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Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening
Published on: September 5, 2018
Establishment of a Three-dimensional Floating Cell Culture System for Screening Drugs Targeting KRAS-mediated
Toshiyuki Tsunoda1, Shuhei Ishikura1, Keiko Doi1
1Department of Cell Biology, Faculty of Medicine Fukuoka University, Fukuoka, Japan Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka, Japan.
Background/Aim:
Oncogenic mutations in the KRAS gene are critically involved in many human tumors but drugs targeting oncogenic KRAS have not yet been clinically developed. Herein, we established a three-dimensional floating (3DF) culture system for screening drugs that target KRAS-mediated signaling molecules.
Materials And Methods:
HKe3 cells, derived from colorectal cancer HCT116 cells and disrupted at mutated (mt) KRAS gene, were infected with a retrovirus expressing wild-type (wt) KRAS or mtKRAS to establish HKe3-derived cells expressing wtKRAS or mtKRAS. Established cells were cultured in 96-well plates with an ultra-low attachment surface and round bottom for 3DF culture.
Results:
HKe3-wtKRAS and HKe3-mtKRAS cells in 3DF culture rapidly assembled into respective single spherical structures (spheroids). Furthermore, mtKRAS but not wtKRAS expression inhibited luminal apoptosis in spheroids indicating that the 3DF culture was compatible with the 3D matrigel culture.
Conclusion:
This 3DF culture system could be useful for screening drugs that target KRAS-mediated signaling molecules.
Insights
A novel three-dimensional floating (3DF) culture system effectively screens drugs targeting KRAS signaling. This system aids in developing therapies for KRAS-mutated cancers by enabling efficient drug discovery.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Oncogenic KRAS mutations are key drivers in numerous human cancers.
- Targeting oncogenic KRAS remains a significant challenge in cancer therapy development.
Purpose of the Study:
- To establish and validate a three-dimensional floating (3DF) culture system for screening drugs targeting KRAS-mediated signaling pathways.
- To provide a novel platform for the development of KRAS-targeted cancer therapies.
Main Methods:
- Developed HKe3-derived cells expressing either wild-type (wt) or mutated (mt) KRAS.
- Utilized a 96-well plate system with an ultra-low attachment surface for 3D floating (3DF) culture.
- Assessed spheroid formation and apoptosis in response to KRAS expression within the 3DF system.
Main Results:
- HKe3 cells expressing wtKRAS or mtKRAS rapidly formed spheroids in the 3DF culture.
- Mutated KRAS expression, but not wild-type KRAS, inhibited apoptosis in spheroids.
- The 3DF culture system demonstrated compatibility with 3D matrigel culture, indicating its robustness.
Conclusions:
- The established 3DF culture system is a viable tool for screening drugs targeting KRAS-mediated signaling molecules.
- This platform holds promise for accelerating the development of novel therapeutics for KRAS-driven cancers.
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