Abstract 2015: Multiparameter flow cytometry analysis of breast cancer cell lines MDA-MB-231 and MDA-MB-468 to simultaneously assess cell proliferation, apoptosis, and DNA damage in response to treatment with camptothecin
Authors: Wilensky Lissette , Roys Stacey , Corselli Mirko , Wang Alice , Emre Nil 
Published: August 2015

Abstract
In cell analysis, separate functional assays are often utilized to monitor multiple functional states. To simultaneously interrogate multiple functional states, we have optimized simple flow cytometric panels that use up to four parameters. As a result, more detailed biological correlations between distinct cellular functions can be obtained from a single sample. In this study, multiparameter panels were used to analyze breast cancer cell lines MDA-MB-231 and MDA-MB-468 for differences in proliferation, apoptosis, and DNA damage in response to drug treatment. Cells were treated in culture with 20 μM camptothecin, and then analyzed on a two-laser BD Accuri™ C6 flow cytometer. In the first panel, we analyzed mitochondrial membrane potential (TMRE), phosphatidylserine exposure (Annexin V), and viability (7-AAD) to quantify early or late apoptotic and necrotic cells. In the second panel, we assessed double-stranded DNA breaks (phosphorylated H2AX), caspase activity (cleaved PARP), DNA synthesis (BrdU incorporation), and DNA content (7-AAD) to quantify DNA damage, apoptosis, and cell cycle status. In the third panel, we performed a more detailed cell cycle analysis by detection of DNA synthesis (BrdU incorporation), DNA content (7-AAD), M-phase associated histone expression (phosphorylated histone H3), and cyclin content (cyclin A or B). In response to camptothecin treatment, both cell lines showed decreased proliferation, increased DNA damage responses, increased apoptosis, and decreased viability. The presence of mitochondrial depolarization, caspase activity, and phosphatidylserine exposure suggests an apoptotic death mechanism for both cell types. Compared to MDA-MB-231 cells, MDA-MB-468 cells showed higher levels of mitochondrial depolarization, phosphatidylserine exposure, caspase activity, and loss of viability indicating increased drug susceptibility. Additionally, while both MDA-MB-231 and MDA-MB-468 cells showed decreased proliferation and an increased DNA damage response, the cell lines showed different cyclin expression across cell cycle compartments, potentially suggesting different cellular responses to drug treatment. Multiparameter flow cytometry enables the simultaneous analysis of cell proliferation, viability status, and other cellular states at the single cell level and serves as an invaluable and tool to understand the response of heterogeneous cancer cells to drug treatment. When combined with an automated platform for sample acquisition, this method can provide an effective tool for high throughput drug screening applications.
Citation Format: Lissette Wilensky, Stacey Roys, Mirko Corselli, Alice Wang, Nil Emre. Multiparameter flow cytometry analysis of breast cancer cell lines MDA-MB-231 and MDA-MB-468 to simultaneously assess cell proliferation, apoptosis, and DNA damage in response to treatment with camptothecin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2015. doi:10.1158/1538-7445.AM2015-2015