Fig 1.
Fixed and stained in vitro images.
A: GFP, RFP channel overlay. B: DAPI, CY5 (Ki-67) channel overlay. C: Individual channels from the inset region shown in 1A. Scalebar = 1 mm.
Fig 2.
Image processing steps for in vitro images.
A: Nuclear staining (DAPI channel). B: Preprocessing steps and distance transform identifies watersheds. C: Segmentation of colonies by watershed algorithm. D: Individual colonies analyzed. E: DsRed and GFP signal captured in RFP and GFP channels respectively. F: DsRed+/GFP+ regions obtained by preprocessing steps and binarization. G: Segmented colonies classified as DsRed+/ GFP+ based on input from images F1 and F2. G: Cell count and area of individual colonies obtained from colony-level DAPI image. I: Ki-67 staining imaged in the CY5 channel. J: Regions of MCF7 cells expressing Ki-67 obtained by binarization. Scale bars: 5 mm (A, E, I, J), 1 mm (G, D).
Fig 3.
A: DAPI Image. B: Segmented Nuclei. C: Ki-67 staining image (CY5 Channel). D: Binarized Ki-67 Stain. E: Overlap of B and D, to identify Ki-67+ nuclei (white arrows) and Ki-67- nuclei (red arrows). Scale bars = 50 microns.
Fig 4.
A: Colony size distributions 2 days and 4 days after plating. The Y-axis shows probability density (relative probability) and the X-axis shows colony size in number of cells. Mean and standard deviations are shown for populations of colonies (n >150, per well) obtained from a set of 3 wells of a 6 well-plate and represent biological replicates. Significance was determined using the two-tailed Wilcoxon rank-sum test. B: Cumulative frequency distribution of Ki-67 index over a population of colonies (n >150, per well) obtained from a set of 3 wells of a 6 well-plate. Dashed lines show Ki-67 index value of 0.8. C: Fraction of cells with Ki-67 index > 0.8, showing mean and standard error over 3 wells. Significance was determined using unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001.
Fig 5.
A: The probability density functions of colony sizes as they change over the duration of the live cell experiments. Mean and standard deviations of probability distribution functions are shown, and represent biological replicates obtained from 3 wells of a 6 well-plate. Number of colonies > 150 per well. The X-axis shows colony area and estimated cell counts. Significance between mean ranks was determined using the Kruskal-Wallis test (*** p<0.001).
Fig 6.
Image processing steps for in vivo images.
A: Overall scheme for segmenting colonies in mice lung tissue sections. B: Flow diagram listing individual steps in the MATLAB script.
Fig 7.
A: Composite RFP/GFP image. B: Metastatic colonies were identified. C: Colony size distribution of DsRed+ and GFP+ metastatic colonies in a single lung. D: Mean Probability Densities and standard deviations of GFP+ and DsRed+ colonies, from a set of three mice. Number of DsRed+ or GFP+ colonies in each mice section are greater than 10. No significant difference was obtained between the median colony sizes using the two-tailed Wilcoxon rank-sum test.
Fig 8.
A: Validation of metastatic counts. Each red/green point represents DsRed+/GFP+ metastatic colonies in one lung section respectively. B: Metastatic colonies and corresponding segmented areas.
Fig 9.
Intra-mouse vs mouse-to-mouse variation.
A: Metastatic counts in intra-mouse (N = 8) and inter-mouse (N = 5) lung tissue sections. Blue data point represents mouse used for generating intra-mouse lung sections. B: Metastatic counts from the intra-mouse set, arranged according to distance across the lung width.