Ahmed, I.

Durante, M.

Hartel, C.

Klein, C.

Ritter, S.

Weyrather, W.

GSI Repository

Effect of hypoxia on the growth of glioma-initiating cellsC. Klein1,2, C. Hartel1, I. Ahmed1,3, S. Ritter1, W.K. Weyrather1, and M. Durante1,41GSI, Darmstadt, Germany; 2Chemistry Department, TU Darmstadt, Germany; 3Biology Department, TU Darmstadt,Germany; 4Condensed Matter Physics, TU Darmstadt, GermanyIntroductionGlioma-initiating cells (GICs) possess stem cell char-acteristics such as the ability for limitless growth, self-renewal and to differentiate into all kinds of cells foundin the original tumour. GICs are held responsible for thestrong radio- and chemoresistance of gliomas.A solid tumour consists of an outer layer that is sur-rounded by a blood vasculature to provide both nutritionand oxygen. In the inner tumor layers, the oxygen concen-tration decreases with increasing distance from the bloodvessels reaching numbers below 0.5 %. This hypoxic coreis referred to as a stem cell niche. Studies concluded thathypoxia regulates glioblastoma stem cell properties andbiomarker expression [1].Materials and MethodsThree glioma cell lines (#10, #10-IR, and U87) were cul-tured as neurospheres in serum-free neurobasal A mediumsupplemented with B27, EGF and FGF. For the growthcurves, 30.000 normoxic cells were seeded in T25 cultureflasks and cultured under normoxia (21 % O2) and hypoxia(1 % O2). In the course of 2-3 weeks they were trypsinisedand counted regularly. For the neurosphere formation assay30.000 cells were seeded in T25 culture flasks then culturedunder hypoxia and normoxia.Figure 1: Percent neurosphere formation under hypoxia(1 % O2), relative to neurosphere formation under nor-moxia. Error bars represent standard deviation, n=3.Results and DiscussionThe results from the neurosphere formation assay aredisplayed in figure1. The U87 cells showed a drasticallyreduced neurosphere formation ability under hypoxic con-ditions (less than 5 % relative to normoxia). For the celllines #10 and #10-IR the neurosphere formation in hypoxiawas 35 % and 53 %, respectively.Cell growth curves were compiled for cell lines #10 and#10-IR and are displayed in figure 2. Due to the reducedgrowth of U87 cells in hypoxic conditions it was not pos-sible to measure growth curves for this cell line. The dou-bling time (td) for #10 is 54 h in normoxic conditions and107 h under hypoxic conditions. #10-IR cells have a td of68 h under normoxia and 79 h under hypoxia.Figure 2: Growth curves of cell lines #10 (left panel) and#10-IR (right panel) under normoxic (closed symbols) andhypoxic (open symbols) culture conditions. Error bars rep-resent standard deviation, n=3.The cell lines # 10 and #10-IR were established as celllines growing in serum-free self-renewal conditions [2]which promote the stem cell properties. The U87 cellline, in contrast, was established decades ago as a (bulk)glioblastoma cell line and is generally cultured in standardcell culture medium containing fetal calf serum. The abilityof the cell lines # 10 and # 10-IR to grow well in a hypoxicenvironment, which is not shared by the U87 cells, could bea hint that the #10 and #10-IR cells are in a more stem-likestate and can be used as a model for cancer stem-like cellswhile U87 cells are not an appropriate model for cancerstem-like cells.References[1] E.E. Bar et al. ”Hypoxia increases the expression of stem-cellmarkers and promotes clonogenicity in glioblastoma neuro-spheres”, Am J Pathol 177 (2010), p. 1491–1502.[2] A. Barrantes-Freer et al. ”Human glioma-initiating cells showa distinct immature phenotype resembling but not identical toNG2 glia”’, J Neuropathol Exp Neurol 72(4) (2013) p.307GSI SCIENTIFIC REPORT 2013 BIOPHYSICS-19doi:10.15120/GR-2014-1-BIOPHYSICS-19 221

Effect of hypoxia on the growth of glioma-initiating cells

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Effect of hypoxia on the growth of glioma-initiating cells

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