TMZ treatment of non-CSCs leads to increased tumorigenicity <i>in vitro</i> which can be diminished by combination treatment with Dox.

<p>Tumorigenicity of GBM cell lines after treatment with TMZ, Dox or a combination of both drugs in vitro, * p<0.05, *** p<0.001, Mann Whitney rank sum test.</p

Mitochondria content of GBM non-CSCs can be affected by temozolomide and doxycycline treatment.

<p>A) Quantification of mitochondria amount in GBM cell lines via qPCR analysis of mitochondrial DNA in relation to nuclear DNA under different treatment conditions, results are depicted as mean values of 3 independent experiments in triplicates, error bars indicate the standard deviation; * p<0.05, *** p<0.001; B) Fluorescence staining of mitochondria in GBM cell lines under different treatment conditions.</p

Temozolomide-induced increase of tumorigenicity can be diminished by targeting of mitochondria in <i>in vitro</i> models of patient individual glioblastoma

<div><p>Glioblastoma multiforme (GBM) is a highly heterogeneous and aggressive brain tumor with a dismal prognosis. Development of resistance towards cytostatic drugs like the GBM standard drug temozolomide is a severe problem in GBM treatment. One potential source of GBM relapse could be so called cancer stem like cells (CSCs). These represent an undifferentiated subpopulation of cells with high potential for tumor initiation. Furthermore, it has been shown that differentiated GBM cells can regain CSC properties when exposed to continuous temozolomide treatment <i>in vitro</i>. In this study, treatment of several primary GBM cell lines with clinically relevant doses of temozolomide increased their tumorigenicity as determined by colony formation assays in soft agar. Increased tumorigenicity is a known property of CSCs. Hence, therapy options that specifically target CSCs are under investigation. CSCs appear to be particularly dependent on mitochondria biogenesis which may represent a useful target for CSC elimination. Toxicity towards mitochondria is a known side effect of several antibiotics. Thus, addition of antibiotics like doxycycline may represent a useful tool to inhibit CSCs in GBM. Here, we show that combining temozolomide treatment of primary GBM cells with doxycycline could counteract the increase of tumorigenicity induced by temozolomide treatment.</p></div

CD15 expression is increased in clinical samples of two relapsed GBM.

<p>IHC staining of nestin and CD15 in clinical samples of two cases pre and post chemotherapy with TMZ, 200x magnification.</p

Nestin and CD15 expression after <i>in vitro</i> treatment with TMZ, Dox and a combination of both drugs.

<p>Upper panels show western blot analysis of GBM non-CSCs under different treatment conditions (50μM TMZ, 50μM Dox or 50μM each), Tubulin represents the loading control. Lower panels are results from densitometric scanning analyses of the western blot signals. Results are given as relative expression to untreated control cells.</p

Sensitivity of patient individual GBM cell lines towards TMZ and Dox.

<p>Results are given as median values of 4 independent experiments in triplicates. Error bars represent the standard deviation, Ctrl: untreated cells, TMZ: 50μM temozolomide, Dox: 50μM doxycycline, T+D: combination treatment with temozolomide and doxycycline (50μM each), *p<0.05, Mann Whitney Rank sum test.</p

IC₅₀ values.

<p>Calculated IC₅₀ values for all cell line pairs and substances tested are listed.</p

Primers used for mutation analyses.

<p>Primers used for mutation analyses.</p

Establishment and Characterization of Primary Glioblastoma Cell Lines from Fresh and Frozen Material: A Detailed Comparison

<div><p>Background</p><p>Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. High molecular heterogeneity of GBM tumors is well recognized, forming the rationale for molecular tests required before administration of several of the novel therapeutics rapidly entering the clinics. One model that has gained wide acceptance is the primary cell culture model. The laborious and time consuming process is rewarded with a relative high success rate (about 60%). We here describe and evaluate a very simple cryopreservation procedure for GBM tissue prior to model establishment that will considerably reduce the logistic complexity.</p><p>Methods</p><p>Twenty-seven GBM samples collected ad hoc were prepared for primary cell culture freshly from surgery (#1) and after cryopreservation (#2).</p><p>Results</p><p>Take rates after cryopreservation (59%) were as satisfactory as from fresh tissue (63%; p = 1.000). We did not observe any relevant molecular or phenotypic differences between cell lines established from fresh or vitally frozen tissue. Further, sensitivity both towards standard chemotherapeutic agents (Temozolomide, BCNU and Vincristine) and novel agents like the receptor tyrosine kinase inhibitor Imatinib did not differ.</p><p>Conclusions</p><p>Our simple cryopreservation procedure facilitates collection, long-time storage and propagation (modeling) of clinical GBM specimens (potentially also from distant centers) for basic research, (pre-) clinical studies of novel therapies and individual response prediction.</p></div

Doubling times.

<p>Mean doubling times for the cell lines are displayed placing the pairs side by side. The doubling time for each cell line was determined three times; the mean doubling time (in h) and standard deviation were calculated and depicted in a bar chart.</p