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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) Confocal images of a spheroid stained with Hoechst taken at indicated distances from the well bottom. As the image plane moves up from the well bottom toward the spheroid center, regions of distinct nuclei appear first in the center (at the surface of spheroid in contact with the plate bottom), then form a ring pattern that increases in diameter until it reaches some maximum associated with the outer surface of the spheroid. The MaxPro image is generated from 11 individual images taken 20 μm apart. (B) Nuclei segmentation shown for corresponding images in A. Individual nuclei are given a random false color. (C) Nuclei counts for a single spheroid measured from the 11 different Z-images taken at indicated distances from the plate bottom. (D) Dependence of nuclei count in the MaxPro image from a single spheroid on the number of individual images in a Z-stack taken over a 200 μm distance. The Z-stack setup was varied from three images taken 100 μm apart to 30 images taken 5 μm apart. (E) Nuclei counts measured from the separate z-images for three representative phenotypes of spheroids: Control (untreated, green), etoposide (200 μM, red), and paclitaxel (400 nM, blue). (F) Comparison between the nuclei counts obtained from the MaxPro images and sum of the nuclei count from all individual images for the three representative phenotypes of spheroids. Error bars in E and F represent ±1 SD (n = 6).
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f3: (A) Confocal images of a spheroid stained with Hoechst taken at indicated distances from the well bottom. As the image plane moves up from the well bottom toward the spheroid center, regions of distinct nuclei appear first in the center (at the surface of spheroid in contact with the plate bottom), then form a ring pattern that increases in diameter until it reaches some maximum associated with the outer surface of the spheroid. The MaxPro image is generated from 11 individual images taken 20 μm apart. (B) Nuclei segmentation shown for corresponding images in A. Individual nuclei are given a random false color. (C) Nuclei counts for a single spheroid measured from the 11 different Z-images taken at indicated distances from the plate bottom. (D) Dependence of nuclei count in the MaxPro image from a single spheroid on the number of individual images in a Z-stack taken over a 200 μm distance. The Z-stack setup was varied from three images taken 100 μm apart to 30 images taken 5 μm apart. (E) Nuclei counts measured from the separate z-images for three representative phenotypes of spheroids: Control (untreated, green), etoposide (200 μM, red), and paclitaxel (400 nM, blue). (F) Comparison between the nuclei counts obtained from the MaxPro images and sum of the nuclei count from all individual images for the three representative phenotypes of spheroids. Error bars in E and F represent ±1 SD (n = 6).

Mentions: At the magnifications used in these studies, taking only one image with a fixed offset does not allow adequate comparison of spheroids of different sizes or shapes, making acquisition of images at multiple focal planes necessary. Imaging protocols were studied using Hoechst-stained spheroids. A series of images was acquired at different planes along the focal axis (Z-stack) and combined into a MaxPro image to enable efficient segmentation and counting of distinct nuclei. The MaxPro algorithm composes a single image by combining maximum intensity pixels from an image stack along lines orthogonal to the image planes. A representative Z-stack of images is shown in Figure 3A. In each image, only a fraction of nuclei are resolved, while other nuclei are either out of focus, too dim, or otherwise obscured (Fig. 3B). As a result, the nuclear count represents only a fraction of the actual nuclei present in each slice. The total cell count from each image in a Z-stack is shown in Figure 3C.


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) Confocal images of a spheroid stained with Hoechst taken at indicated distances from the well bottom. As the image plane moves up from the well bottom toward the spheroid center, regions of distinct nuclei appear first in the center (at the surface of spheroid in contact with the plate bottom), then form a ring pattern that increases in diameter until it reaches some maximum associated with the outer surface of the spheroid. The MaxPro image is generated from 11 individual images taken 20 μm apart. (B) Nuclei segmentation shown for corresponding images in A. Individual nuclei are given a random false color. (C) Nuclei counts for a single spheroid measured from the 11 different Z-images taken at indicated distances from the plate bottom. (D) Dependence of nuclei count in the MaxPro image from a single spheroid on the number of individual images in a Z-stack taken over a 200 μm distance. The Z-stack setup was varied from three images taken 100 μm apart to 30 images taken 5 μm apart. (E) Nuclei counts measured from the separate z-images for three representative phenotypes of spheroids: Control (untreated, green), etoposide (200 μM, red), and paclitaxel (400 nM, blue). (F) Comparison between the nuclei counts obtained from the MaxPro images and sum of the nuclei count from all individual images for the three representative phenotypes of spheroids. Error bars in E and F represent ±1 SD (n = 6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: (A) Confocal images of a spheroid stained with Hoechst taken at indicated distances from the well bottom. As the image plane moves up from the well bottom toward the spheroid center, regions of distinct nuclei appear first in the center (at the surface of spheroid in contact with the plate bottom), then form a ring pattern that increases in diameter until it reaches some maximum associated with the outer surface of the spheroid. The MaxPro image is generated from 11 individual images taken 20 μm apart. (B) Nuclei segmentation shown for corresponding images in A. Individual nuclei are given a random false color. (C) Nuclei counts for a single spheroid measured from the 11 different Z-images taken at indicated distances from the plate bottom. (D) Dependence of nuclei count in the MaxPro image from a single spheroid on the number of individual images in a Z-stack taken over a 200 μm distance. The Z-stack setup was varied from three images taken 100 μm apart to 30 images taken 5 μm apart. (E) Nuclei counts measured from the separate z-images for three representative phenotypes of spheroids: Control (untreated, green), etoposide (200 μM, red), and paclitaxel (400 nM, blue). (F) Comparison between the nuclei counts obtained from the MaxPro images and sum of the nuclei count from all individual images for the three representative phenotypes of spheroids. Error bars in E and F represent ±1 SD (n = 6).
Mentions: At the magnifications used in these studies, taking only one image with a fixed offset does not allow adequate comparison of spheroids of different sizes or shapes, making acquisition of images at multiple focal planes necessary. Imaging protocols were studied using Hoechst-stained spheroids. A series of images was acquired at different planes along the focal axis (Z-stack) and combined into a MaxPro image to enable efficient segmentation and counting of distinct nuclei. The MaxPro algorithm composes a single image by combining maximum intensity pixels from an image stack along lines orthogonal to the image planes. A representative Z-stack of images is shown in Figure 3A. In each image, only a fraction of nuclei are resolved, while other nuclei are either out of focus, too dim, or otherwise obscured (Fig. 3B). As a result, the nuclear count represents only a fraction of the actual nuclei present in each slice. The total cell count from each image in a Z-stack is shown in Figure 3C.

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