Abstract 323: Spheroid-based high throughput screening for identification of molecules targeting different tumor microenvironment characteristics
Authors: Senkowski Wojciech , Zhang Xiaonan , Nygren Peter , Hägg Olofsson Maria , Gustafsson Mats , Linder Stig , Larsson Rolf , Fryknäs Mårten 
Published: August 2015

Abstract
In recent years, cell-based evaluation of drug-induced phenotypic changes has become an important strategy in high throughput drug screening (HTS). However, due to the lack of clinically relevant in vitro models, phenotype-based screening has not resulted in the identification of as many successful compounds as initially assumed. Thus, an application of the long-known multicellular tumor spheroid (MCTS) model for HTS has recently gained attention, as MCTS mimic the complex in vivo situation more closely than standard monolayer cell cultures. However, due to major difficulties with formation of uniform spheroids in a large scale, application of MCTS for HTS has been limited.
We focused our efforts on the development of spheroid-based HTS systems in 384-well format for the identification of molecules targeting different tumor microenvironment characteristics. For spheroid formation we used two colorectal cancer cell lines, HCT116 and HT-29, both constitutively expressing green fluorescent protein (GFP). During the spheroid formation process, culture medium was either changed every 4 days or left unchanged. This approach resulted in the generation of two different spheroid models from the each cell line - one representing tumor regions with the sufficient access to nutrients; the other simulating parts with more harsh conditions. Spheroids formed with medium change were more proliferative and less dependent on oxygen. By measuring spheroid GFP fluorescence intensity we could non-invasively monitor drug action over time. First screens revealed a group of drugs particularly effective in quiescent spheroids compared with proliferating spheroids and monolayer cell cultures. All of the compounds inhibited mitochondrial oxidative phosphorylation. The screen confirmed mitochondrial respiration as a promising target for treatment of dormant cells in nutrient-deprived tumor regions and identified molecules with previously unrecognized potential for further clinical advancement.
Another screening system was developed to identify drugs that directly influence the immune components of tumor microenvironment. Spheroids are formed by co-culturing cancer cells with human primary monocytes. Over the culture period, monocytes differentiate into tumor-associated macrophages. First pilot experiments showed promising results and more extensive screens are planned.
For characterization of drug effects at the transcriptome level, a spheroid-based gene expression profiling method is under development. The method will enable high throughput analysis of transcripts from spheroids treated with chemical libraries.
In conclusion, we here present a novel approach to HTS, which by utilizing diverse MCTS models has a potential to identify molecules directly targeting tumor-specific microenvironment components.
Citation Format: Wojciech Senkowski, Xiaonan Zhang, Peter Nygren, Maria Hägg Olofsson, Mats Gustafsson, Stig Linder, Rolf Larsson, Mårten Fryknäs. Spheroid-based high throughput screening for identification of molecules targeting different tumor microenvironment characteristics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 323. doi:10.1158/1538-7445.AM2015-323