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High-content assays for characterizing the viability and morphology of 3D cancer spheroid cultures.

Sirenko O, Mitlo T, Hesley J, Luke S, Owens W, Cromwell EF - Assay Drug Dev Technol (2015)

Bottom Line: A custom image analysis method was implemented to provide multiparametric characterization of single-cell and spheroid phenotypes.We demonstrated concentration-response effects for different readouts and measured IC50 values, comparing 3D spheroid results to two-dimensional cell cultures.Finally, a library of 119 approved anticancer drugs was screened across a wide range of concentrations using HCT116 colon cancer spheroids.

View Article: PubMed Central - PubMed

Affiliation: 1 Molecular Devices , LLC, Sunnyvale, California.

ABSTRACT
There is an increasing interest in using three-dimensional (3D) spheroids for modeling cancer and tissue biology to accelerate translation research. Development of higher throughput assays to quantify phenotypic changes in spheroids is an active area of investigation. The goal of this study was to develop higher throughput high-content imaging and analysis methods to characterize phenotypic changes in human cancer spheroids in response to compound treatment. We optimized spheroid cell culture protocols using low adhesion U-bottom 96- and 384-well plates for three common cancer cell lines and improved the workflow with a one-step staining procedure that reduces assay time and minimizes variability. We streamlined imaging acquisition by using a maximum projection algorithm that combines cellular information from multiple slices through a 3D object into a single image, enabling efficient comparison of different spheroid phenotypes. A custom image analysis method was implemented to provide multiparametric characterization of single-cell and spheroid phenotypes. We report a number of readouts, including quantification of marker-specific cell numbers, measurement of cell viability and apoptosis, and characterization of spheroid size and shape. Assay performance was assessed using established anticancer cytostatic and cytotoxic drugs. We demonstrated concentration-response effects for different readouts and measured IC50 values, comparing 3D spheroid results to two-dimensional cell cultures. Finally, a library of 119 approved anticancer drugs was screened across a wide range of concentrations using HCT116 colon cancer spheroids. The proposed methods can increase performance and throughput of high-content assays for compound screening and evaluation of anticancer drugs with 3D cell models.

No MeSH data available.


Related in: MedlinePlus

(A) Untreated and treated spheroids were stained with a combination of three dyes: Hoechst 15 μM, EthD-1 3 μM, and calcein AM 1 μM. Images of Hoechst, calcein AM, and EthD-1 were taken using DAPI, FITC, and Texas Red channels, respectively. A composite image of all three channels is shown at the bottom. Images were generated using maximum projection from a Z-stack of seven images 30 μm apart. The resulting object masks from image analyses are shown to the right of each image. (B) Zoomed region of an untreated spheroid showing mixed population of cells. (C). Average fluorescence intensities from spheroids (n = 3) versus incubation time for all three stains. The data are normalized to the 240-min time point. (D) Average fluorescence intensity for spheroids versus dye concentration for all three stains. Error bars not shown; SDs were less than 5% of values.
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f2: (A) Untreated and treated spheroids were stained with a combination of three dyes: Hoechst 15 μM, EthD-1 3 μM, and calcein AM 1 μM. Images of Hoechst, calcein AM, and EthD-1 were taken using DAPI, FITC, and Texas Red channels, respectively. A composite image of all three channels is shown at the bottom. Images were generated using maximum projection from a Z-stack of seven images 30 μm apart. The resulting object masks from image analyses are shown to the right of each image. (B) Zoomed region of an untreated spheroid showing mixed population of cells. (C). Average fluorescence intensities from spheroids (n = 3) versus incubation time for all three stains. The data are normalized to the 240-min time point. (D) Average fluorescence intensity for spheroids versus dye concentration for all three stains. Error bars not shown; SDs were less than 5% of values.

Mentions: Assay staining protocols must minimize disturbances that would cause spheroid degradation or displacement from the well center. We used a previously developed staining protocol with one-step dye mixture addition that eliminates the need for fixing cells or repeated washes.24 Images of representative spheroids stained with a mix of three dyes are shown in Figure 2A and B. Calcein AM was used to measure metabolically active cells, viability, and a variety of morphological parameters. Hoechst was utilized to measure total cell count and nuclear shape. EthD-1 selectively penetrates cells with damaged outer membranes and was used to measure dead or necrotic cells. We optimized dye incubation times and concentrations; results from those studies are shown in Figure 2C and D.


