(683c) Development of a Single-Cell Clonogenic Assay

Standard cell proliferation assays use the bulk media drug concentration to determine a drug’s potency. A more clinically relevant quantity is the amount of drug that is actually taken up by the cell. In this work, we developed an in vitro single-cell clonogenic assay based on flow cytometry. The assay uses a fluorescent drug and the CellTrace^TM Cell Proliferation Kit (Molecular Probes) to evaluate the cytostatic effects of drug exposure. The amount of drug taken up by individual cells can be correlated to the proliferation of that cell. Doxorubicin, a naturally fluorescent chemotherapeutic drug, was used in the development and validation of this assay. Uptake of drug by treated cells was quantified via fluorescence. Various drug concentrations in the media and lengths of exposure to the drug were tested in the following cancer cell lines: BT474, HCT-15, HT-29, IGROV-1, MDA-MB-231, and T47D. Results were also compared to a standard WST-1 assay, which measures bulk levels of cell proliferation based on metabolic activity. In order to demonstrate the utility of measuring actual cellular drug uptake, the effect of inhibiting the multi-drug resistance pump in HCT-15 cells that express the multi-drug resistance pump was shown. This assay can also be generalized to study any fluorescent drug including antibody-drug conjugates which are labeled fluorescently or which carry fluorescent drugs such as Doxorubicin.