  In previous theoretical research, we inferred that cancer stem cells (CSCs),
the cells that presumably drive tumor growth and resistance to conventional
cancer treatments, are not uniformly distributed in the bulk of a tumorsphere.
To confirm this theoretical prediction, we cultivated tumorspheres enriched in
CSCs, and performed immunofluorecent detection of the stemness marker SOX2
using a confocal microscope.
  In this article, we present a method developed to process the images that
reconstruct the amount and location of the CSCs in the tumorspheres. Its
advantage is the use of a statistical criterion to classify the cells in stem
and differentiated instead of setting an arbitrary threshold. From the analysis
of the results of the methods using graph theory and computational modeling, we
concluded that the distribution of Cancer Stem Cells in an experimental
tumorsphere is non-homogeneous. This method is independent of the tumorsphere
assay being useful for analyzing images in which several different kinds of
cells are stained with different markers.