High-content assays for characterizing the viability and morphology of 3D cancer spheroid cultures.

Sirenko O, Mitlo T, Hesley J, Luke S, Owens W, Cromwell EF - Assay Drug Dev Technol (2015)

(A) Untreated and treated spheroids were stained with a combination of three dyes: Hoechst 15 μM, EthD-1 3 μM, and calcein AM 1 μM. Images of Hoechst, calcein AM, and EthD-1 were taken using DAPI, FITC, and Texas Red channels, respectively. A composite image of all three channels is shown at the bottom. Images were generated using maximum projection from a Z-stack of seven images 30 μm apart. The resulting object masks from image analyses are shown to the right of each image. (B) Zoomed region of an untreated spheroid showing mixed population of cells. (C). Average fluorescence intensities from spheroids (n = 3) versus incubation time for all three stains. The data are normalized to the 240-min time point. (D) Average fluorescence intensity for spheroids versus dye concentration for all three stains. Error bars not shown; SDs were less than 5% of values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556086&req=5

f2: (A) Untreated and treated spheroids were stained with a combination of three dyes: Hoechst 15 μM, EthD-1 3 μM, and calcein AM 1 μM. Images of Hoechst, calcein AM, and EthD-1 were taken using DAPI, FITC, and Texas Red channels, respectively. A composite image of all three channels is shown at the bottom. Images were generated using maximum projection from a Z-stack of seven images 30 μm apart. The resulting object masks from image analyses are shown to the right of each image. (B) Zoomed region of an untreated spheroid showing mixed population of cells. (C). Average fluorescence intensities from spheroids (n = 3) versus incubation time for all three stains. The data are normalized to the 240-min time point. (D) Average fluorescence intensity for spheroids versus dye concentration for all three stains. Error bars not shown; SDs were less than 5% of values.
Mentions: Assay staining protocols must minimize disturbances that would cause spheroid degradation or displacement from the well center. We used a previously developed staining protocol with one-step dye mixture addition that eliminates the need for fixing cells or repeated washes.24 Images of representative spheroids stained with a mix of three dyes are shown in Figure 2A and B. Calcein AM was used to measure metabolically active cells, viability, and a variety of morphological parameters. Hoechst was utilized to measure total cell count and nuclear shape. EthD-1 selectively penetrates cells with damaged outer membranes and was used to measure dead or necrotic cells. We optimized dye incubation times and concentrations; results from those studies are shown in Figure 2C and D.

Bottom Line: A custom image analysis method was implemented to provide multiparametric characterization of single-cell and spheroid phenotypes.We demonstrated concentration-response effects for different readouts and measured IC50 values, comparing 3D spheroid results to two-dimensional cell cultures.Finally, a library of 119 approved anticancer drugs was screened across a wide range of concentrations using HCT116 colon cancer spheroids.

View Article: PubMed Central - PubMed

Affiliation: 1 Molecular Devices , LLC, Sunnyvale, California.

ABSTRACT
There is an increasing interest in using three-dimensional (3D) spheroids for modeling cancer and tissue biology to accelerate translation research. Development of higher throughput assays to quantify phenotypic changes in spheroids is an active area of investigation. The goal of this study was to develop higher throughput high-content imaging and analysis methods to characterize phenotypic changes in human cancer spheroids in response to compound treatment. We optimized spheroid cell culture protocols using low adhesion U-bottom 96- and 384-well plates for three common cancer cell lines and improved the workflow with a one-step staining procedure that reduces assay time and minimizes variability. We streamlined imaging acquisition by using a maximum projection algorithm that combines cellular information from multiple slices through a 3D object into a single image, enabling efficient comparison of different spheroid phenotypes. A custom image analysis method was implemented to provide multiparametric characterization of single-cell and spheroid phenotypes. We report a number of readouts, including quantification of marker-specific cell numbers, measurement of cell viability and apoptosis, and characterization of spheroid size and shape. Assay performance was assessed using established anticancer cytostatic and cytotoxic drugs. We demonstrated concentration-response effects for different readouts and measured IC50 values, comparing 3D spheroid results to two-dimensional cell cultures. Finally, a library of 119 approved anticancer drugs was screened across a wide range of concentrations using HCT116 colon cancer spheroids. The proposed methods can increase performance and throughput of high-content assays for compound screening and evaluation of anticancer drugs with 3D cell models.

No MeSH data available.


Related in: MedlinePlus