Fluorescently stained cryo-sections of spheroids in 6 mg/ml collagen matrices revealed that, even for a low invasion rate, cell distribution and polarized morphological profiles were qualitatively similar to cells in spheroids inside 2 mg/ml collagen matrices (Figure 4DC4H). II/ROCK-based cell contractility. This isotropic-to-anisotropic transition corresponds to a shift in migration, from a slow and unpolarized movement at the core, to a fast, polarized and persistent one at the periphery. Our results also show that the ensuing collective invasion of fibrosarcoma cells is induced by anisotropic contractile stresses exerted on the surrounding matrix. model to recapitulate the tumor microenvironment and study the initial steps of invasion from a primary tumor [6C8]. This assay consists of embedding multicellular spheroids inside three-dimensional (3D) extracellular matrices (ECM) such as collagen I, which allows for both cell-cell and cell-ECM interactions. This 3D invasion model has been previously utilized to investigate the molecular mechanisms that govern angiogenic sprouting of endothelial spheroids inside collagen gels  and the role of MMPs in cancer cell invasion . Recent studies have shown that human fibrosarcoma cells that are well-dispersed in a matrix adopt fundamentally different strategies for migration from cells migrating on 2D substrates . However, it is unclear how fibrosarcoma cells within the tumor spheroid C in R406 (Tamatinib) which cells have close cell-cell contacts with their neighbors C may prepare cells at the spheroid periphery to present the correct morphology and polarization for effective invasion into the surrounding matrix. Here, we developed and analyzed a 3D encapsulated spheroid-matrix Lum system to investigate cancer cell invasion into an adjacent collagen matrix. We perfomed dynamic single-cell resolution measurements and report the spatial and temporal kinetics in the morphology and motility behavior of individual cells inside the spheroids. Using this model system, we characterize the invasion profiles of spheroids and identify the role of cell contractility and cell-matrix interactions as essential mediators of cancer cell invasion. We also show that cell invasion in the surrounding matrix requires a large net contractile force exerted by the spheroid on its surroundings before invasion can occur. In addition, cells move persistently toward the invasive front of the spheroid and this behavior is fundamentally different from a homogeneously distributed population of single cells embedded inside similar 3D gels. RESULTS Fibrosarcoma cell invasion and spreading from a spheroid in 3D collagen matrix Collagen I is by far the most abundant component of human connective tissues and is also the main component of the additional extracellular matrix deposited by carcinoma and sarcoma tumors in their periphery [11C13]. Here we focus on fibrosarcoma, a malignant metastatic tumor of fibrous connective tissues  using HT1080 human fibroscarcoma, a cell line used extensively in cell invasion and migration studies [10, 15C19]. To study fibrosarcoma invasion and growth in 3D microenvironments, cell spheroidsaverage initial radius of 174 mwere embedded inside 3D R406 (Tamatinib) collagen I R406 (Tamatinib) matrices (Figure ?(Figure1A1A and Supplementary Figure S2A). Open in a separate window Figure 1 Tumor spheroids are highly invasive inside 3D collagen matricesA. Schematic of the experimental procedure: tumor spheroid preparation, embedding, and invasion inside a 3D collagen matrix. B. Representative stitched phase-contrast images showing human fibrosarcoma HT1080 spheroids at 0, 3, and 6 days after embedding inside 2 mg/ml collagen matrices. The spheroid growing front was manually traced to obtain invasion distance, r. Scale bars, 500 m C. Mean time-dependent invasion distance SEM of HT1080 spheroids as a function of time. The invasion distance of HT1080 tumor spheroids was determined by measuring the distance between spheroid-matrix interface and the spheroid initial radius (= = ~ ~ with an exponent of invasion = 0.83 0.06. This result suggested that cell spheroids were highly invasive and that this invasion process was fundamentally different from the case of homogeneously distributed (individual) cells embedded in 3D collagen gels at low density which undergo the so-called anisotropic random-walk migration  and from the case of cohesively growing spheroids which switch from exponential to linear growth beyond the crossover region (200 m < < 350 m) [22, 23]. Therefore, to investigate how individual fibrosarcoma cells within the spheroid contributed to the overall invasion rate into the surrounding 3D matrix, and to take into account the local cell density, spheroids grown for 3, 5, and 7 days were cryo-sectioned at a thickness of 10 m and analyzed using quantitative fluorescence microscopy (see more details under Methods). Cells and their nuclei were visualized using DAPI staining to detect nuclear DNA and fluorescent labeling of the major cytoskeleton filamentous protein, F-actin (Figure 1D and 1E). Analysis of fluorescent images of the mid-plane sections of spheroids showed an exponential decay.