This paper presents a new 3D culture microtechnology for high throughput production of tumor spheroids and validates its utility for screening anti-cancer drugs. We use two immiscible polymeric aqueous solutions and microprint a submicroliter drop of the “patterning” phase containing cells into a bath of the “immersion” phase. Selecting proper formulations of biphasic systems using a panel of biocompatible polymers results in the formation of a round drop that confines cells to facilitate spontaneous formation of a spheroid without any external stimuli. Adapting this approach to robotic tools enables straightforward generation and maintenance of spheroids of well-defined size in standard microwell plates and biochemical analysis of spheroids in situ, which is not possible with existing techniques for spheroid culture. To enable high throughput screening, we establish a phase diagram to identify minimum cell densities within specific volumes of the patterning drop to result in a single spheroid. Spheroids show normal growth over long-term incubation and dose-dependent decrease in cellular viability when treated with drug compounds, but present significant resistance compared to monolayer cultures. The unprecedented ease of implementing this microtechnology and its robust performance will benefit high throughput studies of drug screening against cancer cells with physiologically-relevant 3D tumor models.

Tumor spheroids are three-dimensional clusters of cancer cells that exhibit characteristics of poorly perfused tumors and hence present a relevant model for testing the efficacy of anti-cancer compounds. The use of spheroids for drug screening is hindered by technological complexities for high throughput generation of consistent size spheroids individually addressable by drug compounds. Here we present and optimize a simple spheroid technology based on the use of an aqueous two-phase system. Cancer cells confined in a drop of the denser aqueous dextran phase are robotically dispensed into a microwell containing the immersion aqueous polyethylene glycol phase. Cells remain within the drop and form a viable spheroid, without a need for any external stimuli. The size of resulting spheroids is sensitive to volume variations of dispensed drops from the air displacement pipetting head of a commercial liquid handling robot. Therefore, we parametrically optimize the process of dispensing of dextran phase drops. For a given cell density, this optimization reproducibly generates consistent size spheroids in standard 96-well plates. In addition, we evaluate the use of a commercial biochemical assay to examine cellular viability of cancer cell spheroids. Spheroids show a dose-dependent response to cisplatin similar to a monolayer culture. However unlike their two-dimensional counterpart, spheroids exhibit resistance to paclitaxel treatment. This technology, which uses only commercially-available reagents and equipment, can potentially expedite anti-cancer drug discovery. Although the use of robotics makes the ATPS spheroid technology particularly useful for drug screening applications, this approach is compatible with simpler liquid handling techniques such as manual micropipetting and offers a straightforward method of 3D cell culture in research laboratories.

Migration of tumor cells is a fundamental event implicated in metastatic progression of cancer. Therapeutic compounds with the ability to inhibit the motility of cancer cells are critical for preventing cancer metastasis. Achieving this goal requires new technologies that enable high-throughput drug screening against migration of cancer cells and expedite drug discovery. We report an easy-to-implement, robotically operated, cell migration microtechnology with the capability of simultaneous screening of multiple compounds. The technology utilizes a fully biocompatible polymeric aqueous two-phase system to pattern a monolayer of cells containing a cell-excluded gap that serves as the migration niche. We adapted this technology to a standard 96-well plate format and parametrically optimized it to generate highly consistent migration niches. The analysis of migration is done automatically using computerized schemes. We use statistical metrics and show the robustness of this assay for drug screening and its sensitivity to identify effects of different drug compounds on migration of cancer cells. This technology can be employed in core centers, research laboratories, and pharmaceutical industries to evaluate the efficacy of compounds against migration of various types of metastatic cancer cells prior to expensive animal tests and thus, streamline anti-migratory drug screening.

Physiologically relevant, in vitro cell-based models are important tools to identify effective anticancer drugs. Three-dimensional (3D) clusters of cancer cells, called tumor spheroids, mimic key characteristics of avascular tumors such as diffusion limitations and hypoxia, and hence serve as a relevant model for compound screening. Nevertheless, current spheroid formation techniques face difficulties to generate large number of uniform sized spheroids, conveniently maintain them in culture and biochemically analyze them using commercially available tools. To address this need, we developed a robotic, high throughput technology for three-dimensional culture of cancer cells that is simple to implement, produces consistent size spheroids in standard microwell plates, and allows convenient robotic media exchange and drug addition, and analysis of cellular responses using microplate readers. The technology is based on the use of an aqueous two-phase system. Cancer cells confined in a drop of the denser aqueous phase are robotically dispensed into a microwell containing the immersion aqueous phase. Cells remain within the drop at the bottom of the microwell and form a viable spheroid. We demonstrate the robust performance of this technology by evaluating the effect of two clinically used drugs against biologically aggressive skin cancer and triple negative breast cancer (TNBC) cells. Skin cancer spheroids treated with paclitaxel and cisplatin show dose-dependent decrease in cell viability, but with a greater resistance compared to the monolayer culture (2D) of cells (paclitaxel: LD50,3D = 178.5 nM, LD50,2D = 22.1 nM and cisplatin: LD50,3D = 131.7 μM and LD50,2D = 29.2 μM. TNBC cell monolayer and spheroids treated with cisplatin displayed a similar LD50 value of ∼13.1 μM. However, unlike the monolayer culture of TNBC cells that showed a dose-response to paclitaxel with LD50,2D = 8.0 nM, TNBC spheroids showed resistance to this drug, even at micromolar-scale concentrations, and maintained a high viability. In conclusion, our tumor spheroid technology enables quick and efficient generation of relevant tumor models in standard microplates to conveniently treat with chemical compounds and analyze. This technology allows investigating the efficacy of drug compounds against cancer cells and reveals cellular responses not captured with widely-used monolayer cultures. The ease of conducting and analyzing high throughput experiments will substantially reduce labor and cost, and expedite discovery of effective anti-cancer compounds with a physiologic tumor model. Citation Format: Stephanie Lemmo, Ehsan Aefi, Gary Luker, Hossein Tavana. High throughput drug screening with engineered tumor spheroids. [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 302. doi:10.1158/1538-7445.AM2015-302