Cell-Matrix Interactions and Migration

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Extracellular matrix molecules, integrin receptors, cytoskeletal motor proteins, proteases, and regulators of these molecular systems contribute to cell migration, signaling, and cancer cell invasion by complex, integrated mechanisms. We are addressing the following specific questions: 1. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how are they coordinated to produce effective cell migration or invasion? 2. What biophysical properties and signaling mechanisms are important for efficient cell migration and invasion? We are using a variety of cell and molecular biology approaches to address these questions, including live-cell wide-field, confocal, and two-photon time-lapse microscopy with fluorescent protein chimeras, biochemical and signal transduction analyses, as well as methods for evaluating local matrix deformations in response to forces from migrating or invading mammalian cells. We use a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins and various configurations of extracellular matrix molecules as part of a long-term program to analyze their functions in integrin-mediated processes. Cancer invasion through basement membranes is the initial step of tumor dissemination and metastasis, and crossing of this tissue barrier is a key pathological sign of invasion. However, relatively little is known about how human cancer cells breach and traverse basement membranes into surrounding extracellular matrix and tissues. We used a three-dimensional in vitro invasion model consisting of human cancer cell spheroids encapsulated by a basement membrane and embedded in 3D type I collagen gels to visualize early events of cancer invasion using microscopy. Human breast cancer cells generated large numbers of basement membrane perforations, or holes, of varying sizes that expanded over time during the process of cell invasion. Treatment with various matrix metalloproteinase (MMP) inhibitors and disrupting actin polymerization strongly suppressed perforation size. Although myosin II-associated actomyosin contractility had been implicated previously in developmental models for enlarging basement membrane perforations to permit breaching, inhibiting contractility in this cancer invasion model had unexpectedly minimal effects on hole expansion. We conclude that human cancer cells can mainly use MMP-type proteolysis and actin polymerization to perforate the basement membrane and expand holes to a size that can permit transit of the nucleus (the largest cell organelle) and the rest of the cancer cell body for invasion across the basement membrane, but with limited roles for actomyosin contractility. This efficient basement membrane breaching process could be demonstrated using the cancer cells alone without the help of accessory cells such as cancer-associated fibroblasts or macrophages. We are extending this study to evaluate the mechanisms by which cancer cells can squeeze through these basement membrane perforations for subsequent migratory invasion in a 3D collagen matrix. We are also completing development of an ex vivo model system for analyzing cell interactions and migration in a physiological 3D environment to complement our studies on cell-matrix interactions in vitro. This latter approach uses mouse fascia as a source of native extracellular matrix. This combined approach involving mechanistic characterization of the regulation of cell migration and cell interactions with various types of extracellular matrix should provide novel approaches to understanding and potentially ameliorating migratory or invasive processes used by cells during abnormal embryonic development and cancer invasion. An in-depth understanding of the precise ways in which cells interact with their extracellular matrix environment should also facilitate tissue engineering studies for future regenerative medicine.