Figure 2
Figure 2. Multiparametric image analysis and high-content screening enables automated analysis of cell viability for CLL patients’ cells exposed to KIs and venetoclax alone and in combination. (A) Correlation of technical replicates for 2S cells from 1 representative patient treated with negative control (DMSO), positive control (STS + venetoclax), and a panel of drugs. Comparison of fluorescence intensity or area threshold analyses to identify positive (dead) cells based on loss of lipid asymmetry (Annexin V, green), loss of mitochondrial membrane potential (TMRE, red), and nuclear condensation (Condensed Nuclei, blue) with a multiparametric classifier (black). Images of cells were acquired as described in Figure 1C. For threshold analysis, cells were classified as in Figure 1D; for multiparametric analysis, cells were classified as nonviable (percentage positive) if they were more similar to the positive control (STS + venetoclax treated) training group than the negative control (DMSO) group based on 8 quantified image features using a random forest classifier. (B) Patient-specific dose response fit for venetoclax (1 pM to 1 µM) in unstimulated (C, black) and 2S CLL cells (2S, red) assessed by multiparametric analysis as in the description of panel A. Data for 9 representative patients were fit using nonlinear least squares with GraphPadPrism 5.03 (GraphPad Software) as mean ± standard deviation (std dev) for cell images from 8 micrographs per condition. (C) Microenvironment-induced resistance to venetoclax is independent of model. Response to 10 nM venetoclax for 72 hours was measured for unstimulated and 2S- or IL4-stimulated CLL cells from 7 patients. Drug response assessed by multiparametric analysis of cells classified as percentage positive as described in panel A. (D) Schematic overview of image-based high-content drug screening in primary CLL cells. Patient-derived CLL cells were stimulated with 2S media, seeded into 384-well plates and treated with 320 KIs at a screening concentration of 1 µM with or without venetoclax (10 nM). Negative (DMSO) and positive controls (STS + venetoclax) used for training of the classifier were included in each plate. Automated fluorescence imaging was performed as described in Figure 1.

Multiparametric image analysis and high-content screening enables automated analysis of cell viability for CLL patients’ cells exposed to KIs and venetoclax alone and in combination. (A) Correlation of technical replicates for 2S cells from 1 representative patient treated with negative control (DMSO), positive control (STS + venetoclax), and a panel of drugs. Comparison of fluorescence intensity or area threshold analyses to identify positive (dead) cells based on loss of lipid asymmetry (Annexin V, green), loss of mitochondrial membrane potential (TMRE, red), and nuclear condensation (Condensed Nuclei, blue) with a multiparametric classifier (black). Images of cells were acquired as described in Figure 1C. For threshold analysis, cells were classified as in Figure 1D; for multiparametric analysis, cells were classified as nonviable (percentage positive) if they were more similar to the positive control (STS + venetoclax treated) training group than the negative control (DMSO) group based on 8 quantified image features using a random forest classifier. (B) Patient-specific dose response fit for venetoclax (1 pM to 1 µM) in unstimulated (C, black) and 2S CLL cells (2S, red) assessed by multiparametric analysis as in the description of panel A. Data for 9 representative patients were fit using nonlinear least squares with GraphPadPrism 5.03 (GraphPad Software) as mean ± standard deviation (std dev) for cell images from 8 micrographs per condition. (C) Microenvironment-induced resistance to venetoclax is independent of model. Response to 10 nM venetoclax for 72 hours was measured for unstimulated and 2S- or IL4-stimulated CLL cells from 7 patients. Drug response assessed by multiparametric analysis of cells classified as percentage positive as described in panel A. (D) Schematic overview of image-based high-content drug screening in primary CLL cells. Patient-derived CLL cells were stimulated with 2S media, seeded into 384-well plates and treated with 320 KIs at a screening concentration of 1 µM with or without venetoclax (10 nM). Negative (DMSO) and positive controls (STS + venetoclax) used for training of the classifier were included in each plate. Automated fluorescence imaging was performed as described in Figure 1.

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