P02.22.B CELLULAR COMPOSITION DETERMINED BY STEMNESS MARKERS IN MONOLAYER GLIOBLASTOMA CULTURES IS TUMOR SPECIFIC, HIGHLY DYNAMIC AND RELATED TO TMZ SENSITIVITY

Abstract

Abstract BACKGROUND Different models are used to study pathophysiology and potential new therapeutic strategies of glioblastoma (GBM) in vitro. Generally, monolayer cultures are chosen because of their high reproducibility, while spheroid cultures are preferred to better recapitulate tumor heterogeneity and effects of the micro-environment. A common assumption is that monolayer cultures display less plasticity than spheroid cultures. In this study we examine the plasticity of stem cell-like cells in monolayer cultures and their relationship to temozolomide (TMZ) sensitivity. MATERIAL AND METHODS Three patient-derived GBM cultures: one TMZ sensitive, one induced TMZ-resistant and one naturally TMZ-resistant were used. All cultures were sorted in four subpopulations (CD133+/CD44+, CD133+/CD44-, CD133-/CD44+ and CD133-/CD44-) and subsequently cultured. Cellular composition was determined at multiple timepoints by immunofluorescent imaging and FACS-analysis. The effect of seeding density on cellular composition was assessed. Furthermore the effect of original culture composition on sphere forming capacity and TMZ sensitivity was assessed. RESULTS Culture composition was tumor-specific and the pre-sorting composition was acquired within two passages after sorting, irrespective of original subpopulation or sorting density. Even the CD133-/CD44- subpopulation was able to rapidly re-acquire the initial tumor composition. Furthermore, sphere-forming capacity was restored four passages after sorting. Interestingly, cells that were inherently resistant to TMZ expressed higher levels of CD44 than TMZ-sensitive cells. There was no relationship between expression of CD133 and sensitivity to TMZ. Sensitivity to TMZ was similar in all subpopulations four passages after sorting. CONCLUSION Monolayer cultures display rapid plasticity with regard to the expression of the stemness markers CD133 and CD44. Culture composition is tumor specific and inherently programmed in each tumor cell. TMZ sensitivity seems to be mostly related to CD44 expression, and not to CD133 expression. Sphere forming capacity and TMZ sensitivity are restored soon after sorting, even in initially differentiated GBM cells.