The Extracellular Matrix in Development and Homeostasis
The extracellular matrix (ECM), defined as the space outside the cell compartment, is a complex micro-ecological setting comprised of proteins and soluble factors that provides scaffolding for the cell. The biophysical and biochemical cues are necessary for cell development, cell-cell communication and growth. The biophysical interaction between the cell and the ECM is primarily guided via integrins; transmembrane heterodimer molecules comprised of α and β subunits with specificity towards diverse matrix ligands such as fibronectin, laminin and collagen. This interaction affects tensional homeostasis, cell polarity and migration within the environment. Furthermore, the downstream intracellular signaling mediated through integrin-ECM interactions plays a significant role in tissue development, differentiation, gene regulation and protein synthesis, and ultimately cell survival.
The composition of the extracellular matrix varies between tissue types as well as within specific tissue as it develops. The development of the murine heart is an example where the spatiotemporal distribution of laminin, collagen and fibronectin as well as the total content between stages of development is highly variable. In fact, the ECM is such a tightly regulated system that disruptions associated with genetic aberrations specific to the ECM composition can significantly affect the physiological behavior and even lead to death. For example inactivation of fibronectin in mouse embryos leads to defects in neural and vascular developments. As another example, in osteogenesis imperfecta (brittle bone disease) mutations of Col1A1 and Col1A2 genes result in abnormal assembly of collagen type I (the most abundant protein in the body) and causes weak bones, stunted growth and defective teeth.
In addition, remodeling of the ECM is a constant process in preserving homeostasis and a necessary process in the event of a wound. The cells constantly remodel their environment through activation of matrix metalloproteases (MMP), a family of enzymes that degrade ECM proteins, as means of removing barriers to facilitate migration to a specific site and repair. Similar to ECM composition, deregulation of the remodeling process can have a significant impact on a person’s health with myriad examples in cardiovascular and neurodegenerative diseases.
In tumors, altered composition and increased remodeling of the ECM results in a permissive and supportive microenvironment that is crucial for tumor progression. The focus of this thesis work is directed at understanding the effects of the biophysical and biochemical modifications to the ECM in cancer biology with specifics to ovarian and breast carcinomas. Our approach to this complex question is to develop and utilize highly mimetic in vitro models as means of bridging the gap between 2D tissue culture and animal studies. To accomplish this, we exploit a fabrication technique analogous to 3D printing that affords sub-micron resolution and free form capabilities to directly mimic biological tissue samples. As a result, we have an experimental construct that is highly repeatable, biologically relevant and relatively affordable in a research setting.
The importance of the ECM in cancer progression
A metastasizing cancer is the true killer for patients who have befallen to this disease. Since 1889, the “seed and soil” hypothesis proposed by Stephen Paget has been a driving force in our understanding of the cancer biology. The stepwise alterations in tumor suppressive genes and accumulations of oncogenes during tumor progression correlate with the aggressiveness of the cancer and its success in metastasizing. Complimentary to the genetic aberrations associated with this disease, significant research over the decades has shown that altered cell-matrix interactions are a crucial step in tumorigenesis and metastasis. Precisely how the cancer cells modify, manipulate and escape their environment determines cancer progression.
One of the hallmarks of cancer progression is the deregulation of migratory mechanisms and transitions to invasive processes. Therefore, significant research efforts have been directed at understanding the cell-matrix interactions as it pertains to enhanced, facilitated cancer migration in vivo.
ECM topography of the stromal microenvironment is believed to be an important contributor to directing and enhancing cancer migratory pathways; where cancerous stroma is permissive to migration while normal stroma is inhibitory. In vivo imaging via multiphoton microscopy has shown that a key difference in migration between in vivo and in vitro is the high degree of persistent linear motion of carcinoma cells in tumors. In accordance with these findings, Second Harmonic Generation (SHG) imaging of stromal collagen has shown that ECM topography of highly aligned fibers are exclusively present in malignant tissues and associated with poor clinical outcome. Well orchestrated intracellular signaling events and migratory modes in response to the topography influence cell migration.
Currently, multiple forms of migration have been identified by which cancer cells interact with their microenvironment. These modes are categorically identified as: amoeboid (blebby or pseudopodial/filopodial), mesenchymal, multicellular streaming and collective. Cancer cells have the ability to shift between modes that are most convenient for effective migration in a given condition. As an example, cancers cells having undergone epithelial to mesenchymal transition (EMT) will transit through the stroma via protrusions that are marked by integrin clustering and proteolytic events mediated predominantly through MT1-MMP. Inhibition of such MMPs and other proteases involved in matrix remodeling results in cancer cell migration in a propulsive squeezing manner termed as amoeboid.
Migration modes are mainly driven by the Rho family of GTPases which control intracellular signaling events associated with modulating cellular polarity, receptor signaling, integrin trafficking and actomyosin contractions. In comparison to 2D substrates, mesenchymal migration can switch between lamellipodia and lobopodia based migration in a 3D scaffold. When operating as lamellipodia, localization of Rac1 and Cdc42 at the leading edge marks this type of migration with an increase in cell-matrix adhesion and inhibition of RhoA-ROCK leading to actin polymerization at the leading edge. Modulation of the ECM environment to a more linearly elastic behavior can drive the switch to lobopodia based migration that is marked by high RhoA –ROCK ex