NEA: Matrigel Overlay Differentiates Invasiveness in vitro. For this technique, artificial wounds were created with a silicone-tipped drill press to leave uniform, circular "nests" within a confluent monolayer of MCF10A or CA1d cells in Petri dishes, and Matrigel (25% or 50%) was added where indicated. Time-lapse images (0, 10, 22, 36 h) of expanding nests were obtained using a Zeiss Axiovert 200 M microscope equipped with a Hamamatsu ORCA-ER CCD camera (2.5×; scale bars = 500 μm). Nest expansion was calculated using ImageJ after applying thresholding and pseudo-color (red) functions to images. All values are presented as the mean ± standard deviation for each cell line, to reflect the fold increase in pixel number of each nest captured at each time point of interest, compared to the same nest at time point 0 h. (A) In the absence of the Matrigel overlay, the non-aggressive MCF10A cell line expanded significantly more than the aggressive, invasive CA1d cells after 10 h (N = 8; P = 0.03). However, time points 22 h and 36 h were immeasurable (results not shown) because all nests fully expanded into the outer ring of the remaining cell monolayer after this duration of incubation. (B/C) In contrast, in the presence of 25% or 50% Matrigel, CA1d nests expanded significantly more than MCF10A nests at all time points measured after 0 h (N ≥ 8; P < 0.05 in all cases). These results suggest that the ECM-like component is key to capturing cell lines' invasive potentials, at least for these cells.