Abstract
A recent decline in the discovery of novel medications challenges the widespread use of 2D monolayer cell assays in the drug discovery process. As a result, the need for more appropriate cellular models of human physiology and disease has renewed the interest in spheroid 3D culture as a pertinent model for drug screening. However, despite technological progress that has significantly simplified spheroid production and analysis, the seeming complexity of the 3D approach has delayed its adoption in many laboratories. The present report demonstrates that the use of a spheroid model may be straightforward and can provide information that is not directly available with a standard 2D approach. We describe a cost-efficient method that allows for the production of an array of uniform spheroids, their staining with vital dyes, real-time monitoring of drug effects, and an ATP-endpoint assay, all in the same 96-well U-bottom plate. To demonstrate the method performance, we analyzed the effect of the preclinical anticancer drug MLN4924 on spheroids formed by VCaP and LNCaP prostate cancer cells. The drug has different outcomes in these cell lines, varying from cell cycle arrest and protective dormancy to senescence and apoptosis. We demonstrate that by using high-content analysis of spheroid arrays, the effect of the drug can be described as a series of EC50 values that clearly dissect the cytostatic and cytotoxic drug actions. The method was further evaluated using four standard cancer chemotherapeutics with different mechanisms of action, and the effect of each drug is described as a unique multi-EC50 diagram. Once fully validated in a wider range of conditions, this method could be particularly valuable for phenotype-based drug discovery.
Highlights
Past decades have witnessed a significant decline in the discovery of novel medications, which challenges the efficacy and even the validity of the modern drug discovery process [1,2,3]
We looked for fluorescent probes that can be used in combination with bright-field microscopy for long-term real-time spheroid imaging
Among the seven prostate cancer cell lines examined in the present work, the majority produced individual spheroids after brief optimization of the culture conditions (Figure S1 in Supplementary Material)
Summary
Past decades have witnessed a significant decline in the discovery of novel medications, which challenges the efficacy and even the validity of the modern drug discovery process [1,2,3]. It is widely accepted that transformed cells growing in a monolayer on plastic dishes have little in common with the complex 3D multicellular organization found in living organisms. Awareness of this discrepancy has led to studies to find more appropriate cellular models to better represent human physiology and disease for drug screening. The increasing complexity of a model inevitably jeopardizes assay robustness, parallelization, integration, and data analysis, which are essential for standardization. It boosts the cost of initial screening, it can be worthwhile in subsequent clinical trials
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