Screening Therapeutic Agents Targeting To Breast Cancer Stem Cells Using A Microfluidic Single-cell Clone Forming Assay

Objective: To develop a single cell analyzing assay on microfluidic chip to screen breast cancer stem cell targeting drugs using the capability of single-cell-derived tumor spheres of cancer stem cells(CSCs).Methods: We designed and fabricated a microfluidic chip with a 3840 single-cell capture array to and optimized operation parameters for single cell manipulations.array to capture single cell and improved its efficiency of single cell controlling.Breast cancer cell lines MCF-7,MDA-MB-231 and T47 D were used for validating the microfluidic assay.The entire process,including single-cell array construction,long-term(14 days)cell culturing,drugs treatment and in situ fluorescence imaging are implemented on the same microfluidic device.The single-cell array were treated with anticancer agents including paclitaxel,doxorubicin,thiostrepton and salinomycin.The CSCs targeting killing effect of an agent is determined as a complete inhibition of single-cell clone forming.Results:We achieved a high single-cell capture rate of ~54.2% thus allowing more than 2000 single cell to be analyzed in a single test.The comparison of size distributions of cells before and after capturing indicated the microfluidic assay has no bias on cell size.on-chip and in bulk were measured and compared.The result shows the capability to capture cells with a wide range of sizes and it has no selectivity of cell sizes.Paclitaxel and doxorubicin showed incomplete inhibitory effects on single-cell clone forming.Their inhibition rates of MDA-MB-231 were 39.10% and32.35% and were 16.77% and 14.79% on MCF-7.In contrast,thiostrepton and salinomycin showed complete CSCs inhibitory effects.The inhibition rates of thiostrepton for MDA-MB-231 and MCF-7 were 100% and 96.41%,and those of salinomycin were all 100% and 100%.Conclusion:The current work developed a microfluidic single-cell clone forming assay for screening breast cancer stem cells targeted agents.The developed microfluidic assay is able to complete the whole analytical process in an integrated manner.The microfluidic assay is confirmed with the capability of screening therapeutic agents targeting to breast cancer stem cells.The CSCs targeting agents showed complete inhibition of single-cell clone forming,while the non-targeting ones showed only incomplete inhibition effects.The developed microfluidic assay with advantages of easy operation and high efficiency is a powerful tool for screening CSCs targeted agents.