Figure 7.

A schematic representation of compound screening with SCESM using relative gene expression and protein expression analysis. A single-cell suspension is seeded with stem medium onto round-bottom ultra-low adherent (ULA) microwell plate and centrifuged to obtain single multi-cellular mass per well. To form spheroids, the cells are cultured for 7 days, with half of the medium changed every 2–3 days. After seven days, half of the stem medium is replaced with treatment medium containing testing compound. Each spheroid can be treated individually with different compounds/concentrations. After five days of treatment, the spheroids are disintegrated by lysis buffer and RNA or protein analysis can be performed. For the RNA analysis, RNA is extracted by spin columns and the isolated RNA can be used for qRT-PCR analysis. For the protein analysis, cells are labeled with fluorescently labeled antibodies and analyzed with flow cytometry. Use of active anti-CSC compounds leads to decreased expression of “stemness” genes/proteins compared to the mock treatment.