ena-STUDY-IRB Barcelona-30-12-2016-09:58:19:026-632
A genome editing approach to study cancer stem cells in human tumors
Many prevalent cancers retain features of the tissue from which they originate including tumor cells that adopt stem- or differentiation-like states. In human cancers, the analysis of stem cell hierarchies has been hampered by the impossibility of identifying or tracking tumor cell populations in their natural environment. To overcome these limitations, we devised a strategy based on editing the genomes of patient-derived tumor organoids using CRISPR/cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry a GFP reporter cassette knocked in the LGR5 locus. Analysis of LGR5-GFP+ cells isolated from organoid-derived xenografts demonstrated that these cells express a gene program similar to that of normal colon stem cells and that upon transplantation into recipient mice, they propagate the disease very efficiently. We also generated CRC organoids that express a tamoxifen-inducible Cre recombinase (creERT2) driven by LGR5 locus and a cre-inducible lox-STOP- lox-Tomato reporter cassette integrated at the AAVS1locus. Using these double edited organoids we performed lineage tracing analysis in human tumors for first time. We showed that LGR5+ CRC cells self-renew and generate progeny over long time periods that undergo differentiation to mucosecreting- and absorptive-like phenotypes. This genetic experiment confirms that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.
Many prevalent cancers retain features of the tissue from which they originate including tumor cells that adopt stem- or differentiation-like states. In human cancers, the analysis of stem cell hierarchies has been hampered by the impossibility of identifying or tracking tumor cell populations in their natural environment. To overcome these limitations, we devised a strategy based on editing the genomes of patient-derived tumor organoids using CRISPR/cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry a GFP reporter cassette knocked in the LGR5 locus. Analysis of LGR5-GFP+ cells isolated from organoid-derived xenografts demonstrated that these cells express a gene program similar to that of normal colon stem cells and that upon transplantation into recipient mice, they propagate the disease very efficiently. We also generated CRC organoids that express a tamoxifen-inducible Cre recombinase (creERT2) driven by LGR5 locus and a cre-inducible lox-STOP- lox-Tomato reporter cassette integrated at the AAVS1locus. Using these double edited organoids we performed lineage tracing analysis in human tumors for first time. We showed that LGR5+ CRC cells self-renew and generate progeny over long time periods that undergo differentiation to mucosecreting- and absorptive-like phenotypes. This genetic experiment confirms that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.