SPOT-006 Stem cell-associated heterogeneity in glioblastoma is a result of intrinsic tumour plasticity shaped by the microenvironment

Introduction To date there is no curative treatment available for patients with Glioblastoma (GBM). The cancer stem cell (CSC) hypothesis posits that GBMs rely on a small subpopulation of cells with stemness properties responsible for tumour progression and recurrence. Recent experimental data from GBM and other cancers however suggest that CSCs may not be a stable entity. The question arises whether such cells are a defined subpopulation of tumour cells or whether they represent a changing entity that any cancer cell can adopt depending on the environmental conditions. Material and methods Tumour cell subpopulations were classified based on their expression of four chosen cell membrane markers (CD133, CD15, A2B5 and CD44). The resulting 16 subpopulations were FACS isolated and analysed for self-renewal and ability to reform the original heterogeneous population in different environments. Mathematical Markov modelling was applied to calculate state transitions between cell states. Intra-tumoral heterogeneity was further interrogated at the single cell transcriptomic level. Results and discussions Similar to patient biopsies, we observed markers to be heterogeneously expressed in patient-derived GBM xenografts and stem-like cell cultures. All analysed tumour cell subpopulations carried stem-cell properties and had the capacity to adapt their marker expression profiles to give rise to the original subpopulations. Mathematical modelling 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 GBM 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. 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 stem cell-associated phenotypic heterogeneity in GBM is a result of intrinsic plasticity, which has important implications for the design of treatment strategies targeting cancer stem-like states.