Abstract LB-053: Tracing redox-regulated breast cancer stem cell state transition using a dual fluorescent reporter system
Authors: Luo Ming , Brooks Michael , Burnett Joseph , Truchan Nathan , Sun Duxin , Wicha Max S. 
Published: August 2018

Abstract
Breast cancer stem cells (BCSCs) display plasticity allowing them to transition between quiescent mesenchymal- (M) and proliferative epithelial-like (E) states. This dynamic equilibrium of BCSCs in distinct M- and E-like states coordinately drives tumorigenesis, metastasis and treatment resistance. Despite the functional significance of CSC plasticity in tumor development and progression, tracing of BCSC state transitions under physiologic conditions is hampered by the lack of a functional dual fluorescent reporter to monitor state dynamics of BCSCs at single cell resolution. To this end, we developed a dual fluorescent reporter to assess EMT-MET state transitions of BCSCs in real-time. As E-BCSCs express high levels of ALDH1A3, we generated an ALDH1A3 fluorescent reporter system utilizing Crisper-Cas 9 mediated knockin of mCherry downstream of the ALDH1A3 promotor. To trace M-BCSCs that express high levels of CD44 and vimentin, we constructed a lentiviral reporter expressing GFP driven by a portion of the vimentin promotor that is specifically expressed in mesenchymal CD44+ cells. Dual reporter cells were generated by infection of ALDH1A3_mCherry expressing cells with the lentivirus expressing Vimentin_GFP. To assess stem cell characteristics of these reporter cells, we first examined their sphere-forming capacity. Both GFP-mCherry+ (mCherry+) and GFP+mCherry- (GFP+) cells have significantly higher capacity to form tumor spheres than GFP-mCherry- (DN) cells while the GFP+mCherry+ (DP) cells harbor the highest sphere-forming ability. To determine the relationship between cells expressing these reporters, we generated tumor spheres from single cells and examined the fluorescent pattern of resultant tumor spheres. While spheres generated from DP cells display mixed phenotypes of cells expressing mCherry and/or GFP, spheres derived from mCherry+ cells maintain mCherry+. Interestingly, spheres derived from GFP+ cells generated three distinct phenotypes with some spheres expressing strong GFP and weak mCherry fluorescence while others expressing strong mCherry but weak or no GFP signal. These distinct phenotypes of tumor spheres derived from GFP+ cells suggest that M-BCSCs possess capacity to transition to a dual (GFP+mCherry+) or epithelial-like (GFP-mCherry+) state in order to form tumor spheres at non-attachment culture conditions that are normally associated with increased oxidative stress. These results suggest that the DP cells are the most primitive, with the greatest tumor sphere forming capacity and multistate differentiation potential followed by the Vimentin_GFP+ cells, ALDH1A3_mcherry+ cells and DN cells. By time-lapse microscopy, we further monitored how metabolic/oxidative stress induced by glycolytic inhibitor 2DG or H2O2 affects BCSC state dynamics at single cell resolution. We found that oxidative stress promotes the transition of GFP+mCherry- M-BCSCs to GFP+mCherry+ (bipotent) and GFP-mCherry+ (E-like) BCSCs. In contrast, the antioxidant N-acetyl cysteine promoted the transition of bipotent and E-like BCSCs to a GFP+mCherry- M-like state. These studies demonstrate that the redox state of the tumor microenvironment regulates state transitions of BCSCs. This dual fluorescent reporter system should prove useful in monitoring CSC state transitions under more physiologically relevant in vivo conditions.
Citation Format: Ming Luo, Michael Brooks, Joseph Burnett, Nathan Truchan, Duxin Sun, Max S. Wicha. Tracing redox-regulated breast cancer stem cell state transition using a dual fluorescent reporter system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-053.