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Updated: Apr 17, 2026
Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications
Published on: March 8, 2024
Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids
Mami Matano1, Shoichi Date2, Mariko Shimokawa1
1Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.
Abstract:
Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-β, TP53 and PI3K pathways. Although these pathways influence intestinal stem cell niche signaling, the extent to which mutations in these pathways contribute to human colorectal carcinogenesis remains unclear. Here we use the CRISPR-Cas9 genome-editing system to introduce multiple such mutations into organoids derived from normal human intestinal epithelium. By modulating the culture conditions to mimic that of the intestinal niche, we selected isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, and in the oncogenes KRAS and/or PIK3CA. Organoids engineered to express all five mutations grew independently of niche factors in vitro, and they formed tumors after implantation under the kidney subcapsule in mice. Although they formed micrometastases containing dormant tumor-initiating cells after injection into the spleen of mice, they failed to colonize in the liver. In contrast, engineered organoids derived from chromosome-instable human adenomas formed macrometastatic colonies. These results suggest that 'driver' pathway mutations enable stem cell maintenance in the hostile tumor microenvironment, but that additional molecular lesions are required for invasive behavior.
Insights
Colorectal cancer pathway mutations enable stem cell growth in tumors. However, additional genetic changes are needed for cancer cells to invade and spread effectively.
Area of Science:
- Gastroenterology and Hepatology
- Oncology
- Genetics and Genomics
Background:
- Colorectal tumors frequently exhibit mutations in key signaling pathways like WNT, MAPK, TP53, and PI3K.
- The precise role of these pathway mutations in driving colorectal carcinogenesis and their impact on intestinal stem cell signaling are not fully understood.
Purpose of the Study:
- To investigate how specific genetic mutations in colorectal cancer pathways contribute to tumor formation and progression.
- To model colorectal carcinogenesis using human intestinal organoids engineered with multiple cancer-associated mutations.
Main Methods:
- Utilized CRISPR-Cas9 genome editing to introduce mutations in APC, SMAD4, TP53, KRAS, and PIK3CA genes into human intestinal organoids.
- Cultured engineered organoids under conditions mimicking the intestinal stem cell niche.
- Implanted mutated organoids into mice to assess tumor formation and metastatic potential.
Main Results:
- Organoids with all five mutations (APC, SMAD4, TP53, KRAS, PIK3CA) grew independently of niche factors and formed tumors in mice.
- These engineered tumors formed micrometastases but failed to colonize the liver.
- Organoids derived from chromosome-unstable adenomas formed macrometastases, indicating the importance of chromosomal instability for invasive spread.
Conclusions:
- Recurrent pathway mutations in colorectal cancer facilitate stem cell maintenance within the tumor microenvironment.
- Additional molecular alterations beyond core pathway mutations are necessary for the invasive and metastatic behavior of colorectal tumors.
