Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors

<p>Abstract</p> <p>Background</p> <p>Although features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed.</p> <p>Methods</p> <p>Following methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells <it>EGFR </it>amplification analysis, LOH/MSI analysis, and <it>P53 </it>nucleotide sequence analysis were performed.</p> <p>Results</p> <p><it>In vitro </it>differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum.</p> <p>Conclusion</p> <p>Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present <it>in vitro </it>multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.</p

Glioblastoma-derived spheroid cultures as an experimental model for analysis of EGFR anomalies

Glioblastoma cell cultures in vitro are frequently used for investigations on the biology of tumors or new therapeutic approaches. Recent reports have emphasized the importance of cell culture type for maintenance of tumor original features. Nevertheless, the ability of GBM cells to preserve EGFR overdosage in vitro remains controversial. Our experimental approach was based on quantitative analysis of EGFR gene dosage in vitro both at DNA and mRNA level. Real-time PCR data were verified with a FISH method allowing for a distinction between EGFR amplification and polysomy 7. We demonstrated that EGFR amplification accompanied by EGFRwt overexpression was maintained in spheroids, but these phenomena were gradually lost in adherent culture. We noticed a rapid decrease of EGFR overdosage already at the initial stage of cell culture establishment. In contrast to EGFR amplification, the maintenance of polysomy 7 resulted in EGFR locus gain and stabilization even in long-term adherent culture in serum presence. Surprisingly, the EGFRwt expression pattern did not reflect the latter phenomenon and we observed no overexpression of the tested gene. Moreover, quantitative analysis demonstrated that expression of the truncated variant of receptor—EGFRvIII was preserved in GBM-derived spheroids at a level comparable to the initial tumor tissue. Our findings are especially important in the light of research using glioblastoma culture as the experimental model for testing novel EGFR-targeted therapeutics in vitro, with special emphasis on the most common mutated form of receptor—EGFRvIII

Eradication of LIG4-deficient glioblastoma cells by the combination of PARP inhibitor and alkylating agent.

Cancer cells often accumulate spontaneous and treatment-induced DNA damage i.e. potentially lethal DNA double strand breaks (DSBs). Targeting DSB repair mechanisms with specific inhibitors could potentially sensitize cancer cells to the toxic effect of DSBs. Current treatment for glioblastoma includes tumor resection followed by radiotherapy and/or temozolomide (TMZ) - an alkylating agent inducing DNA damage. We hypothesize that combination of PARP inhibitor (PARPi) with TMZ in glioblastoma cells displaying downregulation of DSB repair genes could trigger synthetic lethality. In our study, we observed that PARP inhibitor (BMN673) was able to specifically sensitize DNA ligase 4 (LIG4)-deprived glioblastoma cells to TMZ while normal astrocytes were not affected. LIG4 downregulation resulting in low effectiveness of DNA-PK-mediated non-homologous end-joining (D-NHEJ), which in combination with BMN673 and TMZ resulted in accumulation of lethal DSBs and specific eradication of glioblastoma cells. Restoration of the LIG4 expression caused loss of sensitivity to BMN673+TMZ. In conclusion, PARP inhibitor combined with DNA damage inducing agents can be utilized in patients with glioblastoma displaying defects in D-NHEJ

Screening for <i>EGFR</i> Amplifications with a Novel Method and Their Significance for the Outcome of Glioblastoma Patients

