Colorectal cancer (CRC) is one of the most diagnosed cancer worldwide. Though several treatment strategies are available today, around a million people die of this disease each year worldwide. Thus, there is still a pivotal need for new and more effective treatments. Intra-tumor cellular heterogeneity has emerged lately as an intrinsic feature of malignancies and as one of the factors responsible for therapy resistance or tolerance. However, the role that phenotypically distinct subpopulations have on tumor biology is far from being understood. This multi-disciplinary proposal is based on the use of cutting-edge techniques to unravel the basic features of intra-tumor phenotypic heterogeneity and subpopulations interactions. The goals are to (1) identify and characterize phenotypically distinct subpopulations on a set of metastatic CRC human-derived organoids by single-cell RNA sequencing; (2) monitor their plasticity (transitions) by generation of fluorescent reporter lines and live-imaging lineage tracing; (3) study the impact of each subpopulation on tumor growth, and identify underlying molecular pathways by live-imaging. Ambitiously, this project aims at witnessing phenotypic transitions in living cells, a challenging task that has not been fully accomplished yet, with relevant therapeutic consequences. It is expected that this project will provide the scientific community with a deeper understanding on subpopulations dynamics in colorectal cancer, that could ultimately result in the discovery of novel treatment strategies, such as targeting a specific subpopulation or interfering with molecular determinants of transitions. Moreover, it will foster the independent career of the applicant as a researcher by expanding her skills and international network. This project tackles a relevant problem of modern cancer biology by employing innovative techniques, complemented with state-of-the-art facilities and high quality training and mentoring.