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A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids

The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better...

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Autores principales: Demuynck, Robin, Efimova, Iuliia, Lin, Abraham, Declercq, Heidi, Krysko, Dmitri V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140689/
https://www.ncbi.nlm.nih.gov/pubmed/32183000
http://dx.doi.org/10.3390/cells9030703
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author Demuynck, Robin
Efimova, Iuliia
Lin, Abraham
Declercq, Heidi
Krysko, Dmitri V.
author_facet Demuynck, Robin
Efimova, Iuliia
Lin, Abraham
Declercq, Heidi
Krysko, Dmitri V.
author_sort Demuynck, Robin
collection PubMed
description The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment, but detailed cell death assays for 3D cultures (including those for ferroptosis) are scarce. In this work, we show that a new cell death analysis method, named 3D Cell Death Assay (3DELTA), can efficiently determine different cell death types including ferroptosis and quantitatively assess cell death in tumour spheroids. Our method uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. After optimization of Sytox concentration, Triton X-100 concentration and timing, we showed that the 3DELTA method was able to detect signals from all cells without the need to disaggregate spheroids. Moreover, in this work we demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. In conclusion, 3DELTA is a more cost-effective way to identify and measure cell death type in 3D cultures, including spheroids.
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spelling pubmed-71406892020-04-13 A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids Demuynck, Robin Efimova, Iuliia Lin, Abraham Declercq, Heidi Krysko, Dmitri V. Cells Article The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment, but detailed cell death assays for 3D cultures (including those for ferroptosis) are scarce. In this work, we show that a new cell death analysis method, named 3D Cell Death Assay (3DELTA), can efficiently determine different cell death types including ferroptosis and quantitatively assess cell death in tumour spheroids. Our method uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. After optimization of Sytox concentration, Triton X-100 concentration and timing, we showed that the 3DELTA method was able to detect signals from all cells without the need to disaggregate spheroids. Moreover, in this work we demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. In conclusion, 3DELTA is a more cost-effective way to identify and measure cell death type in 3D cultures, including spheroids. MDPI 2020-03-13 /pmc/articles/PMC7140689/ /pubmed/32183000 http://dx.doi.org/10.3390/cells9030703 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Demuynck, Robin
Efimova, Iuliia
Lin, Abraham
Declercq, Heidi
Krysko, Dmitri V.
A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids
title A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids
title_full A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids
title_fullStr A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids
title_full_unstemmed A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids
title_short A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids
title_sort 3d cell death assay to quantitatively determine ferroptosis in spheroids
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140689/
https://www.ncbi.nlm.nih.gov/pubmed/32183000
http://dx.doi.org/10.3390/cells9030703
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