<div><p>Glioblastoma is a highly aggressive tumour of the central nervous system, characterised by poor prognosis irrespective of the applied treatment. The aim of our study was to analyse whether the molecular markers of glioblastoma (<i>i.e. TP53</i> and <i>IDH1</i> mutations, <i>CDKN2A</i> deletion, <i>EGFR</i> amplification, chromosome 7 polysomy and <i>EGFRvIII</i> expression) could be associated with distinct prognosis and/or response to the therapy. Moreover, we describe a method which allows for a reliable, as well as time- and cost-effective, screening for <i>EGFR</i> amplification and chromosome 7 polysomy with quantitative Real-Time PCR at DNA level. In the clinical data, only the patient’s age had prognostic significance (continuous: HR = 1.04; p<0.01). At the molecular level, <i>EGFRvIII</i> expression was associated with a better prognosis (HR = 0.37; p = 0.04). Intriguingly, <i>EGFR</i> amplification was associated with a worse outcome in younger patients (HR = 3.75; p<0.01) and in patients treated with radiotherapy (HR = 2.71; p = 0.03). We did not observe any difference between the patients with the amplification treated with radiotherapy and the patients without such a treatment. Next, <i>EGFR</i> amplification was related to a better prognosis in combination with the homozygous <i>CDKN2A</i> deletion (HR = 0.12; p = 0.01), but to a poorer prognosis in combination with chromosome 7 polysomy (HR = 14.88; p = 0.01). Importantly, the results emphasise the necessity to distinguish both mechanisms of the increased <i>EGFR</i> gene copy number (amplification and polysomy). To conclude, although the data presented here require validation in different groups of patients, they strongly advocate the consideration of the patient’s tumour molecular characteristics in the selection of the therapy.</p></div

Selected results of the statistical analyses.

<p>Cox’s Proprtional Hazard values pertain to the univariate analysis for age and to the multivariate analysis (adjusted for age) for other analyses. HR values refer to the presence of given feature.</p><p>For example:</p><p>In the group of patients younger than 60 years old, the risk of death over given time is 3.745 times higher in those with <i>EGFR</i> amplification than in those without the amplification.</p><p>Abbreviations:</p><p><i>TP53</i>– <i>TP53</i> mutation;</p><p><i>EGFR</i> – <i>EGFR</i> amplification;</p><p>Poly 7– chromosome 7 polysomy;</p><p><i>EGFRvIII</i> – <i>EGFRvIII</i> expression;</p><p><i>CDKN2A</i> – <i>CDKN2A</i> deletion;</p><p>y. o. – years old.</p

Kaplan-Meier diagrams depicting differences in survival times related to the clinical aspects.

<p>The attached table presents statistical data for each diagram. Cox's proportional hazard refers to univariate analysis for diagram A and to multivariate analysis for diagrams B, C, D. The calculated HR values pertain to the second subgroup listed (“total” subgroup for diagram B), while the HR values of the first subgroup (cumulatively of “partial” and “subtotal” subgroups for diagram B) equal to 1. ♦ - complete responses; Δ - censored responses. A. age of the patient, the threshold of 60 years included in the “younger” subgroup; B. extent of resection; C. radiotherapy; D. radio-chemotherapy.</p

Kaplan-Meier diagrams depicting differences in survival times related to the <i>EGFR</i> amplification and clinical aspects.

<p>The attached table presents statistical data for each diagram. Cox’s proportional hazard refers to multivariate analysis. The calculated HR values pertain to the second subgroup listed, while the HR values of the first subgroup equal to 1. ♦ - complete responses; Δ - censored responses. A. <i>EGFR</i> amplification in patients aged 60 years and less; B. <i>EGFR</i> amplification in patients treated with radiotherapy; C. comparison of patients not treated with radiotherapy with those with the <i>EGFR</i> amplification treated with radiotherapy; D. <i>EGFR</i> amplification in a cumulative group of younger patients and those treated with radiotherapy.</p

Kaplan-Meier diagrams depicting differences in survival times related to the molecular aspects.

<p>The attached table presents statistical data for each diagram. Cox’s proportional hazard refers to multivariate analysis. The calculated HR values pertain to the second subgroup listed, while the HR values of the first subgroup equal to 1. ♦ - complete responses; Δ - censored responses. A. <i>EGFRvIII</i> expression; B. <i>CDKN2A</i> deletion; C. The combination of <i>CDKN2A</i> deletion with <i>EGFR</i> amplification; D. the combination of chromosome 7 polysomy with <i>EGFR</i> amplification.</p