Proliferation and pathway-modulation by Sunitinib.

<p>(A) Western Blot analysis to analyze AKT phosphorylation (Ser473) was performed with 18 BTIC lines of which 2 representatives are shown. To evaluate distinct phosphorylation patterns under treatment, BTIC were treated with 1 μM Sunitinib or 0.00025% DMSO for 6 hours with growth factor supplementation (25 ng/ml) as outlined. GAPDH was used as protein loading control. (B) BTICs were incubated with 1 μM Sunitinib or 0.00025% DMSO (control), and the XTT proliferation assay was performed after 96 h. The relative difference of the mean proliferation relative to control is blotted in a dot blot graph (y-axis) against the corresponding BTIC line (x-axis). Each data point indicates the result of an individual experiment. (C) Growth pattern in a responding (BTIC-25) and a non-responding (BTIC-26) BTIC line. Representative pictures are shown for two differently responding BTIC lines.</p

Gene expression pattern and prediction of proliferation.

<p>(A) Heat map of the 300 most differentially expressed genes when comparing Sunitinib treated with untreated samples. The samples are nicely separated into treated vs. untreated samples. (B) The FC (fold change) expression difference between DMSO and Sunitinib treated samples were calculated for each gene with expression values obtained from construction and validation data sets, respectively. The correlation of FC values is shown as a scatterplot (correlation coefficient, 0.7; p<0.001). (C) Proliferation inhibition after 96 hours treatment was predicted by calculating the sum of weighted expression of 6 signature genes (CLK4, BCLAF1, LOC100130581, ACTG, VAV3, DPF3). Predicted proliferation inhibition was plotted against the average relative proliferation inhibition (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151312#pone.0151312.g001" target="_blank">Fig 1B</a>) (correlation coefficient, -0.257; p = 0.304).</p

Clinical and biological information of parental tumor specimen from the analyzed BTIC panel vs. BTIC lines.

<p>Clinical and biological information of parental tumor specimen from the analyzed BTIC panel vs. BTIC lines.</p

Cellular growth and proliferation under Sunitinib treatment.

<p>BTICs were incubated with 1 µM Sunitinib or 0.00025% DMSO (control), and the XTT proliferation assay was performed after 96 h. Each individual assay was performed with five replicates per treatment group. The assay was repeated at least three times for each cell line. (A) Growth pattern in a responding (BTIC-5) and a non-responding (BTIC-16) BTIC line. Representative pictures are shown for two differently responding BTIC lines. (B) The mean absorbance of Sunitinib treated cells relative to control cells obtained in an individual assay was assessed after 24 h, 96 h and 144 hours incubation period and is plotted in a dot blot graph (y-axis) against incubation time (x-axis). (C) The relative difference of the mean proliferation relative to control is blotted in a dot blot graph (y-axis) against the corresponding BTIC line (x-axis). Each data point indicates the result of an individual experiment. The assay shows the variety of effects in the investigated lines. (D) Prediction of proliferation based on gene expression 6 h after treatment in vivo. The x-axis shows cross validated predictions of proliferation response after 96 hours based on gene expression levels monitored 6 hours after treatment, while the y-axis shows the actual proliferation measurements after 96 hours. The correlation between predicted and measured proliferation is significant (p<0.01, chi-square test). (E) Failed prediction of proliferation using expression values from untreated samples. There is no significant correlation between predictions and measurements (p = 0.98).</p

Heterogeneity of expression response to Sunitinib treatment.

<p>(A) Shown is a heat map of the 300 most differentially expressed genes when comparing Sunitinib treated with untreated samples. The samples are nicely separated into treated vs. untreated samples. However, the pronounced stripes in the heat map indicate that the vast majority of genes change expression only in subsets of cases. (B) The mean logFCs between control and Sunitinib treated samples for the predictive genes (CLK4, BCLAF1, LOC100130581, ACTG2, VAV3, DPF3) of 6 BTIC lines was calculated using (i) Microarray data and (ii) β-Actin normalized expression values assessed by qPCR for each individual gene.</p

Phosphorylation pattern of signaling molecules downstream of Sunitinib target receptor tyrosine kinases.

<p>Western Blot analysis was performed with 18 BTIC lines of which 3 representatives are shown. (A) To evaluate distinct phosphorylation patterns under treatment, BTIC-1 was treated with 1 µM Sunitinib or 0.00025% DMSO for 6 hours with growth factor supplementation (25 ng/ml) as outlined. (B) To evaluate a dose curve for Sunitinib, BTIC-14 cells were incubated with different Sunitinib-concentrations or 0.001% DMSO in media supplemented with 25 ng/ml of each VEGFA and PDGF-AB. (C) After definition of growth factor supplementation and Sunitinib dose, Western Blot analysis for changes in phosphorylation after treatment with Sunitinib was performed with 18 BTIC lines of which 3 representatives are shown. Cells were treated with 1 µM Sunitinib or 0.00025% DMSO with (+) or without (–) growth factors (GF) PDGF-AB and VEGFA (25 ng/ml) for 6 h after incubation in growth factor free medium for 16 h. The asterisks (*) indicates the corresponding loading control. GAPDH was used as protein loading control.</p

Phosphorylation pattern of signaling molecules after treatment.

<p>To evaluate changes in the phosphorylation of Sunitinib targets, cell lines were treated with growth factors (GF; 25 ng/ml of hPDGF-AB and hVEGF-A), 1 µM Sunitinib, or a combination of Sunitinib plus growth factor in 18 BTIC lines of high-grade gliomas. Changes in the phosphorylation intensity of pSTAT3, pAKT and pERK1/2 compared to the untreated control sample were graded as follows by three independent investigators: ++ strong increase, + moderate increase, 0 no change, – moderate decrease, – – strong decrease. Some samples were unevaluable (u.e.) or showed no signal (n.S.).</p><p>Phosphorylation pattern of signaling molecules after treatment.</p

Metformin inhibits proliferation and migration of glioblastoma cells independently of TGF-β2

<p>To this day, glioblastoma (GBM) remains an incurable brain tumor. Previous research has shown that metformin, an oral anti-diabetic drug, may decrease GBM cell proliferation and migration especially in brain tumor initiating cells (BTICs). As transforming growth factor β 2 (TGF-β₂) has been reported to promote high-grade glioma and is inhibited by metformin in other tumors, we explored whether metformin directly interferes with TGF-β₂-signaling. Functional investigation of proliferation and migration of primary BTICs after treatment with metformin+/−TGF-β₂ revealed that metformin doses as low as 0.01 mM metformin thrice a day were able to inhibit proliferation of susceptible cell lines, whereas migration was impacted only at higher doses. Known cellular mechanisms of metformin, such as increased lactate secretion, reduced oxygen consumption and activated AMPK-signaling, could be confirmed. However, TGF-β₂ and metformin did not act as functional antagonists, but both rather inhibited proliferation and/or migration, if significant effects were present. We did not observe a relevant influence of metformin on TGF-β₂ mRNA expression (qRT-PCR), TGF-β₂ protein expression (ELISA) or SMAD-signaling (Western blot). Therefore, it seems that metformin does not exert its inhibitory effects on GBM BTIC proliferation and migration by altering TGF-β₂-signaling. Nonetheless, as low doses of metformin are able to reduce proliferation of certain GBM cells, further exploration of predictors of BTICs' susceptibility to metformin appears justified.</p