Dataset Information

An image-based assay to quantify changes in proliferation and viability upon drug treatment in 3D microenvironments.

ABSTRACT:

Background

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.

Methods

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.

Results

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.

Conclusion

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.

SUBMITTER: Murali VS

PROVIDER: S-EPMC6537405 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Publications

An image-based assay to quantify changes in proliferation and viability upon drug treatment in 3D microenvironments.

Murali Vasanth S VS Chang Bo-Jui BJ Fiolka Reto R Danuser Gaudenz G Cobanoglu Murat Can MC Welf Erik S ES

BMC cancer 20190528 1

<h4>Background</h4>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.<h4>Methods</h4>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 rel ...[more]

PMID: 31138163

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Project description:PurposeMagnetic resonance imaging (MRI) is increasingly used in radiation oncology for target delineation and radiotherapy treatment planning, for example, in patients with gynecological cancers. As a consequence of pelvic radiotherapy, a part of the bowel is irradiated, yielding risk of bowel toxicity. Existing dose-effect models predicting bowel toxicity are inconclusive and bowel motion might be an important confounding factor. The exact motion of the bowel and dosimetric effects of its motion are yet uncharted territories in radiotherapy. In diagnostic radiology methods on the acquisition of dynamic MRI sequences were developed for bowel motility visualization and quantification. Our study aim was to develop an imaging technique based on three-dimensional (3D) cine-MRI to visualize and quantify bowel motion and demonstrate it in a cohort of gynecological cancer patients.MethodsWe developed an MRI acquisition suitable for 3D bowel motion quantification, namely a balanced turbo field echo sequence (TE = 1.39 ms, TR = 2.8 ms), acquiring images in 3.7 s (dynamic) with a 1.25 × 1.25 × 2.5 mm3 resolution, yielding a field of view of 200 × 200 × 125 mm3 . These MRI bowel motion sequences were acquired in 22 gynecological patients. During a 10-min scan, 160 dynamics were acquired. Subsequent dynamics were deformably registered using a B-spline transformation model, resulting in 159 3D deformation vector fields (DVFs) per MRI set. From the 159 DVFs, the average vector length was calculated per voxel to generate bowel motion maps. Quality assurance was performed on all 159 DVFs per MRI, using the Jacobian Determinant and the Harmonic Energy as deformable image registration error metrics. In order to quantify bowel motion, we introduced the concept of cumulative motion-volume histogram (MVH) of the bowel bag volume. Finally, interpatient variation of bowel motion was analyzed using the MVH parameters M10%, M50%, and M90%. The M10%/M50%/M90% represents the minimum bowel motion per frame of 10%/50%/90% of the bowel bag volume.ResultsThe motion maps resulted in a visualization of areas with small and large movements within the bowel bag. After applying quality assurance, the M10%, M50%, and M90% were 4.4 (range 2.2-7.6) mm, 2.2 (range 0.9-4.1) mm, and 0.5 (range 0.2-1.4) mm per frame, on average over all patients, respectively.ConclusionWe have developed a method to visualize and quantify 3D bowel motion with the use of bowel motion specific MRI sequences in 22 gynecological cancer patients. This 3D cine-MRI-based quantification tool and the concept of MVHs can be used in further studies to determine the effect of radiotherapy on bowel motion and to find the relation with dose effects to the small bowel. In addition, the developed technique can be a very interesting application for bowel motility assessment in diagnostic radiology.

Dataset Information

An image-based assay to quantify changes in proliferation and viability upon drug treatment in 3D microenvironments.

Background

Methods

Results

Conclusion

Publications

An image-based assay to quantify changes in proliferation and viability upon drug treatment in 3D microenvironments.

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