A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin.
Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell's ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 10(5) -10(10) doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell's ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4-12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug's single-cell potency and can be used for any fluorescent or fluorescently labeled drug, including nanoparticles or antibody-drug conjugates.
Department of Chemical Engineering, Massachusetts Institute of Technology; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology.
Katie F
KF
Maass
Oncology Medicinal Chemistry, Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development.
Chethana
C
Kulkarni
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology.
Mohiuddin A
MA
Quadir
Department of Chemical Engineering, Massachusetts Institute of Technology; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology.
Paula T
PT
Hammond
Translational Research Group, Department of Pharmacokinetics Dynamics and Metabolism, Pfizer Worldwide Research and Development.
Alison M
AM
Betts
Department of Chemical Engineering, Massachusetts Institute of Technology; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology; Department of Biological Engineering, Massachusetts Institute of Technology. Electronic address: wittrup@mit.edu.
Karl Dane
KD
Wittrup
D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003114:Colony-Forming Units Assay / Q000379:methods; D004396:Coloring Agents / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000494:pharmacology; D019073:HT29 Cells; D006801:Humans; D053758:Nanoparticles / Q000008:administration & dosage; D014700:Verapamil / Q000494:pharmacology
D004396:Coloring Agents; D005456:Fluorescent Dyes; D004317:Doxorubicin; D014700:Verapamil
cancer chemotherapy; cell lines; drug effects; efflux pumps; pharmacodynamics