The extracellular matrix (ECM), the noncellular component of the microenvironment, influences cell growth, survival, migration and tissue-specific differentiation through a repertoire of receptors including integrins, syndecans and discoidin receptors. My group is exploring the molecular mechanisms whereby ECM receptors modulate cell fate and tumorigenesis. We investigate how mechanical and topological properties of the ECM, which are related to its composition and organization, alter integrin expression and function to modify stem cell fate and tumor progression.
One part of our research program focuses on clarifying how mechanical force (ECM stiffness and cell contractility) modulate breast tumor progression and treatment efficacy with a focus on signaling and epigenetics using two and three dimensional (2D/3D) monolayer and organotypic culture models, xenograft/syngeneic and transgenic mouse models and clinical specimens and a repertoire of approaches to measure and manipulate mechanical force. The second part of our research is directed towards characterizing the role of and molecular mechanisms whereby mechanical force regulates human embryonic stem cell fate using 2D/3D natural and synthetic matrices and a force reactor with a focus on adhesion signaling and epigenetics.
Analysis of MCF10A mammary epithelial cell acinar morphogenesis within well-defined 3-dimensinal systems
Epithelial tissue morphogenesis proceeds within the context of a three dimensional (3D) extracellular matrix (ECM). Accordingly, to clarify the molecular basis of tissue-specific differentiation and disease, a variety of 3D systems exploiting natural ECMs have been developed, such as reconstituted basement membrane (rBM) and purified collagen hydrogels. These natural hydrogels recapitulate epithelial tissue architecture and behaviors in vitro with reasonable fidelity. Nevertheless, natural matrices suffer from considerable preparation variability and remain poorly defined biochemically and biophysically. To understand epithelial cell biology requires defined biomaterials in which biochemical, topological and biophysical properties can be systematically varied. Towards this goal we use the nonmalignant MCF10A mammary epithelial cell (MEC) line and are conducting a systematic analysis of acinar morphogenesis using the natural hydrogels collagen type I and rBM and three synthetic matrices: rBM-conjugated polyacrylamide gels, self assembling peptide gels (PuraMatrix) with and without rBM and hyaluronic acid gels with and without rBM. Rigorous, morphometric and quantatitive analysis as well as immunohistochemistry, and electron microscopy are in progress.
Biomechanical Regulation of Breast Cancer Migration
The transition of the mammary epithelium to a metastatic state is an important event that drastically increases mortality during breast cancer progression. Metastatic progression depends on the ability of tumor cells to migrate to nearby blood vessels and lymphatics before they can successfully metastasize and colonize into other tissues. An understanding of how biomechanical and biochemical signals influence directional migration to these sites of tissue escape could help develop therapies to slow or halt metastasis. To understand how the mechanical properties of the tumor microenvironment may affect metastasis, we study cell motility of mammary epithelia cells migrating on substrates with gradients of stiffness and with different matrix conjugation.
Exploring functional links between matrix stiffness, micro RNAs and HoxA9 dependent regulation of BRCA1 and mammary cell survival and tumorigenesis.
Stromal-epithelial interactions drive development and maintain tissue homeostasis through a network of soluble and insoluble factors that operate within a three-dimensional (3D) tissue. Genetic and epigenetic changes in mammary epithelial cells (MECs) cooperate with a modified tissue microenvironment to drive malignant transformation of the breast. We have been studying how altered expression of developmental regulators contributes to breast tumorigenesis and have specifically focused on investigating their influence on integrin expression and/or adhesion activity. Homeobox genes play a critical role in tissue development, are frequently lost in tumors, and can regulate integrin and extracellular matrix (ECM) expression. Global expression analysis of matched tumor/normal breast tissue revealed that HoxA9 expression was significantly lower in the tumors. HoxA9 represses the malignant phenotype of breast cancer cells in vivo as well as in 3D rBM and this reversion is coincident with BRCA1 induction and normalization of adhesion and integrin expression. Accordingly, we are exploring functional and mechanistic links between HoxA9, BRCA1 and integrin-dependent tissue behavior. In addition, we are studying how changes in the biomechanical properties of the ECM during normal development and mammary tumorigenesis can influence HoxA9 expression.
