Fractal Analysis of Mixed Culture MCF10A and CA1d Cell Lines. (A) Nests were created with MCF10A-GFP alone, MCF10A-GFP mixed with either unlabeled MCF10A or CA1d cells (1:1), and with CA1d alone. Nests were then overlaid with 50% Matrigel and allowed to expand in either the presence or absence of serum for 36 or 28 h, respectively. Scale bar is 1000 μm. (B) In the presence of serum, CA1d nests expanded significantly more than MCF10A after 36 h (N = 8; P < 0.001, in all cases), and 1:1 MCF10A+CA1d colonies expanded at an intermediate rate. Similar results were observed in the absence of serum. (C) Magnified images (10×) of nests with fluorescent labels for visualization of individual cell types (pre-labeled MCF10A-GFP (green); final nests were fixed/stained with rhodamine-phalloidin to mark actin filaments and visualize CA1d cells (red). Importantly, in the mixtures of MCF10A-GFP and CA1d in normal culture conditions (+ serum), there is clear trapping of groups of MCF10A cells in the inner portion of the nests by expanding CA1d cells (upper panel). Serum deprivation in the space-constrained environment gives a similar result, except that the difference between CA1d and MCF10A-GFP expansion is amplified (lower panel). Scale bar is 600 μm. (D) Fractal dimension measures confirm the differences observed at nest borders across MCF10A and CA1d cells in the two conditions tested. Data is presented to include only the change in Df from 0 h to the final end point measured. In normal tissue culture conditions, MCF10A-GFP, MCF10A-GFP+ MCF10A (1:1), and MCF10A-GFP+CA1d (1:1) nests displayed comparable changes in Df measures from 0 h to 36 h (N = 8; P > 0.05, in all cases). CA1d cells alone led to somewhat larger measures than other nest types. In serum-deprived conditions, the separation between MCF10A and CA1d nests increased dramatically. MCF10A-GFP, MCF10A-GFP+MCF10A (1:1), and MCF10A-GFP+CA1d (1:1) nests again displayed comparable Df measures from 0 h to 28 h (N = 8; P > 0.05, in all cases). However, in the absence of serum, CA1d nests had drastically larger measures than all other nest types (N = 8; P < 0.001, in all cases).