Final Report Summary - EDITCRC (A genome editing-based approach to study the stem cell hierarchy of human colorectal cancers)
A hallmark of cancer is tumor cell heterogeneity, which results from combinations of multiple genetic and epigenetic alterations within an individual tumor. In contrast, most human colorectal cancers (CRCs) are composed of mixtures of phenotypically distinct tumor cells organized into a stem cell hierarchy that displays a striking resemblance to the healthy colonic epithelium. To analyze the organization of CRCs without the constraints imposed by tumor cell transplantation experiments, we have developed a method that allows for the first time tracking and manipulating the fate of specific cell populations in whole human tumors. This technology is based on editing the genomes of primary human CRCs cultured in the form of tumor organoids using CRISPR/Cas9 to knock-in either lineage tracing or cell ablation alleles in genes that define colorectal cancer stem cells (CRC-SCs). Edited tumor organoids generate CRCs in mice that reproduce the tumor of origin while carrying the desired genetic modifications. This technological advance has opened the gate to perform classical genetic and developmental analysis in human tumors. We have exploited this approach to investigate the contribution of tumor stem cells to growth and dissemination of human CRCs. Analysis of LGR5-EGFP+ CRC cells isolated from organoid-derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage tracing experiments showed that LGR5+ CRC cells self-renew and generate progeny over long time periods that undergo differentiation to mucosecreting- and absorptive-like phenotypes. Yet, we also observed that differentiated cells contribute to tumor growth as well and that there is extensive tumor cell plasticity which will complicate the development of therapies targeting CRC-SCs. Targeting essential functions of CRC-SCs may prove more effective than targeting their identity. In addition, we have characterized the existence of a subset of slow proliferating LGR5+ stem cells in normal intestinal mucosa and in CRC that resist chemotherapy and regenerate the disease after this insult. Finally, the acquisition of successive mutations is crucial to potentiate the metastatic capacity of CRC-SCs. Analyses of metastatic dissemination in quadruple mutant CRC-SCs models led us to discover a new mechanism of immune evasion driven by elevated levels of TGF-beta in the tumor microenvironment. These findings are having an immediate implementation in the clinical management of colorectal cancer patients.