Screening Therapeutic Agents Specific to Breast Cancer Stem Cells Using a Microfluidic Single‐Cell Clone‐Forming Inhibition Assay

Screens of cancer stem cells (CSCs)-specific agents present significant challenges to conventional cell assays due to the difficulty in preparing CSCs ready for drug testing. To overcome this limitation, developed is a microfluidic single-cell assay for screening breast cancer stem cell-specific agents. This assay takes advantage of the single-cell clone-forming capability of CSCs, which can be specifically inhibited by CSC-targeting agents. The single-cell assay is performed on a microfluidic chip with an array of 3840 cell-capturing units; the single-cell arrays are easily formed by flowing a cell suspension into the microchip. Achieved is a single cell-capture rate of ≈60% thus allowing more than 2000 single cells to be analyzed in a single test. Over long-term suspension culture, only a minority of cells survive and form tumorspheres. The clone-formation rate of MCF-7, MDA-MB-231, and T47D cells is 1.67%, 5.78%, and 5.24%, respectively. The clone-forming inhibition assay is conducted by exposing the single-cell arrays to a set of anticancer agents. The CSC-targeting agents show complete inhibition of single-cell clone formation while the nontargeting ones show incomplete inhibition effects. The resulting microfluidic single-cell assay with the potential to screen CSC-specific agents with high efficiency provides new tools for individualized tumor therapy.