Cell-Surface Interactions in Pathogenesis

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Our goal in this project is to characterize new roles for cellular interactions in disease pathogenesis. A major current focus is on tumor cell invasion and metastasis, with a goal of identifying novel molecular regulators and mediators. A fundamental question in cancer biology is the relationship of the local microenvironment to cancer progression. We have been evaluating the roles of the extracellular matrix in cancer cell migration and invasion. The two major forms of matrix we examine in these interactions have been the 3D fibrillar extracellular matrix and the flat sheet-like basement membrane. Current models of cancer cell migration and invasion have increasingly used 3D extracellular matrix systems. These model systems differ in molecular composition and fibril organization, which may produce conflicting findings. We directly compared five different 3D extracellular matrix systems for studying cancer cell migration and invasion using automated computer-based cell tracking. We chose HT-1080 fibrosarcoma and MDA-MB-231 breast carcinoma cell lines as examples of cancers of mesenchymal versus epithelial origin. The first three 3D model systems examined involved reconstituted 3D fibrillar gels consisting of two types of collagen hydrogel and tissue matrix gel (TMG). The last two model systems consisted of cell-derived extracellular matrices extracted from densely cultured primary human or cancer-associated fibroblasts. HT-1080 cancer cells displayed rounded morphologies in all three reconstituted 3D matrices, but they became spindle-shaped in the two cell-derived matrices. MDA-MB-231 breast cancer cell morphologies were elongated in all matrices. Quantitative measures of key parameters of cell migration differed markedly between the different types of 3D matrix. For cells within the three reconstituted matrices, cells migrated most rapidly and furthest in TMG. However, comparing the three reconstituted matrices with the cell-derived matrices, cells migrated even more efficiently in the two cell-derived matrices. The most notable differences involved directionality (directional persistence) of migration, which was greatest in the two cell-derived matrices, which had characteristic aligned fibrils. We conclude that measurements of cancer cell morphology, speed of translocation, and directional persistence of migration can differ dramatically depending on the type of 3D matrix system being employed. Consequently, conclusions about cancer cell migration and invasion need to specify the exact type of matrix used, since conclusions about the mode of 3D cell migration can differ depending on the type of matrix employed in the study. One problem with tumor spheroid models of cancer is that they may not replicate a key feature associated with cancer progression: interactions with the basement membrane. Basement membranes can confine and organize cells in tissues, and a cardinal feature of cancer is the breaching of basement membrane barriers by tumor cells. We established a cancer spheroid model system in which a basement membrane can be generated to fully surrounded the spheroid by incubating cells with low concentrations of a basement membrane protein extract. The cells at the spheroid surface were able to use the protein components of the extract to assemble a basement membrane. Transplantation of these spheroids encapsulated by a basement membrane into a 3D collagen gel resulted in cancer cell breaching of the basement membrane and migratory streaming of invading cancer cells outward into the collagen hydrogel by single-cell or collective-cell migration. This system can provide an approach to study the initial steps of penetration and breaching of the sheet-like basement membrane, followed by subsequent 3D cell migration into fibrillar extracellular matrix. Tumor progression can be influenced by multiple external microenvironmental factors, including immune cells. A collaborative study involving a joint postdoctoral fellow in the laboratory of Dr. Alberto Baena at Oxford is evaluating the interplay between Drosophila tumors and immune cells. The roles of Drosophila macrophages appear to depend on regulation from caspases that is separate from their well-known roles in apoptosis. There is surprisingly widespread non-apoptotic caspase activation in Drosophila tumors engineered to mimic human tumors by activation of the EGF receptor and JAK/STAT signaling. This tumor-associated caspase activation provides partial suppression of JNK signaling and tumor expansion by limiting tumor cell proliferation and cell fate alteration in processes independent of classical apoptosis. An unexpected additional finding has been the effect of caspase activity on macrophage proliferation near the tumors associated with tumor expansion. Because we have extensive expertise in real-time imaging of cell behavior in vitro and in organ explants, we have an ongoing research collaboration involving a joint postdoctoral fellow with Dr. Ashok Kulkarni's laboratory to use the GCaMP6 mouse system for direct visualization of calcium signaling in pain-sensing neurons. Current research has focused on the regulatory kinase Cdk5 in characterizing orofacial pain signaling and mechano-sensation in the mouse trigeminal ganglion. Quantification of calcium signaling responses has permitted detailed characterization of trigeminal neuronal responses during the sensing of acute and inflammatory pain, as well as sensing light touch and its pathological conversion to allodynia. The ability to compare signaling in the same neurons during responses to different stimuli is providing an approach to be able to distinguish polymodal from more stimulus-specific responses of individual trigeminal neurons. This system also permits a demonstration of the effects of inhibiting Cdk5 activity on real-time trigeminal neuronal signaling. In other collaborations, our laboratory members contributed to studies of mechanisms of organ-specific targeting of tumor cells in zebrafish, as well as to determining the role of the fibronectin receptor integrin alpha5-beta1 in human trophoblast functions dependent on pregnancy-specific glycoprotein 1. Our various primary and collaborative studies are providing new insights into the ways in which normal physiological cell interactions crucial for embryonic development are altered or subverted in disease pathogenesis.