Abstract
BackgroundEvery biological experiment requires a choice of throughput balanced against physiological relevance. Most primary drug screens neglect critical parameters such as microenvironmental conditions, cell-cell heterogeneity, and specific readouts of cell fate for the sake of throughput.MethodsHere we describe a methodology to quantify proliferation and viability of single cells in 3D culture conditions by leveraging automated microscopy and image analysis to facilitate reliable and high-throughput measurements. We detail experimental conditions that can be adjusted to increase either throughput or robustness of the assay, and we provide a stand alone image analysis program for users who wish to implement this 3D drug screening assay in high throughput.ResultsWe demonstrate this approach by evaluating a combination of RAF and MEK inhibitors on melanoma cells, showing that cells cultured in 3D collagen-based matrices are more sensitive than cells grown in 2D culture, and that cell proliferation is much more sensitive than cell viability. We also find that cells grown in 3D cultured spheroids exhibit equivalent sensitivity to single cells grown in 3D collagen, suggesting that for the case of melanoma, a 3D single cell model may be equally effective for drug identification as 3D spheroids models. The single cell resolution of this approach enables stratification of heterogeneous populations of cells into differentially responsive subtypes upon drug treatment, which we demonstrate by determining the effect of RAK/MEK inhibition on melanoma cells co-cultured with fibroblasts. Furthermore, we show that spheroids grown from single cells exhibit dramatic heterogeneity to drug response, suggesting that heritable drug resistance can arise stochastically in single cells but be retained by subsequent generations.ConclusionIn summary, image-based analysis renders cell fate detection robust, sensitive, and high-throughput, enabling cell fate evaluation of single cells in more complex microenvironmental conditions.
Highlights
Every biological experiment requires a choice of throughput balanced against physiological relevance
We find that cells grown in 3D spheroids exhibit very similar drug responses to single cells in a 3D microenvironment, in contrast to cells grown on 2D plastic plates which are less sensitive than either single cells in 3D collagen or cells in 3D spheroids
Procedure to measure the effects of drug treatment on cell proliferation and viability at single cell resolution When designing an assay to quantify the effects of drug treatments, we identified three critical design criteria: (1) single cell resolution, which enables identification of cell subtypes that respond differentially to treatment, (2) ability to distinguish cytostatic from cytotoxic effects, and (3) ability to measure cell fates in complex 3D microenvironments
Summary
Every biological experiment requires a choice of throughput balanced against physiological relevance. Emerging evidence suggests that the three-dimensional characteristics of the cell microenvironment, including matrix composition, dimensionality, and stiffness affect cancer progression [3,4,5,6] and it is unknown how the effects of drug perturbation on 2D assays would translate to more complex assays that in principle represent in vivo microenvironment more fully. These complex assays are often considered too Murali et al BMC Cancer (2019) 19:502 expensive or difficult to implement for pharmaceutical candidate screening [7,8,9]. There is a need for an assay that is capable of increasing microenvironmental complexity while retaining the simplicity necessary for high throughput drug screening studies
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