Plate-based drug and toxin screening using multiplexed benchtop flow cytometry

Drug-based screening, identification of positive ‘hit’ compounds, and evaluation of mode of action require an understanding of how the drug modulates critical cellular parameters and activities which eventually lead to cytotoxicity or alter cell proliferation.¹

This is particularly important in oncology research, where combinatorial strategies using drug mixtures can have additive/synergistic effects on proliferation and apoptosis. Given the increased need for assays with statistically-rich informational content and mechanism-based data, drug researchers and toxicologists have turned to flow cytometry assays. With its potential for precise and quantifiable comparisons between cell populations, flow cytometry enhances dose-response calculations with the statistical power afforded by large sample size.²

The guava easyCyte™ benchtop flow cytometry systems (EMD Millipore) enable plate-based compound screening because they require significantly fewer cells per well, and because its powerful InCyte™ software enables multiparametric comparisons of concurrent responses.The addition of a violet laser in the easyCyte™12 instrument permits the simultaneous measurement of up to 12 parameters, offering flexibility and more powerful analysis in multiparametric screening studies.

This article demonstrates the power of the guava® system and accompanying software to provide enriched data during drug screening processes. Using just two fundamental assays, plate-based screening was performed for various cytotoxic compounds and identified key ‘hits’. The same assays were used to further evaluate select compounds in greater detail with dose-response and time-response studies. These assays also provided information on the mechanism of action of the compounds gambogic acid, celastrol and thimerosal, and their modulation of apoptotic and proliferative responses.

Multiparameter analysis

The ability to obtain multiparametric information during compound screening is invaluable to the drug discovery process. Screening multiple compounds while multiplexing cell health markers leads to many data points and often makes ‘hit’ identifi cation complex. The guava easyCyte™ benchtop flow cytometry systems simplify this process by analyzing up to six parameters simultaneously, whether a single parameter from up to six plates, or six parameters from each sample in a plate, as demonstrated here.

The relationship between changes in mitochondrial membrane potential (MMP) and cell health permits the use of a powerful assay for apoptosis. Combining EMD Millipore’s MitoSense Red for assessment of MMP with Caspase 3/7 FAM detection reagent, Annexin V Brilliant Violet™ 421 (BioLegend) and 7-AAD enables multiparametric assessment of apoptosis with simultaneous discrimination of necrotic cells in a single, simplified assay.

Upon treatment with gambogic acid, Jurkat cells showed a decrease in MMP, increased caspase activity, increased Annexin V staining and increased cell death, as expected. Combining multiple cell health markers in the manner described permits discrimination of the stage of apoptosis. For example, not all cells that were positive for Annexin V were also found to be simultaneously positive for Caspase 3/7.

Monitoring proliferation

Assessment of proliferation is critical to cell analysis, and is of particular importance when screening for bioactivity or cytotoxicity. Because its expression is required for progression through the cell cycle, measuring expression of the nuclear protein Ki67 has become the prototypical assay for cell proliferation.

A robust Ki67 signal was observed in untreated Jurkat cells. In contrast, a subpopulation of cells treated with the anti-inflammatory compound celastrol demonstrated the diminished Ki67 expression that is indicative of a reduction in proliferation. Celastrol has been shown to inhibit proliferation in numerous tumor cell types via downregulation of cyclins D1 and E3.³

Plate-based screening

The assays described above were next applied to the plate-based screening of cytotoxic compounds. A panel of 80 cytotoxic, immunosuppressive, anti-proliferative and anti-inflammatory compounds was obtained from Microsource Discovery Systems, Inc. at a concentration of 10 mM in dimethyl sulfoxide (DMSO). Compounds were diluted in complete growth medium to either 10 μM or 40 μM. Plate setup included negative controls containing 0.2 per cent DMSO, and positive controls treated with 1 μM staurosporine, a known protein kinase inhibitor.

The population percentage altered for each parameter was compared in heat map format using the InCyte™ software. The software allows for quick identification of ‘hit’ compounds and comparison of all parameters simultaneously, as shown in the pie graphs and population percentages in Figure 1. While the number of hits between the 10 μM (1A) and 40 μM (1B) plates look similar, the percentage of cells showing higher percentages of effects varied, as shown by the darker shades of blue in hit wells.

From the results shown in Figure 1, autofluorescent compounds and those treatments causing less than 20 per cent change compared with negative controls were eliminated to focus on compounds that showed cytoactivity at either concentration. Twenty compounds induced cell response at 10 μM while one additional compound induced response at 40 μM only. Some compounds, such as gambogic acid, induced a large change in MMP, with a lower percentage of cells being Annexin V positive and even fewer showing Caspase 3/7 activity. These results were consistent with published reports that gambogic acid induces apoptosis specifically in tumour cells while having less impact on normal
cells.^4-6

Other compounds, such as the anti-tumour agent juglone,⁷ induced a change in mitochondrial depolarization and an almost identical degree of caspase response at 10 μM, with changes in Annexin V binding only occurring at the 40 μM concentration. Several compounds showed large impacts on MMP changes with a lower percentage for the other markers, while others demonstrated impact on all assays in parallel. From these screening results, a subset of compounds was selected for dose and time-response analysis.

