Abstract

Abstract BACKGROUND Cellular heterogeneity has been well established within numerous cancer types, including malignant brain tumours. Initially, cancer stem cells (CSC) have been accounted for formation of phenotypic heterogeneity and tumor progression in glioblastoma (GBM). Recent data, however, suggest that CSCs may not represent a stable entity and intrinsic plasticity plays a key role in tumor adaptation to changing microenvironments. The question arises whether CSCs are a defined subpopulation of tumor cells or whether they represent a changing entity that any cancer cell can adopt depending on the environmental conditions. MATERIAL AND METHODS Intra-tumoral phenotypic heterogeneity was interrogated at the single cell transcriptomic and proteomic level in GBM patient-derived orthotopic xenografts (PDOXs) and stem-like cultures. Tumor cell subpopulations were further classified based on expression of four stem cell-associated membrane markers (CD133, CD15, A2B5 and CD44). The resulting 16 subpopulations were FACS isolated and functionally analyzed. Mathematical Markov modelling was applied to calculate state transitions between cell states. RESULTS GBM patient biopsies, PDOXs and stem-like cell cultures display remarkable stem cell-associated intra-tumoral heterogeneity. Independent of marker expression, all analysed tumor subpopulations carried stem-cell properties and had the capacity to recreate phenotypic heterogeneity. Mathematical modeling revealed a different propensity in reforming the original heterogeneity over time, which was independent of the proliferation index but linked to tumorigenic potential. Although subpopulations varied in their potential to adapt to new environments, all were able to reach a steady state microenvironment-specific equilibrium. CONCLUSION Our results suggest that phenotypic heterogeneity in GBM results from intrinsic plasticity allowing tumor cells to effectively adapt to new microenvironments. Cellular states are non-hierarchical, reversible and occur via stochastic state transitions of existing populations, striving towards an equilibrium instructed by the microenvironment. Our data provides evidence that CSCs do not represent a clonal entity defined by distinct functional properties and transcriptomic signatures, but rather a cellular state that is determined by environmental conditions, which has implications for the design of treatment strategies targeting CSC-like states.