Cancer cell spheroids present a biologically relevant model of avascular tumors and a unique tool for anti-cancer drug discovery. Despite being used in research laboratories for several decades, spheroids are not routinely used in the mainstream drug discovery pipeline primarily due to the difficulty of mass-producing uniformly sized spheroids and intense labor involved in handling, drug treatment, and analyzing of spheroids. We overcome this barrier using a novel technology to robotically microprint spheroids in standard 384-microwell plates. An aqueous drop containing cancer cells is dispensed into a bath of a second, immiscible aqueous phase to allow generation of a single spheroid. Using several cancer cells, we establish that this approach results in spheroids of well-defined size with less than 8% deviation from the mean diameter in 384-microwell plates. We demonstrate the feasibility of robotic, high throughput compound screening against tumor spheroids using of a collection of 25 standard chemotherapeutics and molecular inhibitors against three different cancer cells, HT-29 colon cancer cells, U-87 MG brain cancer cells, and MDA-MB-157 breast cancer cells. Dose-dependent experiments are done with each drug at a wide range of concentrations. After six days of incubation, viability of cancer cells in drug-treated spheroids is measured using a PrestoBlue assay that we have optimized for three-dimensional cell cultures. Post-treatment morphological changes are used as a secondary measure for analysis.
This screening identifies specific MEK inhibitors including Trametinib and Selumetinib that potently inhibit growth of HT-29 spheroids at nanomolar concentrations, whereas several PI3K inhibitors such as pictilisib significantly block proliferation of U-87 MG spheroids at micromolar concentrations. Spheroids of MDA-MB-157 cells show resistance to most of the drugs. We identify few compounds such as a Survivin inhibitor, YM155, and doxorubicin that disintegrate spheroids and induce significant cell death in all three cells. To generate a scoring system, we use half-maximum inhibitory concentration (IC50), maximum inhibition (Emax), and area under the dose-response curve (AUC) to present a multi-parametric approach that takes into account both potency and efficacy parameters for evaluating drug responses in tumor spheroids. In conclusion, our robotic technology offers a low cost and convenient platform for high throughput screening of large collections of chemical compounds against tumor spheroids. Incorporation of this technology in drug discovery applications will make drug testing and screening with realistic tumor models a routine laboratory technique prior to expensive and tedious in vivo analyses, dramatically improving testing throughput and efficiency and reducing costs.
Citation Format: Pradip Thakuri, Gary Luker, Hossein Tavana. High throughput robotic drug screening against microprinted tumor spheroids. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 616.