The influence of the fibrillar tumor microenvironment on the cell-cell contact response of migrating cancer cells

The breast tumor microenvironment is a dynamic niche in which cells interact with each other and remodel protein fibers to form an environment conducive to tumor cell invasion. Metastatic breast cancer cells invade along protein fibers, using them as a physical guidance cue out of the primary tumor. The alignment of these protein fibers promotes invasion and was recently identified as a prognostic indicator of metastatic disease. How migrating cancer cells interact with each other and with other cells in the confined, fibrillar tumor microenvironment remains unclear. Whether cancer cells are able to circumnavigate other cells and maintain an invasive migratory trajectory in a spatially constrained environment has important implications for understanding and treating metastatic breast cancer.;To emulate the fibrillar tumor microenvironment, we utilized microcontact printing to confine cells on narrow adhesive lines varying in width from 6 to 33 microns to capture the physiological range of fibers observed in vivo. By confining cells on these high-aspect ratio lines, pairwise interactions involving migrating cells could be observed in real-time and quantitatively analyzed using a binary collision response. Our results showed metastatic breast cancer cells undergo invasive "sliding" collisions across all line widths with high efficiency while non-transformed epithelial cells exhibit only moderate levels of sliding on broad micropatterns.;To investigate the potential correlation between sliding ability and metastatic potential, we used a series of known cancer-promoting molecular perturbations and measured the resulting contact response behavior of cells on our fiber-like platform. To quantitatively compare the relative influence of these perturbations, we defined the characteristic fiber-like dimension (CFD) as the hypothetical line width required to achieve intermediate levels of sliding. Our results demonstrate the accumulation of cancer-promoting perturbations in epithelial cells leads to a progressive acquisition of sliding ability and thus a reduction in CFD toward the metastatic phenotype. In addition, we identified transforming growth factor beta-1 (TGFbeta) as a potent inducer of sliding ability on narrow and intermediate line widths. While an overt EMT was not required for sliding, we found TGFbeta-induced overt EMT had a superimposable effect on sliding. Varying the dose and duration of TGFbeta treatment, we show the extent of EMT as measured by E-cadherin expression was correlated with sliding ability and was characterized using the CFD. These results provide further evidence that hybrid epithelial/mesenchymal states that occur throughout EMT are correlated with the progressive acquisition of an invasive phenotype.;We next investigated the heterotypic contact response behavior of migrating cancer cells on fiber-like micropatterns. As cancer cells disseminate from the primary tumor they encounter a variety of cells within the TMEN. In addition, the fibrillar dimensions of the breast TMEN are most relevant at the tumor-stromal boundary where the likelihood of a heterotypic interaction is higher. To maintain an invasive trajectory, cancer cells must circumnavigate non-transformed epithelial cells, fibroblasts, macrophages, and immune cells found within the TMEN. To study heterotypic pairwise interactions we first tuned the surface chemistry used to prepare our fiber-like micropatterns. A silane-based, covalent surface chemistry approach enabled fluorescence microscopy so that labeled non-transformed cells could be distinguished from unlabeled metastatic cancer cells during heterotypic collisions. Quantitative analysis of these heterotypic interactions found metastatic breast cancer cells preferentially maintain their migratory trajectory and slide around non-transformed mammary epithelial cells in confined microenvironments. Furthermore, we analyzed pairwise interactions involving metastatic ovarian cancer cells and observed the same collision response as breast cancer cells. These results led us to conclude that breast and ovarian cancer cells are particularly well tuned to the fibrillar microenvironment in which they are accustomed to invading.;Collectively, the results presented within this thesis advance our understanding of how the fibrillar TMEN contributes to local invasion during metastasis. Our work shows the extent of fiber maturation within the TMEN conspires with metastasis-promoting molecular perturbations to enhance the invasive phenotype of cancer cells. We show that the accrual of multiple molecular perturbations enhances this invasive phenotype in non-transformed cells and propose the characteristic fiber-like dimension (CFD) as a novel metric to quantify and compare metastatic potential. The controlled molecular perturbations confirmed a direct correlation between the ability of cells to slide in vitro and the ability of cells to metastasize in vivo , thus validating this platform as a potential preclinical drug-screening tool to guide future therapeutic strategies to treat metastatic breast cancer.