Dose and time-response experiments

The mechanism of celastrol, a known antioxidant and anti-inflammatory compound, was investigated by evaluating its dose-dependent impact on Jurkat cells. Population percentages for cell health markers at various concentrations are plotted in Figure 2. The degree of MMP decrease and Annexin V staining was similar, with a lower percentage of cells showing caspase activity across the concentration range. The loss of cell proliferation increased with increased celastrol concentration. Proliferation recovery was observed at the two highest concentrations, but this can likely be attributed to the high percentage of cells exhibiting cell death at the same concentrations. The study demonstrated that treatment with celastrol caused mitochondrial depolarization, apoptosis and proliferation loss at lower concentrations than those causing any significant cell death.

To assess the effect of increasing compound exposure time on cell health, Jurkat cells were treated with gambogic acid, an antitumour compound, and thimerosal, an antifungal and antiseptic compound, for various durations. Time-dependent responses to gambogic acid (Figure 3A) demonstrated mitochondrial and apoptotic impacts. Results obtained with gambogic acid demonstrate that a rapid and significant loss of MMP occurred at earlier time points when compared to other cell health markers tested. Annexin V- and Caspase 3/7-positive population percentages showed a steady increase at three and six hours with maximal percentages at 16 hours. Significant cell death and proliferation loss were not seen until 16 hours.

Time-dependent responses to thimerosal underscored different cell health impacts (Figure 3B). Even with short incubation times, thimerosal caused almost complete depolarization of the mitochondrial membrane by three hours. At three hours, this was also accompanied by significant changes in Annexin V binding, with a continued increase at six, 16 and 24 hours. A high percentage of cells showing caspase activity and cellular death was not seen until the six-hour time point, with maximal caspase activity attained at 16 hours. Low levels of proliferation inhibition were seen starting at six hours, with a gradual decrease in proliferation over time. While equivalent cell death and proliferation loss responses were seen with gambogic acid, an increased percentage of cells displayed death responses to thimerosal.

Conclusion

Multiparametric analysis at a single-cell level provides comprehensive mechanistic and cell health information, which greatly facilitates the assessment of cytoactive compounds. These studies showed that plate-based screening using the guava easyCyte™ 12 benchtop flow cytometer
enabled uncomplicated comparison of mechanisms of action of diverse compounds while also providing the sample-conserving benefits of microcapillary flow cytometry. Specifically, gambogic acid induced significant and rapid mitochondrial depolarization, celastrol acted by inhibiting proliferation and inducing apoptosis, and thimerosal triggered rapid cell death.

The ease of these multiplexed assays, coupled with plate-based microcapillary cytometry and the heat mapping functionality in the InCyte™ software, enables researchers to obtain insights into how compounds modulate apoptotic and proliferation processes and their relationship to mitochondrial dysfunction and cell death pathways.

References

1. Wesierska-Gadek, J., Gueorguieva, M., Ranftler, C., Zerza-Schnitzhofer, G. (2005, Sep.) A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death. J Cell Biochem, 1;96(1):1-7.
2. Lombardo, T., Anaya, L., Kornblihtt, L., Blanco, G. (2012) Median effect dose and combination index analysis of cytotoxic drugs using flow cytometry. In Flow Cytometry – Recent Perspectives, M.Sc. Ingrid Schmid (Ed.) ISBN: 978-953-51-0626-5, InTech, DOI: 10.5772/38214.
3. Kannaiyan, R., Manu, K.A., Chen, L., Li, F., Rajendran, P., Subramaniam, A., Lam, P., Kumar, A.P., Sethi, G. (2011, Oct.) Celastrol inhibits tumor cell proliferation and promotes apoptosis through the activation of c-Jun N-terminal kinase and suppression of PI3 K/Akt signaling pathways. Apoptosis, 16(10), 1028-41.
4. Shi, X., Chen, X., Li, X., Lan, X., Zhao, C., Liu, S., Huang, H., Liu, N., Liao, S., Song, W., Zhou, P., Wang, S., Xu, L., Wang, X., Dou, Q.P., Liu, J. (2010, Jan. 1) Gambogic acid induces apoptosis in imatinib-resistant chronic myeloid leukemia cells via inducing proteasome inhibition and caspase-dependent Bcr-Abl down regulation. Clin Cancer Res., 20(1):151-63.
5. Zhao, L., Guo, Q.L., You, Q.D., Wu, Z.Q., Gu, H.Y. (2004) Gambogic acid induces apoptosis and regulates expressions of Bax and Bcl-2 protein in human gastric carcinoma MGC-803 cells. Biol. Pharm. Bull., 27, 998–1003.
6. Li, X., Liu, S., Huang, H., Liu, N., Zhao, C., Liao, S., et al. (2013) Gambogic acid is a tissue-specific proteasome inhibitor in vitro and in vivo. Cell Rep., 3(1), 211–22.
7. Xu, H.L., Yu, X.F., Qu, S.C., Zhang, R., Qu, X.R., Chen, Y.P., Ma, X.Y., Sui, D.Y. (2010, Oct. 25) Anti-proliferative effect of Juglone from Juglans mandshurica Maxim on human leukemia cell HL-60 by inducing apoptosis through the mitochondria-dependent pathway. Eur. J. Pharmacol., 645(1-3), 14-22.

About the Authors

Katherine Gillis, Applications Scientist, katherine.gillis@emdmillipore. com; Julie Clor, Applications Scientist, julie.clor@emdmillipore. com; Kamala Tyagarajan, R&D Manager, [email protected]