Multiparametric Analysis of Oncology Drug Screening with Aqueous Two-Phase Tumor Spheroids

Abstract

Spheroids present a biologically relevant three-dimensional model of avascular tumors and a unique tool for discovery of anticancer drugs. 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 spheroids. We overcome this barrier using a polymeric aqueous two-phase microtechnology to robotically microprint spheroids of well-defined size in standard 384-microwell plates. We use different cancer cells and show that resulting spheroids grow over time and display characteristic features of solid tumors. We demonstrate the feasibility of robotic, high-throughput screening of 25 standard chemotherapeutics and molecular inhibitors against tumor spheroids of three different cancer cell lines. This screening uses over 7000 spheroids to elicit high quality dose-dependent drug responses from spheroids. To quantitatively compare performance of different drugs, we employ a multiparametric scoring system using half-maximum inhibitory concentration (IC), maximum inhibition (E), and area under the dose-response curve (AUC) to take into account both potency and efficacy parameters. This approach allows us to identify several compounds that effectively inhibit growth of spheroids and compromise cellular viability, and distinguish them from moderately effective and ineffective drugs. Using protein expression analysis, we demonstrate that spheroids generated with the aqueous two-phase microtechnology reliably resolve molecular targets of drug compounds. Incorporating this low-cost and convenient-to-use tumor spheroid technology in preclinical drug discovery will make compound screening with realistic tumor models a routine laboratory technique prior to expensive and tedious animal tests to dramatically improve testing throughput and efficiency and reduce costs of drug discovery.

Cell‐Inspired All‐Aqueous Microfluidics: From Intracellular Liquid–Liquid Phase Separation toward Advanced Biomaterials

“…The cultivated spheroids show spherical morphology in high reproducibility (Figure 26b) and excellent viability (Figure 26c) to proliferate into larger spheroids (Figure 26d). The patterned tumor spheroids partially addressed challenges in the heterogeneity of spheroid culture in vitro, which is therefore used as promising platforms for executing high‐throughput anticancer compounds screening. The ability to scale‐up 3D spheroid culture in microfluidic devices offers new opportunity for fast testing of multiple anticancer drugs and analyze their half‐maximum (IC 50 ) and maximum ( E max ) inhibitory concentrations, which facilitates the multiparametric analysis of cellular responses to drug compounds and identify novel chemical probes with chemotherapeutic benefits, as demonstrated in Figure 26e …”

“…Copyright 2016, Wiley‐VCH. e) Reproduced with permission . Copyright 2016, American Chemical Society.…”

Biomaterials‐Based Approaches to Tumor Spheroid and Organoid Modeling

“…[125] The potential of this approach for high throughput drug screening was demonstrated by single- and dual-agent testing of a collection of anti-cancer compounds against spheroids of brain, breast, and colon cancer cells to identify treatments that effectively induce cytotoxic or cytostatic effects. [35, 36] …”

Optical Coherence Tomography Detects Necrotic Regions and Volumetrically Quantifies Multicellular Tumor Spheroids

“…This failure rate in the late stages of clinical trials is in large part due to the use of overly simplistic in vitro cell assays and in vivo animal models with limited predictive value during various stages of drug discovery. Recently, three-dimensional (3D) tumor spheroid models have been explored to provide novel physiological and pharmacological data that are more predictive of drug efficacy and toxicity in the clinic (2–24). These studies show that 3D tumor spheroid models are expected to have an immediate and long-lasting impact in shortening drug discovery timelines, reducing costs of investment, and bringing new medicines to more patients.…”

“…These studies show that 3D tumor spheroid models are expected to have an immediate and long-lasting impact in shortening drug discovery timelines, reducing costs of investment, and bringing new medicines to more patients. To characterize 3D tumor models in high-throughput screening, bright field imaging or phase contrast imaging are routinely employed (7,9,22,24). However, these modalities can only provide 2D projections of tumor spheroids and are unable to resolve their 3D structures.…”