The image analysis allowed us to perform a multiscale analysis (16) of the MBs. For each chip, single images of the anchors were acquired automatically with the motorized stage of the microscope. The analysis was conducted on a montage of the detected anchors using a custom MATLAB code (r2016a, MathWorks, Natick, MA). Two distinct routines were used: one with bright-field detection and one for the fluorescence experiments.

For the bright field-detection described previously (16), the cells were detected in each anchor as pixels with high values of the intensity gradient. This allowed for each cell aggregate to compute morphological parameters such as the projected area A and the shape index SI that quantifies the circularity of an objectSI=4π×APwhere P is the perimeter. Shape index values range from 0 to 1, with 1 being assigned for perfect disk.

The MB detection with fluorescence staining (DAPI/Casp3/COX-2, DAPI/phalloidin, DAPI/N-cadherin, or LIVE/DEAD) was performed as described previously (16). First, morphological data were extracted at the MB level, such as the equivalent diameter of the MBs or the shape index. Also, the mean fluorescence signal of each MB was defined as the subtraction of the local background from the mean raw intensity.

At the cellular level, two different methods were used, both relying on the detection of the nuclei centers with the DAPI fluorescence signal. On the one hand, each cell location could be assigned to a normalized distance from the MB center (r/R) to correlate a nuclear fluorescence signal with a position in the MB, as previously described (16). On the other hand, the cell shapes inside the MBs were approximated by constructing Voronoi diagrams on the detected nuclei centers. Basically, the edges of the Voronoi cells are formed by the perpendicular bisectors of the segments between the neighboring cell centers. These Voronoi cells were used to quantify the cellular cytoplasmic signal (COX-2, F-actin and N-cadherin, VEGF and RUNX-2). In detail, to account for the variability of the cytoplasmic signal across the entire cell (nucleus included), the fluorescence signal of a single cell was defined as the mean signal of the 10% highest pixels of the corresponding Voronoi cell.

Image processing was also used to get quantitative data on 2D cultures, as previously described (16). Last, different normalization procedures were chosen in this paper. When an effect was quantified compared with a control condition, the test values were divided by the mean control value, and the significance was tested against 1. For some other data, the values were simply normalized by the corresponding mean at the chip level to discard the interchip variation from the analysis.

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