Fig. 5

Matrix reorganization associated with malignant cell invasion. (a) Maximum intensity z-projections (40×) of multicellular tumor spheroids composed of epithelial MCF-10A cells (red) and malignant MDA-MB-231 cells (green), embedded within 1.5 mg ml⁻¹ collagen matrix (white fibers imaged by confocal reflectance microscopy) and imaged immediately after embedding (0 h, left panels) and following culture (48 h, middle panels). Regions of interest at 48 h are indicated in middle panels by white boxes and are magnified 3× in right panels. Confocal reflectance images in right panels were colorized with OrientationJ as described in Materials and Methods. White arrowheads highlight areas of collagen fiber alignment. Inset in center panel is cross-sectional view along the invasive strand. Black arrowheads indicate boundaries of matrix microtrack generated by invading malignant leader cells. (b) Quantitative analysis of collagen fiber alignment relative to the spheroid surface. Briefly, local collagen fiber orientations (θ_Fiber) were determined with OrientationJ and compared to the angle given by the original spheroid surface (θ_Sph), resulting in alignment distributions representative of all fibers in the regions of interest. Scale bars = 50 μm