Role of Cytoskeleton in Regulating the Organoid Formation of Human Colorectal Cancer

Yu-Ching Huang黃郁晴1, Yi-Ting Lee 2, Jui-I Chao 1, 2, 3

1 Institute of Molecular Medicine and Bioengineering, National Yang Ming Chiao Tung University

 2Department of Biological Science and Technology, National Yang Ming Chiao Tung University

3Center For Intelligent Drug Systems and Smart Bio-devices, National Yang Ming Chiao Tung University 

Colorectal cancer (CRC) is one of the most common malignancies with high mortality. Organoid model is currently being an important platform for cancer research that can mimic morphology, intercellular communicating situation, and organization in original tissues. Cytoskeleton and cell adhesion play crucial roles in maintaining the cell morphology and cell polarity that may regulate organoid formation. However, the mechanism of cytoskeleton in regulating organoid formation remains unclear. In this study, we established a 3D culture cell model of CRC patient-derived organoid to explore the role of cytoskeleton in the organoid formation. The CRC organoids were separated into single cells and then cultured in Matrigel to observe the progression of organoid formation. The single cell formed small cyst and premature organoid with small lumen at day 7, and matured with large and hallow lumen at day 14. The protein levels of β-tubulin, E-cadherin, and β-catenin were increased gradually during the lumen formation, but the β-actin protein levels did not altered. Interestingly, β-actin proteins were located on the apical membrane that near the lumen of the organoid. Moreover, the levels of Rho/ROCK pathway-related proteins which mediate actin filament stabilization were increased. Various cytoskeleton inhibitors were investigated on the effects of organoid formation. Paclitaxel caused the loose organoid structure with polymerized microtubule accumulation. Besides, colchicine destroyed the organoid lumen structure, and induced apoptotic cells in the lumen. PT262, a ROCK inhibitor, induced the structure alteration of organoids. Together, these findings demonstrate that inhibition of cytoskeleton function can inhibit cancer organoid formation, which may provide the potential strategy for CRC therapy.