Related Experiment Video
Updated: Mar 3, 2026
Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells
Published on: March 10, 2023
A genome editing approach to study cancer stem cells in human tumors
Carme Cortina1, Gemma Turon1, Diana Stork1
1Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain.
Abstract:
The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, 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 EGFP and lineage-tracing cassettes knocked in the LGR5 locus. Analysis of LGR5-EGFP+ 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 toward mucosecreting- and absorptive-like phenotypes. These genetic experiments confirm 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.
Insights
Researchers engineered colorectal cancer (CRC) organoids to track cancer stem cells. This method reveals CRC hierarchical organization, similar to normal intestinal stem cells, aiding tumor heterogeneity studies.
Area of Science:
- Cancer Biology
- Stem Cell Research
- Genomic Engineering
Background:
- Identifying and tracking cancer stem cell populations in intact human tumors is challenging.
- Understanding tumor heterogeneity and hierarchical organization is crucial for effective cancer therapy.
Purpose of the Study:
- To develop a novel strategy for analyzing stem cell hierarchies in human cancers.
- To engineer patient-derived colorectal cancer (CRC) organoids for tracking tumor cell populations.
- To investigate the self-renewal and differentiation potential of LGR5+ CRC stem cells.
Main Methods:
- Utilized CRISPR/Cas9 gene editing technology to engineer patient-derived tumor organoids.
- Integrated EGFP and lineage-tracing reporter cassettes into the LGR5 locus of CRC organoids.
- Analyzed LGR5-EGFP+ cells from organoid-derived xenografts and performed lineage-tracing experiments.
Main Results:
- Engineered LGR5-EGFP+ CRC organoids exhibit gene expression patterns similar to normal intestinal stem cells.
- LGR5+ CRC cells efficiently propagate disease in recipient mice.
- Lineage tracing demonstrated self-renewal and differentiation of LGR5+ CRC cells into mucosecreting and absorptive phenotypes.
Conclusions:
- Human colorectal cancers exhibit a hierarchical organization analogous to the normal colonic epithelium.
- The developed genomic engineering strategy provides a powerful tool for studying cell heterogeneity in human tumors.
- This approach has broad potential applications in cancer research and therapeutic development.