Exploring the link between human embryonic stem cell organization and fate using tension-calibrated extracellular matrix functionalized polyacrylamide gels
Human embryonic stem cell (hESc) lines are likely the in vitro equivalent of the pluripotent epiblast. hESc express high levels of the extracellular matrix (ECM) laminin integrin receptor α6β1 and consequently can adhere robustly and be propagated in an undifferentiated state on tissue culture plastic coated with the laminin rich basement membrane preparation, MatrigelTM, even in the absence of supporting fibroblasts. Such cultures represent a critical step in the development of more defined feeder free cultures of hESc; a goal deemed necessary for regenerative medical applications, and have been used as the starting point in some differentiation protocols. However, on standard non deformable tissue culture plastic hESc either fail or inadequately develop the structural/morphological organization of the epiblast in vivo. By contrast, growth of hEScs on appropriately defined mechanically deformable polyacrylamide substrates permits recapitulation of many of these in vivo features. We suggest this strategy as a prospective in vitro model of the genetics, biochemistry, and cell biology of pre and early gastrulation stage human embryos and the permissive and instructive roles that cellular and substrate mechanics might play in early embryonic cell fate decisions. Such knowledge should inform regenerative medical applications aimed at enabling or improving the differentiation of specific cell types from embryonic or induced embryonic stem cells.
Force characterization of tissue from normal, pre-invasive and invasive breast cancer
Historically, cancer research has focused on understanding the genetic and biochemical regulation of tumor progression while the biomechanical influences have only recently been studied. Clear evidence has emerged indicating that mechanical forces are closely associated with tumor progression. In fact, biomechanical cues are integrated with biochemical and genetic cues at every step. To investigate how micro-environment stiffness may affect progression in human breast tumors, we use atomic force microscopy (AFM) to probe the mechanical properties of tissue from patients diagnosed with various stages of breast cancer. Using this technique we are able to assess the elastic modulus of the tissue focusing on the border between the tumor epithelium and the surrounding stroma. Preliminary data indicates that the extracellular matrix surrounding tumors have varying stiffness. These stiffer areas may be predictive of tumor type and invasiveness. Coupling these results with immunohistochemistry allows us to determine the nature of these mechanically distinct regions by drawing a correlation between stiffness and collagen cross-linking with localization of LOX. We are also determining how the tumor epithelium responds to mechanical cues by examining integrin activation and stress fiber formation.
Roles of collagen crosslinking and ECM remodeling in mammary tumor malignant transformation.
In vivo, cells are maintained in mechanical balanced microenvironments. We showed that ECM stiffness alters cell proliferation, survival and polarity via integrin clustering, focal adhesion maturation, and cell-generated force. Increased tissue stiffness, changes of ECM (e.g. collagen) remodeling and ECM remodeling enzymes (such as MMPs, lysyl oxidase LOX) are strongly associated with breast cancer progression. We therefore hypothesis ECM remodeling affects tumor progression via increasing tissue stiffness. Since crosslinking of collagen I increases its mechanical strength, we tested if collagen crosslinking by Lysyl Oxidase (LOX) affects tumor progression. We have shown that breast transformation is accompanied by elevated levels LOX, collagen I deposition, and significant collagen cross-linking as well as a pronounced stiffening of the breast and its surrounding extracellular matrix. We tested and confirmed ECM stiffness can modulate PTEN level and PI3K activity in the culture system. Thus, collagen crosslinking and substrate stiffness can modulate oncogene effects through PTEN and integrin dependent pathways and thus affect breast cancer progression. To this end we employ three dimensional organotypic culture models, xenograft and syngeneic mouse models, transgenic animals and fresh and fixed clinical samples.
