The choice of gene targets (as well as stromal cells) thus should conform to the cancer model of interest, and will have significant impact on the drug discovery. == Epithelial cancer progression is associated with an evolving tissue interface of direct epithelial-stromal interactions1. In human tumor biopsies, extensive gene expression changes are correlated with cancer staging on both sides of the tumor-stroma interface2,3; importantly, some epithelial-mesenchymal transition (EMT) signatures are preferentially expressed NSC-207895 (XI-006) by cancer cells close to the interface, while interfacial stromal fibroblasts promote EMT more effectively than those extracted from the bulk millimeters away4,5. It is however extremely difficult to NSC-207895 (XI-006) clarify the exact, cell-specific contribution of tumor-stromal interactions in the development of this structure-function relationship in cancer progressionin vivobecause of a lack of experimental control6. Conventionalin vitromodels use random or transwell co-cultures to study contact- or soluble factor-mediated tumor-stromal signaling and screen for new drugs7,8. However, in real tumors, cells at the tissue bulk and interface can be simultaneously and differentially influenced by the extent of heterotypic cell-cell contact and the long/short-range diffusion of soluble factors9. These models that indiscriminately mix two or more cell types cannot resolve this critical spatial perspective of tumor-stromal interactions, nor accurately assess drug action mechanisms in the heterogeneous cell compartments in the bulk and at the interface. Micro-engineered cell cultures have emerged as powerful platforms to model processes in tissue microenvironments at appropriate length scales and identify their impact on cell morphogenesis and differentiation10,11,12,13. Yet, the downstream analysis of micro-engineered cultures (as well as conventional cultures) has largely relied on resource-demanding immunocytochemistry, or mechanochemical cell isolation to understand cell-specific phenomena which introduces additional experimental artifacts and results in a loss of information on cells original location. Microscopy-based laser capture has been used to retrieve cells in micropatterns for gene expression analysis14. However, the spatial resolution of the technique was not fully leveraged, and its combined use with micro-engineered cell co-cultures to understand spatially-defined signaling in cancer progression and drug actions has not been demonstrated to-date. A micropatterned tumor-stromal assay (TSA) is established to organize tumor and stromal cells into distinct, spatial compartments with a defined heterotypic cell interface. By integrating TSA with microscopy and laser capture microdissection (LCM), we enable cell-specific analysis of phenotypes and gene expressionin situwith precise spatial resolution. Using TSA, we reveal a preferential instigation of malignant tumor-stromal signaling by bone marrow fibroblasts. Tumor cell expression profiles in Rabbit Polyclonal to FGFR2 TSA are benchmarked against human ER+ breast cancer tissue and found to have 63% concordance using a defined set of genes related to cancer progression. The co-culture system is further adapted to evaluate a new mechanism of action by known cancer therapeutics to disrupt tumor-stromal interfacial interactions with prediction of TSA observationsin NSC-207895 (XI-006) vivo. Below, we present TSA as a tool to explore a new frontier of tumor-stromal interfacial interactions with the underlying hypothesis that bulk and interfacial tumor cells can be differentially influenced by the type of neighboring stromal cell or a pharmacological intervention. == Results == == Micropatterning to control a tumor-stroma interface == Micropatterned tumor-stromal assays (TSAs) were designed to organize tumor and stromal cells into distinct, spatial compartmentsin vitrowith a defined heterotypic cell interface by a stencil micropatterning technique12,15(Fig. 1a), mimickingin vivoconstraints on contact- and paracrine-signaling in the context of a growing tumor-stroma boundary layer. A cell-repellent, silicone mask was created with circular apertures that were cut by laser to form a cell culture stencil. The stencil mask defined the shape and size of areas where cancer cells initially.