Cell-Surface Interactions in Pathogenesis

National Institute Of Dental & Craniofacial Research

Investigators

Linked publications, trials & patents

Abstract

Our goal in this project is to characterize new roles for cellular and cell-extracellular interactions in disease pathogenesis with a goal of identifying novel molecular regulators and mediators. Pathologists use cancer cell invasion across the basement membrane as a diagnostic feature of malignant invasion. Proteases and physical mechanisms produce initial basement membrane perforations, but how the first cells squeeze through initially small holes into matrix environments is not well understood. We use a 3D invasion model consisting of cancer-cell spheroids encapsulated by a basement membrane and embedded in collagen to characterize early steps in invasion across this barrier. This organoid-type NAM (New Approach Methodology) using primarily human cancer cell lines has permitted characterization of microscopic features otherwise too difficult to visualize in vivo. We found that certain cancer cells extend exceptionally long (~30-100 microns in length) protrusions through basement membranes via actin and microtubule cytoskeletal function. These long, slender protrusions use integrin adhesion and myosin II-based contractility to pull cells through the basement membrane for initial invasion. These long, organelle-rich protrusions concurrently pull surrounding collagen inward toward the spheroid while they are pulling cancer cells outward through perforations in the basement-membrane barrier. These unusual long, thin, contractile cellular protrusions can facilitate initial breaching of the basement-membrane barrier as a first step in cancer metastasis. We are continuing to explore the regulation of the formation of these long, prehensile adhesions. Aberrant integrin signaling contributes to the pathogenesis of osteoarthritis. Activation of the alpha5-beta1 integrin by a proteolytic fragment of fibronectin, but not by the intact protein, was found to lead to reactive oxygen species (ROS) production initially at the cell surface, but then in endocytosed vesicles termed early endosomes. These ROS-producing endosomes, termed redoxosomes, contained a molecular complement of fibronectin and integrin, a ROS-producing enzyme, and SRC, a redox-regulated kinase that promotes production of the protease MMP-13. Articular cartilage from patients with osteoarthritis showed increased amounts of SRC and a component involved in ROS production. As in vitro, immunolocalization analyses could identify redoxosomes in vivo that could sustain integrin signaling in response to matrix damage. This signaling mechanism both induces a matrix-degrading protease and sets up a self-perpetuating cycle contributing to ongoing degradation of cartilage matrix in osteoarthritis. We collaborated to write a review for Nature Reviews on these and multiple other published studies further implicating integrins – including alpha5-beta1 – in not only pathogenesis of osteoarthritis, but also in mediating normal joint development and homeostasis. There is a delicate balance of signaling needed to maintain joint tissue function, with input from both integrin and growth factor signaling pathways. Integrins and their signaling may provide an approach to alleviating osteoarthritis. We collaborated in a study of the role of collagen matrix organization mimicking fibrosis in vascular capillary function in a microphysiological model of capillaries on a chip to identify roles for perivascular cells in pathological responses to altered collagen fibrillar matrix. We are also continuing our research collaboration with Prof. Simon Tran in the Faculty of Dental Medicine and Oral Health at McGill University to provide approaches to prevent the loss of salivary gland function in patients receiving therapeutic radiation for head and neck cancer. We are collaborating on RNA-sequencing analyses of various salivary gland models. In all our research projects, our Section emphasizes rigorous and responsible conduct of research to conduct investigations that are reproducible and transparent, e.g., by describing in detail our specific methods, reagents, and the instrument settings we use, to be able to ensure that our work can be replicated. We perform at least 3 independent biological repeats (vs. technical repeats) for each experiment we report, as well as using orthogonal approaches with skepticism to test any important conclusion. A key component of rigorous and reproducible research, we feel, is the continuous conscientious use of electronic lab notebooks that provide forensic tracking and backups (we use the Federal version of LabArchives), as well as retaining two backups of all primary data, of which one copy is retained at NIH after a lab member leaves. We evaluate and communicate error and uncertainty by using quantification, unbiased data analysis approaches, and appropriate statistical tests. In our publications, we discuss reservations, caveats, and limitations of our work and the assumptions on which it is based. We aim to construct experimental tests to obtain yes/no and/or quantitative answers in order to rigorously test our hypotheses. We welcome negative results as opening new avenues of exploration into areas that had not been predicted. We avoid conflicts of interest in our research, as well as in our reviewing for journals and grant funding agencies, and we take care to submit our research to journals with unbiased peer review, as well as avoiding “predatory” journals. We encourage collaboration and interdisciplinary research, which enriches and broadens our approaches. This approach includes sharing our underlying primary data as much as practical, e.g., by deposition in public repositories after publication of our research. Public availability of the raw data underlying research allows others to evaluate and replicate our findings. We also continue to emphasize the training and mentoring of the next generation of biomedical researchers.