Abstract B183: Formation of adherent multicellular spheroids through migration of cells without use of the extracellular matrix components: Spheroids characterization and application in the development of antitumor agents
Authors: Waki Atsuo , Yoshii Yukie , Yoshida Kaori , Ozawa Satoshi , Hiroyama Masami , Kakezuka Anna , Sakamoto Ruriko , Shimomura Manami , Tanaka Satoru , Fujibayashi Yasuhisa , Nakatsura Tetsuya 
Published: June 2012

Abstract
Recently, multicellular spheroids (MCS) of tumor cells have been considered to be very useful in vitro system for the study of tumor biology. Two the most used methods for MCS formation include either use of gels (such as Matrigel, agar) or non-adhesive plates, which can be additionally coated (such as collagen, poly(HEMA)). Although these methods are very useful for tumor studies, they may not be suitable for the development of antitumor agents because of the low reproducibility, complex handling, and low cell viability.
We introduce a new method to obtain adherent MCS without the use of the extracellular matrix. Selected nano-size structures on the bottom of the plates (nano culture plate, NCP) enable formation of MCS with consistent quality. Spheroids formed on the NCP were characterized and compared to a monolayer cultures.
Findings are summarized as follows: i) Formation of MCS on NCP: over 60 types of cells successfully formed MCS on the NCP. The time-lapse imaging revealed that MCS on the NCP were formed by cell migration and merge of cells and small size spheroids into MCS. SEM observation of HeLa cells showed increase of extracellular matrix expression on the surface of MCS and revealed formation of the spheroid “foot” attached to the plate pattern. ii) Morphology: MCF7 showed lumen-like structure on day 2–3, which gradually became occupied with cells. TEM analysis of MCF7 indicated that polarized cells formed lumen. iii) Growth: cells cultured on the NCP proliferate at almost the same rate as monolayer culture. Proliferation is abolished on day 10 and the cell number remains constant at least on day 14. iv)Viability: the HT-29 cell viability of MCS was compared on the NCP and other 3D cell culture systems. It was found that the viability was the highest in a monolayer culture, followed by spheroids on the NCP and significantly lower in spheroids on U-bottom and ultra-low adhesion plate. v)Gene expression analysis: Gene expression analysis of HCT116 MCS on day 10 were performed and compared to monolayer cells on day 3. Over 1450 genes had 2-fold or higher expression in MCS on the NCP and expression of 1,350 genes decreased by at least 0.5 fold. MCS of HT-29 cells transfected with HRE-GFP reporter gene revealed the activation of hypoxia-inducible factor inside the MCS cultured under normoxic condition. vii) Drug sensitivity: Different drug sensitivity was observed between a monolayer and the MCS on the NCP; MCS of human breast cancer cell lines on NCP showed higher sensitivity to Herceptin, but lower to Paclitaxel. PI-3kinase inhibitors, PX866 and Wortmannin were more effective on MCS compared to the monolayer.
Conclusion: Adherent multicellular spheroids were obtained from over 60 types of cancer cell lines cultured on the NCP. Heterogeneous microenvironment inside MCS and the tight cell-cell interaction are considered to be the cause of the difference in gene expression, resulting in the different sensitivity of antitumor drugs, compared to monolayer. This method might enhance the use of MCS in the screening of antitumor agents as well as in the analysis of tumor biology and molecular targets of tumors.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B183.