3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

Abstract

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.

In Vitro Modeling of Tissue-Specific 3D Microenvironments and Possibile Application to Pediatric Cancer Research

“…Since chemotherapeutic drugs often target the dividing cells, this quiescence may justify the "resistance" to cytostatic drugs observed in MSs [89,98]. Furthermore, in vivo, drug bioavailability and delivery are also regulated by the ECM's attributes (configuration, composition, structure), that can alter/disrupt cell signaling [99,100]. Heterotypic MSs more closely mimic the heterogeneity of tumors and the heterotypic inter-relations between cells, and, in addition to monotypic culture, they allow the investigation of important tumor endpoints such as angiogenesis, tumor cell migration and invasion, and drug responsiveness [101].…”

“…Poly(lactidecoglycolide), poly(lactic acid), poly(vinyl alcohol), poly( -caprolactone) or polyethylene glycol) (e.g. Poly(lactide-coglycolide), poly(lactic acid), poly(vinyl alcohol), poly( -caprolactone) or polyethylene glycol) [77,78,99,[115][116][117][118][119][120][121]. One advantage of scaffold-based strategies is that it is possible to better manage matrix stiffness/compliance, porosity, and biochemical composition.…”

“…Scaffolds formed of chitosan and alginate's mixture were shown to better favor the tumor progression of human-derived glioma cells than does each component alone [125]. Moreover, scaffolds/ hydrogels can be functionalized to favor more native cell behavior and more tissue-like function [99] . Hence, the choice of natural matrix-derivatives and configurations is also of importance, since, as in in vivo tumors, it undoubtedly influences cell behavior, from cancer reversion to promotion steps.…”

Invited Review Article: Advanced light microscopy for biological space research

“…132,133 Parallelization and miniaturization of cell-based assays is now common practice in two dimensions but is also becoming more feasible in 3D. Indeed, microspace plates have been used to establish three-dimensional cultures of primary hepatocytes, 134 agarose-coated 384-well plates have been used to perform high-throughput compound screens on spheroids from primary colon cancer cells, 135 and even a perfusion-based microfluidic chip has been conceived for parallelized 3D culture of primary chondrocytes. 136 Compatibility with LSFM will require alternative well plate geometries as well as a convenient interface for rapid exchange of samples.…”

Fully Automated One-Step Production of Functional 3D Tumor Spheroids for High-Content Screening

“…Table S1) rely on solutions developed in-house based on the overlay culture technique. [19][20][21][22][23] Spheroids are grown on a semisolid agarose bed or in/on 100% Matrigel deposited in flat-bottomed glass plates. The agarose culture method leads to the formation of a large round spheroid possessing the preferred size necessary for analysis of tumor spheroids (diameter 300-1000 µm), but only one spheroid is produced per well, which poorly supports statistical evaluation.…”