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Monitoring the Activation of the DNA Damage Response Pathway in a 3D Spheroid Model.

Mondesert O, Frongia C, Clayton O, Boizeau ML, Lobjois V, Ducommun B - PLoS ONE (2015)

Bottom Line: Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation.Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation.We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response.

View Article: PubMed Central - PubMed

Affiliation: Université de Toulouse; ITAV-USR3505, F-31106 Toulouse, France; CNRS; ITAV-USR3505, F-31106 Toulouse, France.

ABSTRACT
Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation. Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation. We show that cells expressing the DDR-Act-FP biosensor efficiently report activation of the DDR pathway after DNA damage and its pharmacological manipulation using ATM kinase inhibitors. We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response. Finally, using multicellular spheroids expressing the DDR-Act-FP we demonstrate that DDR activation and its pharmacological manipulation with inhibitory and activatory compounds can be efficiently monitored in live 3D spheroid model. This study paves the way for the development of innovative screening and preclinical evaluation assays.

No MeSH data available.


Related in: MedlinePlus

Use of DDR-Act-FP for the dynamic monitoring of DDR activation in live 3D spheroids.Spheroids were prepared with HCT116 cells expressing the DDR-Act reporter in 96 well plates. Relative fluorescence intensity to the untreated control was monitored on live spheroids at the indicated time and in the presence of the indicated compounds. The results are the average of determination on 6 spheroids for each experimental condition. (A) Image acquisition were automatically performed on an ArrayScan (Cellomics) using the Thermo Cellomics Compartimental Analysis V4 software. The bright field channel was used to perform segmentation and to create a mask used to extract the mRFP average fluorescence intensity from the second channel. A spheroid treated for 24 hours with 5μM etoposide was used in this illustration. (B) Bar graph shows the mean relative fluorescence intensity+SD after 48h and 72h of growth in the presence of the indicated concentrations of Etoposide (3 to 5 independent experiments, with 6 samples per condition). ***: P<0.005, ****: P<0.001. (C) Mean relative fluorescence intensity+SD after 24h in the presence of the indicated compounds. (D) Mean relative fluorescence intensity+SD after treatment for 24 hours with increasing concentration of the ATM inhibitor CP-466722 prior to incubation in the presence of 10μM Etoposide for 48 hours (3 independent experiments with 6 samples per condition for each).
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pone.0134411.g004: Use of DDR-Act-FP for the dynamic monitoring of DDR activation in live 3D spheroids.Spheroids were prepared with HCT116 cells expressing the DDR-Act reporter in 96 well plates. Relative fluorescence intensity to the untreated control was monitored on live spheroids at the indicated time and in the presence of the indicated compounds. The results are the average of determination on 6 spheroids for each experimental condition. (A) Image acquisition were automatically performed on an ArrayScan (Cellomics) using the Thermo Cellomics Compartimental Analysis V4 software. The bright field channel was used to perform segmentation and to create a mask used to extract the mRFP average fluorescence intensity from the second channel. A spheroid treated for 24 hours with 5μM etoposide was used in this illustration. (B) Bar graph shows the mean relative fluorescence intensity+SD after 48h and 72h of growth in the presence of the indicated concentrations of Etoposide (3 to 5 independent experiments, with 6 samples per condition). ***: P<0.005, ****: P<0.001. (C) Mean relative fluorescence intensity+SD after 24h in the presence of the indicated compounds. (D) Mean relative fluorescence intensity+SD after treatment for 24 hours with increasing concentration of the ATM inhibitor CP-466722 prior to incubation in the presence of 10μM Etoposide for 48 hours (3 independent experiments with 6 samples per condition for each).

Mentions: The HCT116 DDR-Act-FP cell line was used to produce multicellular tumor spheroids in 96-well plates (as described in the methods section) thus allowing automated monitoring of multiple parameters including DDR-Act-FP-dependent fluorescence in live spheroids. Image acquisitions were performed on an ArrayScan (Cellomics) setup and analyses were carried out using the Thermo Cellomics Compartimental Analysis V4 software. Spheroids were segmented using the bright field channel, allowing extraction of area and determination of relative fluorescence intensity on the mRFP channel (Fig 4A).


