Figure 2.

Download video file ^{(1.4MB, mov)}

Characterization and quantification of growth behavior from time lapse microscopy correlated with analysis of longitudinal darkfield image data (shown in Fig. 1). (a) Individual frames from time-lapse microscopy acquired from 4 h following plating until 4 days following plating (the full time-lapse sequence is included as a video). Yellow arrows indicate clusters of cells that rapidly migrate over the Matrigel surface and assemble to form larger multicellular acini. The green box indicates a representative cell that does not participate in assembly events in the immediate vicinity, but does undergo cell division. Scale bars=100 μm. (b) Schematic representation of the bimodal growth kinetics described in Fig. 1 and (a). Approximately 30% of cells (gray), exhibit low motility and little potential to migrate toward other clusters of cells. The other 70% of cells (beige cells) exhibit a much more aggressive growth behavior, rapidly forming larger and larger multicellular aggregates. The sequential numbering 1, 2, and 3 in the diagram indicates (1) single cells following plating that (2) form small clusters and (3) subsequently form larger multicellular clusters. Inset: confocal immunofluorescence section from the center of a nodule fixed and stained for E-cadherin (red) and DAPI (blue) at day 10 of growth to reveal large multicellular 3-D structures formed from assembly and division of individual cells on the gel surface. Scale bar=20 μm. (c) Assembly events lead to an overall decay in the spatial density of 3-D acini with a decay constant of 5.9 days⁻¹. Error bars in (c) are standard deviations of density measurements from five fields, each of three to nine independent prepared culture dishes imaged at a given time point. (d) Exponential decay in density is concomitant with an increase in mean diameter over time of rapidly growing nodules [corresponding to s_c2 from Gaussian fits in Fig. 1 and Eq. 1 of the text]. Mean diameter remains constant in the remaining nodules [s_c1 in Eq. 1]. Error bars in (d) are derived from the widths of Gaussian fits shown in Fig. 1. (QuickTime, 1.45 MB) .