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• W2947104118 abstract "Every 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.Here 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.We 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.In 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." @default.
• W2947104118 created "2019-06-07" @default.
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• W2947104118 date "2019-05-28" @default.
• W2947104118 modified "2023-10-12" @default.
• W2947104118 title "An image-based assay to quantify changes in proliferation and viability upon drug treatment in 3D microenvironments" @default.
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• W2947104118 doi "https://doi.org/10.1186/s12885-019-5694-1" @default.
• W2947104118 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6537405" @default.
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• W2947104118 hasPublicationYear "2019" @default.
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