Background <p>Phenotypic plasticity is a major factor in tumor heterogeneity and treatment resistance. In particular, cancer stem cells (CSCs) represent a small subpopulation within tumors that possesses self-renewal and tumor-forming capabilities. Understanding reprogramming, maintenance, and lineage properties of CSCs requires dedicated tools to disentangle the respective influences of phenotypic inheritance and cell-cell interactions.</p> Methods <p>Here, we set up ultra-wide field microscopy to image breast cancer cell lines expressing a stemness fluorescent reporter over several days. The fluorescent reporter distinguishes three phenotypes: CSCs, cancer differentiated cells (CDCs), and intermediate/transiting cancer cells (iCCs).</p> Results <p>Spatial statistics indicate significant zonation in which CSCs cluster together and are spatially separated from CDCs, forming patterns resembling niches. Surprisingly, single-cell time series reveal spontaneous reprogramming events from CDC to CSC even in unperturbed populations. We identify that such transitions are prone to arise during the cell cycle. Moreover, lineage analysis shows that the phenotype is partially inherited from ancestor cells. However, such heredity is not sufficient to explain the spatial properties of the cell population, which also depend on cell-cell interactions. Indeed, we find that phenotypic transitions of cancer cells are influenced by the phenotypic state of neighboring cells. Reprogramming into CSCs is respectively promoted and inhibited by the presence of CSCs and CDCs in the neighborhood.</p> Conclusions <p>Altogether, our results disentangle how phenotypic inheritance and intercellular interactions orchestrate the spatio-temporal self-organization of cancer cell heterogeneity, maintaining a subpopulation of CSCs within niches.</p>