Pathway to metastasis: carcinoma dissemination via organized collagen tracks

University of Minnesota Ph.D. dissertation.December 2017. Major: Biomedical Engineering. Advisor: Paolo Provenzano. 1 computer file (PDF); xv, 185 pages + supplementary video filesA vital part of the metastatic cascade that leads to cancer-related deaths is the initial dissemination of cancer cells from a confined lesion into neighboring tissue by migration and invasion. Breast and pancreatic carcinomas are often associated with increased deposition of collagen-I, which can assemble into aligned fiber tracks in mature breast tumors. Such aligned tracks provide contact guidance cues for directed cancer cell migration and dissemination leading to increased invasion, metastasis and decreased disease-free survival in breast cancer patients. Using multiphoton imaging, we demonstrate that organized collagen architectures develop in the pancreatic ductal adenocarcinoma (PDA) stroma even at the pre-invasive stage with single cells and multicellular clusters interacting with aligned collagen tracks in vivo. Mimicking the collagen patterns with microfabricated substrates, we show that for single cells, aligned architectures induce constrained focal adhesion maturation and associated F-actin alignment, consequently orchestrating anisotropic traction stresses that drive cell orientation and directional migration. While such interactions allow single mesenchymal-like cells to spontaneously “sense” and follow topographic alignment, intercellular interactions within epithelial clusters counteract anisotropic cell-substratum forces, resulting in substantially lower directional response. Indeed, anisotropic cell-substratum interactions from organized periductal collagen may contribute to cell extrusion and dissemination from pre-invasive ductal epithelia in PDA. Such contact guided spreading of cancer cells may be inhibited by dismantling the fiber architecture, diminishing its density or by abrogating cell-ECM interactions. To validate our findings in 3D we engineered novel in vitro substrates using a simple method to align 3D collagen gels by guided cellular compaction, to produce highly aligned, acellular collagen constructs as a controlled microenvironment in vitro. Additionally, we integrated the aligned collagen matrices to cell dense tumor-like plugs, allowing tracking of the temporal evolution of the advancing invasion fronts over several days. Live cell imaging and analysis of 3D cell migration revealed profoundly enhanced motility in aligned collagen matrices for the putative cancer stem cell subpopulation. Heterogeneity in cell migration behavior was also observed between cells at the leading edge and those within the tumor boundary, thus demonstrating the versatility of these platforms in capturing the dynamics of contact guided carcinoma dissemination