Computer simulations based on the Hybrid Discrete-Continuous (HDC) mathematical model of cancer invasion (Anderson et al., Cell. 2006,127:905) predict that the degree of severity of the tumor microenvironment (tmE) directly impacts on the emergence of invasion. More precisely, harsh ME conditions (e.g., hypoxia, discontinuous matrix, inflammation) select for dominant aggressive clones that grow into a fingering, infiltrating mass. In contrast, in mild ME conditions (normoxia, homogenous matrix) selection for dominant aggressive clones is relaxed, so that they coexist with less aggressive ones and, together, they grow into a smooth-margin, noninvasive tumor mass. To populate HDC simulations with experimental data, we use a panel of cell lines, derived from the breast epithelial cell MCF10A. We have established a collection of variants of this platform cell line with distinct invasive potential, generated by transfection of oncogenes or passaging in vivo. We measured the following parameters: oxygen consumption/hypoxia, tumor cell proliferation, tumor cell survival, metabolic consumption, and matrix degrading enzyme activity. Initial simulations with these homogenous data indicate that invasion may require competition between phenotypes with distinct adaptive traits to the tmE. To validate these predictions in vitro, we have developed a novel Island Invasion Assay (IIA), which closely mimics the spatial arrangements of the HDC model. Preliminary results suggest that IIA supports the HDC predictions concerning invasion (fingering) under stressful tmE conditions. In addition, some unexpected results point to novel features that could be included in the HDC model to increase realism. This is an excellent example of synergistic interactions between modeling and experimentation, which will hopefully produce novel insights in the mechanisms underlying cancer invasion. For in vivo validation, we are comparing orthotopic (“mild ME”) versus subcutaneous (“harsh ME”) human breast cancer xenografts in mice (in vivo VICBC group, headed by Lisa McCawley). We utilized MCF-10 variant cell lines, CA1a and CA1d, shown to have distinct and consistent tumorigenic properties through repeated passage in immunocompromised mice. Several in vivo imaging modalities are being exploited to provide quantitative analysis of cellular parameters of the same tumor over time: Magnetic Resonance Imaging (MRI) analysis to distinguish tumor volume and necrotic tumor areas (i.e. nonoxygenated states) from viable (i.e. oxygenated) tissue; positron emission topography (PET) scan imaging of 18F-labeled-fluorodeoxyglucose(FDG) for metabolism; Optical imaging analysis of fluorogenic probes termed “proteolytic beacons” for matrix degrading protease activity (i.e. substrate hydrolysis). Tumor specimens are also biopsied at fixed volumes (0.5, 1 and 1.5 cm diameters) for further ex vivo analyses, including histology to assess invasion, immunohistochemistry and immunofluorescence to measure cellular proliferation (BrdU incorporation), apoptosis (TUNEL) and hypoxia (hypoxiprobe). Initial results from these analyses reveal advantages and limits of in vivo parameterization and validation. The main advantage is that relevant variables and parameters for tumor invasion can only be truly identified in a living organism, at least until convincing in vitro surrogates for spatial invasion, such as the IIA, are developed. The limits reflect largely the fact that conventional experimental biology has been seldom used for model validation, so that tools and approaches must be refined and adapted. These broad issues as well as intitial specific data will be discussed.