We hypothesize that related biological processes are involved not only in normal morphogenesis and tissue repair, but also in pathological processes such as tumor cell invasion and HIV pathogenesis. Previous studies by numerous laboratories including our own have identified overlapping pathological mechanisms that include changes in cell-matrix and cell-cell adhesion, migration, and associated signal transduction pathways. Understanding these processes should help to clarify mechanisms of pathogenesis and to identify novel potential targets for therapeutic intervention. ? ? In cancer metastasis, normal cell behavior is subverted by processes that modify the formation of cell adhesions, migration, and invasion. We are investigating mechanisms of invasion in hopes of identifying targets for potential therapeutic intervention in the pathogenic process, e.g., key steps and target molecules such as specific matrix metalloproteinases or interactions of components of integrin adhesion and signaling complexes. Previous studies by numerous laboratories including our own have identified mechanisms involved in invasion that include choreographed changes in cell-matrix and cell-cell adhesion, migration, and associated signal transduction pathways. Understanding these processes should help to clarify mechanisms of pathogenesis and potentially design novel therapies. ? ? Both tumor cells and certain HIV-infected cells can migrate and invade tissues. The role of proteases in these processes is currently uncertain. We are focusing on the following three facets of this problem relevant to both developmental and cancer cell migration. ? 1. Precisely where on the cell surface do tumor cells degrade the matrix during cell motility, and is proteolysis essential for migration?? 2. How do invadopodia - tiny cell surface structures mediating proteolysis - initiate and function?? 3. How do normal cells such as fibroblasts migrate through 3D matrices, and do they require proteases? ? We have focused on developing new tools to address these questions; a long-term collaboration on HIV disease is being phased out. One approach has been to develop cutting-edge microscopy and adapting TIRF (total internal reflection fluorescence) microscopy to study the initial steps of formation of invadopodia by the use of fluorescent protein chimeras and immunolocalization. A second approach is to develop means of determining the initial sites of focal proteolysis by tumor cells in two-dimensional and ultimately three-dimensional matrix microenvironments. ? ? The third approach is an ongoing effort to develop very thin 3D matrices for high-resolution microscopy. Although three-dimensional matrices can be used readily to study invasion by phase-contrast microscopy, they have proven difficult to use for high-resolution immunolocalization of the many proteins involved in the formation of cell adhesion complexes, migration, and invasion. We are experimenting with various thin-film approaches to permit localization of multiple proteins at the same time in cells in three-dimensional environments.
