PMC

IC₅₀ values of 38 compounds from the small library screen of 119 approved anticancer drugs obtained from 4-parameter fits to calcein AM-positive cell readout over the range of tested concentrations.

Representative images of spheroids treated with different compound concentrations. Composite images of Hoechst (blue), calcein AM (green), and EthD-1 (red). Note the dose-dependent decrease of spheroid size and also increase in the number of dead cells (in red) across all treatments. Spheroids treated with high concentrations of some compounds (e.g., paclitaxel and staurosporine) appear to disintegrate.

Concentration-dependent effects and 4-parameter curve fits of selected compounds in 3D spheroid culture using (A) Live cells (EthD-1 negative) per spheroid or (B) total cells per spheroid as determined by Cell Scoring analysis of MaxPro image. Red circles—paclitaxel; dark red squares—staurosporine; blue diamonds—doxorubicin; green triangles—mitomycin C; teal open circles—etoposide; purple open diamonds—fluoroadenine. Error bars represent  ±1 SD (n = 8).

Apoptosis assay. Spheroids were treated with indicated compounds for 42 h, then stained with Hoechst (blue) and caspase 3/7 (green) reagents. (A) Representative images and analysis of control and sample treated with 1 μM staurosporine. Nuclei are identified by a blue mask; Apoptotic cells are identified by a pink mask. (B) Dose-dependent increase of number of apoptotic cells in spheroids treated with 3 compounds: paclitaxel (green circles, IC₅₀ = 9.5 nM), staurosporine (purple triangles, IC₅₀ = 41.5 nM), and mitomycin C (red squares, IC₅₀ = 6.01 μM). The content of apoptotic cells was ∼50% in the treated samples. Error bars represent ± 1 SD (n = 3).

(A) Representative spheroid phenotypes (top) and their corresponding masks (bottom) showing image analysis and segmentation. (B) Image analysis readouts derived as a result of Nuclei Count and Cell Scoring analysis for the following compounds: control (0.1% DMSO), paclitaxel 150 nM, etoposide 200 μM, staurosporine 300 nM, mitomycin C 1 μM, doxorubicin 1 μM, and fluoroadenine 100 μM. (C) Image analysis readouts were derived from geometric or average intensity readouts. The values in C are normalized to DMSO controls (set to 1,000). Error bars in B and C represent ± 1 SD (n = 8).

(A) Spheroids from HCT116 cells plated at different densities. Images were acquired 48 h postplating. Smaller spheroids (500 cells/well or less) were observed to have less consistent spheroidal shape. (B) Example of image analysis and spheroid mask (blue) using the TL image. (C) Dependence of spheroid width as determined by image analysis on cell plating density (n = 12) for three cell types: HCT116 cells (green); HepG2 cells (purple); DU145 cells (blue). The response was modeled using a spherical volume model with a single-cell diameter variable. A fit of the HCT116 data to the theoretical spherical volume model using a cell diameter of 32 μm is shown in red. Error bars not shown; SDs were less than 5% of values.

(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.

(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).