and stemness properties. CXCR2 silencing in CSCs abolished the tumor-promoting effects of endothelial cells in vivo, confirming a critical role for this signaling pathway in GMB pathogenesis. Together, our results reveal synergistic interactions between endothelial cells and CSCs that promote the malignant properties of CSCs in an IL-8-dependent manner. Furthermore, our findings underscore the relevance of tissue-engineered cell culture platforms to fully analyze signaling mechanisms in the tumor microenvironment.