Abstract 5152: Cancer stem cells arise directly from differentiated cancer cells: A new model of dynamic equilibrium modulated by the Akt pathway

Abstract

Abstract Introduction: Cancer stem cells (CSCs) are undifferentiated, highly tumorigenic, and therapy-resistant tumor cells which play a central role in cancer formation and progression. To date, CSCs have been widely conceptualized as a small population of self-renewing founder cells from which all other cancer cells arise, an intuitive but unproven model. Here we propose a novel alternative model based on our discovery of a dynamic two-way equilibrium: CSCs indeed generate differentiated cancer cells, but the differentiated cancer cells in turn de-differentiate and replenish the CSC population. Methods: Subpopulations of CSCs and differentiated cancer cells within the J82 human bladder cancer cell line were tracked over serial time points using FACS with Hoechst dye staining, a method that identified a small side population (SP) of Hoechst negative CSCs versus the majority of Hoechst positive differentiated cancer cells (non-SP). To rule out artifactual cross-contamination during sorting, the Hoechst/FACS experiments were also conducted with GFP labeled SP and non-SP subpopulations. Results: We identified an SP subpopulation and confirmed its CSC phenotype relative to the non-SP subpopulation (increased progenitor gene transcripts, clonogenicity, and tumorigenicity). Surprisingly, we observed that the SP subpopulation cycled in size over time, comprising ∼1% of all cells after seeding, then rising to >20% of all cells as the culture neared confluence, and then falling to ∼1% again after passaging to a new plate, and so on through serial passages. Moreover, non-SP cells that were isolated by FACS and cultured independently gave rise to a new SP subpopulation over several days. To validate this phenomenon, we grew GFP+ non-SP cells combined with GFP− SP cells (80:20 ratio) in vitro and in vivo. Remarkably, both in culture and in tumor xenografts the previously GFP− SP subpopulation turned progressively GFP+ as it expanded over time, confirming that the SP subpopulation was arising from conversion of non-SP cells. This conversion was significantly reduced in culture by LY294002, an Akt pathway inhibitor. Conclusion: Our studies demonstrate for the first time that CSCs are not a small and static group of founder cells; rather, they are continuously depleted and repleted in an ongoing state of dynamic equilibrium with differentiated cancer cells. Much like microbial populations, the cancer cells do not proliferate randomly but rather interact in a highly coordinated manner that is governed by environmental factors and mediated at least in part by the Akt pathway. Specifically, under permissive conditions, a rapidly-dividing, differentiated state predominates, but in response to space and nutrient depletion the differentiated cells reconstitute a resistant, dormant CSC population which preserves and eventually regenerates the cancer when favorable conditions return. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5152.