Toshiyuki Tsunoda1, Shuhei Ishikura1, Keiko Doi1, Yuri Iwaihara2, Hiromasa Hidesima2, Hao Luo2, Yumiko Hirose1, Senji Shirasawa3. 1. Department of Cell Biology, Faculty of Medicine Fukuoka University, Fukuoka, Japan Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka, Japan. 2. Department of Cell Biology, Faculty of Medicine Fukuoka University, Fukuoka, Japan. 3. Department of Cell Biology, Faculty of Medicine Fukuoka University, Fukuoka, Japan Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka, Japan sshirasa@fukuoka-u.ac.jp.
Abstract
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. Copyright
BACKGROUND/AIM: Oncogenic mutations in the KRAS gene are critically involved in many humantumors 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. Copyright
Authors: Theodosia Charitou; Sriganesh Srihari; Miriam A Lynn; Mohamed-Ali Jarboui; Erik Fasterius; Max Moldovan; Senji Shirasawa; Toshiyuki Tsunoda; Marius Ueffing; Jianling Xie; Jin Xin; Xuemin Wang; Christopher G Proud; Karsten Boldt; Cristina Al-Khalili Szigyarto; Walter Kolch; David J Lynn Journal: Br J Cancer Date: 2019-05-28 Impact factor: 7.640