High-Content Assays for Characterizing the Viability and Morphology of 3D Cancer Spheroid Cultures

Abstract

There is an increasing interest in using three-dimensional (3D) spheroids for modeling cancer and tissue biology to accelerate translation research. Development of higher throughput assays to quantify phenotypic changes in spheroids is an active area of investigation. The goal of this study was to develop higher throughput high-content imaging and analysis methods to characterize phenotypic changes in human cancer spheroids in response to compound treatment. We optimized spheroid cell culture protocols using low adhesion U-bottom 96- and 384-well plates for three common cancer cell lines and improved the workflow with a one-step staining procedure that reduces assay time and minimizes variability. We streamlined imaging acquisition by using a maximum projection algorithm that combines cellular information from multiple slices through a 3D object into a single image, enabling efficient comparison of different spheroid phenotypes. A custom image analysis method was implemented to provide multiparametric characterization of single-cell and spheroid phenotypes. We report a number of readouts, including quantification of marker-specific cell numbers, measurement of cell viability and apoptosis, and characterization of spheroid size and shape. Assay performance was assessed using established anticancer cytostatic and cytotoxic drugs. We demonstrated concentration–response effects for different readouts and measured IC50 values, comparing 3D spheroid results to two-dimensional cell cultures. Finally, a library of 119 approved anticancer drugs was screened across a wide range of concentrations using HCT116 colon cancer spheroids. The proposed methods can increase performance and throughput of high-content assays for compound screening and evaluation of anticancer drugs with 3D cell models.

High-Content Analysis Provides Mechanistic Insights into the Testicular Toxicity of Bisphenol A and Selected Analogues in Mouse Spermatogonial Cells

“…Several studies demonstrated that data obtained with HCA are concordant with drug-induced toxicity in humans (O'Brien et al, 2006;Schoonen et al, 2005;Xu et al, 2008). HCA has been widely used to assess chemical-induced mechanistic effects, and has been applied to drug discovery using specific cell type or 3D cell culture models (Anguissola et al, 2014;Kameoka et al, 2014;Radio et al, 2008;Ramery and O'Brien, 2014;Sirenko et al, 2015). In this study, we developed and validated a number of HCA-based assays and phenotypic read-outs, including characterizations of nuclear morphology, DNA content, cell cycle, cytoskeleton integrity and DNA damage responses using a germline cell line.…”

Three-dimensional culture systems in cancer research: Focus on tumor spheroid model

“…However, this technique is incompatible with endpoint analysis that requires viable spheroids, as cell viability is poorly affected following destruction of spheroid integrity. High Content Assay (HCA) is a nondestructive live-cell imaging technique that allows simultaneous quantitative analysis of total cell count, density, dimensions, growth kinetics, nuclear mass, and mitochondrial membrane potential of live MCTS (Sirenko et al , 2015). HCA also allows high throughput screening of anti-cancer drug candidates (Arora et al , 2014).…”

A High‐Throughput Automated Confocal Microscopy Platform for Quantitative Phenotyping of Nanoparticle Uptake and Transport in Spheroids

“…Quantitative imaging approaches afford the opportunity of providing rich biological information about how nanomaterials interact with cells growing in three dimensions. To date, the majority of HCS/HCA platforms applied in the field of drug discovery, including targeted drug delivery, have been utilized to detect variations in spheroid size and shape or through the use of simple fluorescence‐based assays to assess spheroid viability upon various treatments (Table S6, Supporting Information). Our study is unique in combining a series of methodological, image acquisition and image analysis innovations that allow not only efficient quantitative morphometric profiling of large numbers of 3D spheroids, but also preserves high‐resolution detail that allows individual subcellular compartments and passage of NPs through cellular pathways to be dissected.…”