Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

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Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions and therefore represent a promising model system for discovery of phenotypes specifically expressed in stressed, hypoxic and nutrient-depleted conditions. We here show the setup of a 3D cell culture compatible high-content screening system for the identification of substances that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. This setup was used in a pilot screen to identify tool compounds and subsequently in a larger screening approach covering approximately 500000 compounds from the Bayer substance library. The resulting hit list could be subdivided into two different categories based on the identification of two distinct phenotypes. Mode of action studies identified the first category as inhibitors of the mitochondrial respiratory chain, being critically dependent on extracellular glucose concentrations. On the other hand the second hit category could be identified as modifiers of fatty acid metabolism. The identified hits offer the opportunity for further characterization in vivo, particularly in combination with drugs targeting the well perfused, proliferating tumor areas, such as chemotherapy. Taken together the data presented here show for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. Citation Format: Carsten Wenzel, Sven Christian, Carolyn Algire, Wolfgang Schwede, Roland Neuhaus, Judith Guenther, Ningshu Liu, Sebastian Raese, Karsten Parczyk, Stefan Prechtl, Patrick Steigemann. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions. [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 317. doi:10.1158/1538-7445.AM2015-317

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