(177h) One Step Microfluidic Immunomagnetic Separation of Tumor Initiating Cells Based on Multiple Markers

There has been increasing proposition and observation that cancer growth is driven and sustained by tumor initiating cells (TICs, also known as cancer stem cell (CSC)), which are capable of self-renewal and aberrant differentiation. TICs usually express abnormal high or low level of proteins, but their detection and isolation present unique challenges due to their extremely low frequency (<1%) in most human tumors. Microfluidics has been used for rare cell separation because of their minimal sample requirements. However, current methods are limited in sorting of TICs based on single marker in one step, which is not sufficient for high purity selection; or multiple markers in multiple steps, which increases the risk of cell loss. Here, we propose a new strategy that allows isolation of TICs in one step using two beads. Breast cancer TICs are identified as expression of CD44⁺/CD24^-. In our method, CD24⁺ cells are excluded by firstly occupying their surface with CD24 antibody linked nonmagnetic beads, so that CD44 antibody linked magnetic beads are only able to bind to CD24^- cells. Finally, CD44⁺/CD24^- cells are enriched after one step microfluidic immunomagnetic separation. We tested our technology with SUM 149 cell line. Cells enriched with two beads method generate 2.4 times tumorspheres compared to unsorted cells, and they showed 30% more efficiency in tumorsphere formation compared to one beads method. We also test unsorted and sorted cells with flow cytometry. CD44 antibody coated magnetic beads select cells with high expression of CD44, while cells sorted with our two beads method shift to both CD44⁺ direction and CD24^- direction. We envision that our approach will be useful for highly specific selection of TICs with two markers of opposite preferences. Our method allows the isolation of TICs with high purity from small samples, thus will provide a useful tool for cancer diagnostic and therapeutic researches on precious samples (e.g.  primary cells from animals and patients).