Monitoring the Activation of the DNA Damage Response Pathway in a 3D Spheroid Model.

Mondesert O, Frongia C, Clayton O, Boizeau ML, Lobjois V, Ducommun B - PLoS ONE (2015)

Use of DDR-Act-FP for the dynamic monitoring of DDR activation in live 3D spheroids.Spheroids were prepared with HCT116 cells expressing the DDR-Act reporter in 96 well plates. Relative fluorescence intensity to the untreated control was monitored on live spheroids at the indicated time and in the presence of the indicated compounds. The results are the average of determination on 6 spheroids for each experimental condition. (A) Image acquisition were automatically performed on an ArrayScan (Cellomics) using the Thermo Cellomics Compartimental Analysis V4 software. The bright field channel was used to perform segmentation and to create a mask used to extract the mRFP average fluorescence intensity from the second channel. A spheroid treated for 24 hours with 5μM etoposide was used in this illustration. (B) Bar graph shows the mean relative fluorescence intensity+SD after 48h and 72h of growth in the presence of the indicated concentrations of Etoposide (3 to 5 independent experiments, with 6 samples per condition). ***: P<0.005, ****: P<0.001. (C) Mean relative fluorescence intensity+SD after 24h in the presence of the indicated compounds. (D) Mean relative fluorescence intensity+SD after treatment for 24 hours with increasing concentration of the ATM inhibitor CP-466722 prior to incubation in the presence of 10μM Etoposide for 48 hours (3 independent experiments with 6 samples per condition for each).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4520595&req=5

pone.0134411.g004: Use of DDR-Act-FP for the dynamic monitoring of DDR activation in live 3D spheroids.Spheroids were prepared with HCT116 cells expressing the DDR-Act reporter in 96 well plates. Relative fluorescence intensity to the untreated control was monitored on live spheroids at the indicated time and in the presence of the indicated compounds. The results are the average of determination on 6 spheroids for each experimental condition. (A) Image acquisition were automatically performed on an ArrayScan (Cellomics) using the Thermo Cellomics Compartimental Analysis V4 software. The bright field channel was used to perform segmentation and to create a mask used to extract the mRFP average fluorescence intensity from the second channel. A spheroid treated for 24 hours with 5μM etoposide was used in this illustration. (B) Bar graph shows the mean relative fluorescence intensity+SD after 48h and 72h of growth in the presence of the indicated concentrations of Etoposide (3 to 5 independent experiments, with 6 samples per condition). ***: P<0.005, ****: P<0.001. (C) Mean relative fluorescence intensity+SD after 24h in the presence of the indicated compounds. (D) Mean relative fluorescence intensity+SD after treatment for 24 hours with increasing concentration of the ATM inhibitor CP-466722 prior to incubation in the presence of 10μM Etoposide for 48 hours (3 independent experiments with 6 samples per condition for each).
Mentions: The HCT116 DDR-Act-FP cell line was used to produce multicellular tumor spheroids in 96-well plates (as described in the methods section) thus allowing automated monitoring of multiple parameters including DDR-Act-FP-dependent fluorescence in live spheroids. Image acquisitions were performed on an ArrayScan (Cellomics) setup and analyses were carried out using the Thermo Cellomics Compartimental Analysis V4 software. Spheroids were segmented using the bright field channel, allowing extraction of area and determination of relative fluorescence intensity on the mRFP channel (Fig 4A).

Bottom Line: Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation.Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation.We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response.

View Article: PubMed Central - PubMed

Affiliation: Université de Toulouse; ITAV-USR3505, F-31106 Toulouse, France; CNRS; ITAV-USR3505, F-31106 Toulouse, France.

ABSTRACT
Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation. Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation. We show that cells expressing the DDR-Act-FP biosensor efficiently report activation of the DDR pathway after DNA damage and its pharmacological manipulation using ATM kinase inhibitors. We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response. Finally, using multicellular spheroids expressing the DDR-Act-FP we demonstrate that DDR activation and its pharmacological manipulation with inhibitory and activatory compounds can be efficiently monitored in live 3D spheroid model. This study paves the way for the development of innovative screening and preclinical evaluation assays.

No MeSH data available.


Related in: MedlinePlus