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Establishment of a Three-dimensional Floating Cell Culture System for Screening Drugs Targeting KRAS-mediated Signaling Molecules.

Anticancer research (2015-07-15)
Toshiyuki Tsunoda, Shuhei Ishikura, Keiko Doi, Yuri Iwaihara, Hiromasa Hidesima, Hao Luo, Yumiko Hirose, Senji Shirasawa
ABSTRACT

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. 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. 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. This 3DF culture system could be useful for screening drugs that target KRAS-mediated signaling molecules.

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