Computational analysis of image-based drug profiling predicts synergistic drug combinations: applications in triple-negative breast cancer

Abstract

An imaged-based profiling and analysis system was developed to predict clinically effective synergistic drug combinations that could accelerate the identification of effective multi-drug therapies for the treatment of triple-negative breast cancer and other challenging malignancies. The identification of effective drug combinations for the treatment of triple-negative breast cancer (TNBC) was achieved by integrating high-content screening, computational analysis, and experimental biology. The approach was based on altered cellular phenotypes induced by 55 FDA-approved drugs and biologically active compounds, acquired using fluorescence microscopy and retained in multivariate compound profiles. Dissimilarities between compound profiles guided the identification of 5 combinations, which were assessed for qualitative interaction on TNBC cell growth. The combination of the microtubule-targeting drug vinblastine with KSP/Eg5 motor protein inhibitors monastrol or ispinesib showed potent synergism in 3 independent TNBC cell lines, which was not substantiated in normal fibroblasts. The synergistic interaction was mediated by an increase in mitotic arrest with cells demonstrating typical ispinesib-induced monopolar mitotic spindles, which translated into enhanced apoptosis induction. The antitumour activity of the combination vinblastine/ispinesib was confirmed in an orthotopic mouse model of TNBC. Compared to single drug treatment, combination treatment significantly reduced tumour growth without causing increased toxicity. Image-based profiling and analysis led to the rapid discovery of a drug combination effective against TNBC in vitro and in vivo, and has the potential to lead to the development of new therapeutic options in other hard-to-treat cancers.

Keywords: Compound profiling; High-content screening; KSP/Eg5 inhibitors; Microtubule-targeting agents; Synergy; Triple-negative breast cancer.

Figure 1

Workflow for the image–based discovery of potential synergistic drug combination. High‐content image analysis and compound profiling using a vector machine formed the basis for comparison of phenotypic outcome due to compound treatment. Combination pairs were selected for validation if compound profiles proved to be consistently similar or dissimilar. Subsequently, the effect of identified combination pairs on cell growth inhibition was measured. The assessment of the most promising synergistic combination pair was extended to an in vivo model of TNBC.

Figure 2

Treatment–induced alterations of cellular phenotypes, image segmentation, and dissimilarities of compound profiles. A) Fluorescence staining of microtubule and DNA (channels overlaid), revealing some phenotypic alterations between untreated, 0.1 μM doxorubicin or 0.1 μM vinblastine treated MDA‐MB‐231‐luc cells after 24 h. B) Delineation of single cells was conducted after conversion of RGB to grayscale images followed by image‐segmentation using a seeded watershed algorithm. C) Dot plot representation of relative profile dissimilarity for compound pairs. Compound profiles were derived using an SVM. Profile dissimilarities were computed for each concentration pair using a distance measure and then normalized to the average dissimilarity of all compound profiles. D) Dot plot representation of the combination index (CI) for each compound pair. CI values were calculated for each concentration pairs following the Chou and Talalay method.

Figure 3

Combination effects of KSP/Eg5 inhibitors and vinblastine on the growth of TNBC cells and normal fibroblasts. Effects of combination treatments were tested for 25 concentration pairs 72 h post‐treatment. (A, B) Surface plots of the Excess Over Highest Single Agent (EOHSA) show the difference in cell growth inhibition between KSP/EG5 inhibitors/vinblastine in combination and the most potent single treatment at the corresponding concentration in three independent TNBC cell lines. Shades of blue depict positive values and thus stand for a greater effect of the combination compared to single agents. (B, C) Dot plot representations of the combination index (CI) for the combinations monastrol/vinblastine and ispinesib/vinblastine for all 25 concentration pairs each. CI values were calculated following the Chou and Talalay method. (D) Surface plots showing the cell viability of MRC‐5 fibroblasts after treatment with ispinesib/vinblastine alone or in combination. Single compound concentrations are denoted on the x‐ and y‐axis, and cell viability is plotted on the z‐axis relative to control. (E) Surface plot of the Excess Over Highest Single Agent (EOHSA) shows the difference in cell viability between ispinesib/vinblastine in combination and the most potent single treatment at the corresponding concentration in MRC‐5 cells.

Figure 4

Combination effects of ispinesib and vinblastine on cell cycle, mitotic spindle and apoptosis induction. (A) Representative cell cycle profiles of MDA‐MB‐231 cells incubated for 24 h with 0.5 nM vinblastine (red), 0.32 nM ispinesib (orange), or the combination (blue) measured using PI staining, and (B) quantification of the fraction of cells arrested in G2/M phase. Columns, means of at least 3 individual experiments; Bars, SD. Statistical analysis was performed using one‐way ANOVA (**p < 0.01, ***p < 0.001). (C) Representative photographs of fluorescent staining of microtubules and nuclei in MDA‐MB‐231 cells 24 h post‐treatment with 4 nM of ispinesib or vinblastine or their combination. Arrows and arrowheads denote mitotic cells with monoploar and bipolar spindles, respectively. Scale bars, 20 μm. (D) Quantification of mitotic cells with monopolar or bipolar mitotic spindles. Columns, average of at least 350 cells; Bars, SD. Statistical analysis was performed by comparing the number of mitotic cells with bipolar spindles using one‐way ANOVA (****p < 0.0001). (E) Representative flow cytometry profiles of MDA‐MB‐231 cells and (F) quantification of apoptotic cells after 48 h treatment with 0.32 nM ispinesib and 0.5 nM vinblastine alone or in combination measured using Annexin V‐FITC and PI staining. Columns, means of at least 3 individual experiments; Bars, SD. Statistical analysis was performed using one‐way ANOVA (***p < 0.001).

Figure 5

Change in animal weight and antitumour effect in vivo. Nude mice (BALB/c) were injected with 1*106 MDA‐MB‐231 cells to establish an orthotopic TNBC model. Treatment with ispinesib (5 mg/kg) and Vinblastine (1 mg/kg) alone or in combination was initiated when the mean tumour volume reached about 70 mm3 and was administered i.p. weekly for three weeks. Mice were monitored for a period of 8 weeks. (A) Change in animal weights for the untreated control and treatment groups. (B) Antitumour effect of combination treatment and single treatments. Statistical analysis of the tumor volume between vinblastine only and the combination treatment was performed using the Student's t‐test (*p < 0.05). Points, mean values for 8 mice per group; Bars, SE.