Novel screening approach for stem cell selective inhibitors and their possible translational therapeutic potential for endometriosis

Endometriosis is an estrogen-dependent benign disease characterized by growth of the endometrial tissue outside the uterine wall. Several reports suggest the possibility of the pathogenesis and recurrence of endometriosis being related to functions of stem/progenitor cells of the endometrium. The drawback of the widely used method of using Hoechst 33342, a fluorescent dye, to collect stem cell-like populations, is the requirement of an ultraviolet (UV) excitation source not commonly provided on standard flow cytometers. Here, we aimed to overcome this hurdle by establishing a novel method that uses DyeCycle Green (DCG), a cell-permeable DNA dye, for collecting a significantly higher fraction of stem cell-like side population (SP) from HHUA cells (human endometrial cancer cell line) with standard equipment without a UV laser. Furthermore, subculturing the DCG-SP cells expanded their population remarkably. The DCG-SP cells possessed stem cell-like characteristics with high expression of stem cell markers such as aldehyde dehydrogenase 1 A (ALDH1A), sushi domain containing 2 (SUSD2), increased colony formation ability, and high tumorigenicity in vivo, although the expression of some stem cell markers varied during expansion. We screened inhibitors for selective proliferation of the DCG-SP cells over immortalized endometrial cells (EM-E6/E7/hTERT-2 cells) and identified two effective compounds disulfiram and NSC319726. In addition, these compounds inhibited the colony formation and invasiveness of the DCG-SP cells. Our DCGmediated screening of SP cells would possibly be translational to identify compounds that selectively target stem cells for the treatment and inhibition of recurrence of endometriosis.